ANNUAL
REPORT
Division of Intramural Research Programs
National Institute of Mental Health
October 1, 1986 - September 30, 1987
VOLUME II PART 1
INDIVIDUAL PROJECT REPORTS
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVtCES
Public Health Service
Alcohol, Drug Abuse, and Mental Health Administration
National Institute of Mental Health
Division of Intramural Research Programs
JAN 1 9 1993
National Institutes of Health
_ANm[AL REPORT
niVISION OF INTRAMURAL RFSFARCH PROGRAMS
NATIONAL INSTITUTE OF MENTAL HEALTH
It
October 1, 1PR6 - September 30, 199,7
VOLUME II PART I
INniVinilAL PROJECT REPORTS
110. U
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ANNUAL RRPORT
DIVISION OF INTRAMURAL RESEARCH PROfiRAMS
NATIONAL INSTITUTE OF MENTAL HEALTH
October 1, 1986 - September 30, 1987
TABLE OF CONTENTS
VOLUME II - INniVinilAL PROJECT REPORTS
PART I
OFFICE OF THE DIRECTOR
Office of the Clinical Director
ZOl MH 02188-03 OCD Biological Studies of Borderline
Personality Disorder 1
BIOLOGICAL PSYCHIATRY BRANCH
Office of the Chief
ZOl MH 00183-02 BP Biology and Behavior of Aggression
and Suicide 7
Section on Psychobiology
ZOl MH 00070-14 BP Psychological and Biological
Interactions in the Mood and
Anxiety Disorders 15
Unit on Anxiety and Affective Disorders
ZOl MH 00071-07 BP Psychobiol ogical Correlates and
Treatment of Pane and Related
Mood Disorders 37
Section on Clinical Neuroendocrinology
ZOl MH 00452-12 BP Neuroendocrine Studies of Major
Psychiatric Disorders 69
ZOl MH 01090-11 BP Studies of Central Nervous Sys-
tem Functional Anatomy 95
I
Unit on Peptides
zni m ooiftn-n5 bp
zni MH nni8i-04 rp
NIMH Consultation Liaison Service
zni MH nni«?-n4 rp
Psychobiology and Treatment of Men-
strual ly-Related Mood Disorders. .101
Hormonal Studies of Affective
Disorders 107
Rehavioral Medicine Ill
Unit on Rehavioral Pharmacology
ZOl MH nni47-l? RP
Rehavioral and Physiological
Effects of Rrain Peptides and
Other Psychoactive Compounds. .. .117
Unit on Neurochemistry
ZOl MH 00400-05 BP
ZOl MH 01R31-11 RP
ZOl MH 01833-07 BP
Clinical Psychobiology Branch
ZOl MH 00450-13 CP
ZOl MH 0?iq3-05 CP
ZOl MH 0?iq7-01 CP
ZOl MH 0??01-05 CP
ZOl MH 0??05-0? CP
ZOl MH 0??06-03 CP
Protein Phosphorylation in
Brain 127
Basic and Clinical Studies of
Neuronal and Glial Enolases 131
Asenosine Receptors in the CNS..137
Riological Rhythms In Affective
Illness 145
Clinical Studies of Insomnia 149
Study of the Effects of Light and
Triazolam on Delayed Sleep Phase
Synd rome 151
Early Versus Late Partial Sleep
Deprivation In the Treatment of
Depression 155
Antidepressant Effects of Light
In Seasonal Affective Disorder
and Normal Controls 157
Neurobiology of Seasonal Affective
Disorder and Light Therapy 165
II
zni MH n??2^-()i cp
zni MH n???3-n4 cp
Zni MH 07225-OA CP
zni MH n?290-n3 cp
zoi MH n2;?92-n3 cp
zni MH n?294-n3 cp
zni MH n23n3-n? cp
zni MH n?324-n? cp
zni MH n?325-n? cp
zni MH n2326-n? cp
zni MH n23?7-n2 cp
zni MH n232fi-n? cp
zni MH n2382-ni cp
zni MH n2383-ni cp
zni MH n24n2-ni cp
The Treatment of Rapid-Cycling Manic
nepressives with Thyroxine 173
Pentobarbital and Ethanol Toxicity:
Relation to the Benzodiazepine
Receptor 17fi
Studies on the Role of Calcium
Flux in the Sleep-Inducing Effects
on Flu Tazepam 177
Melatonin Analysis of Clinical
Samples 179
Melatonin Effects on Hormone-
Stimulated Cell Growth 181
Antidepressant Pharmacology of the
Rodent Circadian System 183
Studies of Sleep in Psychiatric
Illness 187
Neuroendocrine Modulation of Cel-
lular Immune Response 189
Light and Lymphocyte Activity:
Basic and Clinical Studies 191
Behavioral Modulation of the Cel-
lular Immune Response 193
Direct Effect of Lymphokines on
Cultured Human Breast Cancer
Cells 19f^
Direct Effects of IL-? on Cultured
Anterior Pituitaries 199
An Animal Model for Human Sleep
Apnea 2ni
Effects on Sleep of a Microinjec-
tion of Triazolam to Discrete
Brain Loci ?0^
Causes and Treatment of Summer
Depression 2n9
III
Zni MH n24n3-ni CP Mechamsm of Action of
Mel at on i n ?13
Zni MH 0?405-ni CP Chemical Antidepressant Effects
on Rody Mass and Body Composition
i n Hampsters J'lB
LARnPATORY OF CLINICAL SCIENCE
Section on Analytical Riochemistry
Zni MH nn?74-13 LCS Methods of Ionization in Mass
Spectrometry ?1^
Zni MH nn?76-n8 ICS Metabolism of Melatonin ?23
ZOl MH 00277-08 LCS Synthesis of Stable Isotope
Label ed Compounds 22%
ZOl MH 0027P-05 LCS Pharmacology of Neurotoxins 221
ZOl MH 02384-01 LCS Brain Ouinolinic Acid Metabolism:
Role in Neuropathology 233
Section on Biomedical Psychiatry
ZOl MH 00351-13 LCS Clinical Pharmacology of the
Central Nervous System 237
ZOl MH 02289-03 LCS Psychobiology of Eating Dis-
orders 241
Section on Clinical Neuropharmacology
ZOl MH 00326-14 LCS Clinical Neuropharmacology and
Psychobiology of Repression and
Mania 251
ZOl MH 00332-OQ LCS Animal Models for the Study of
Neuropharmacologic Effects 255
ZOl MH 00336-08 LCS The Phenomenology and Treatment
of Obsessive-Compulsive Disorder
in Adults 259
IV
zni MH nn337-nR LCS Neuropharmacology of Neuroendo-
crine and Neurotransmitter
Regulatory Mechanisms ?fi3
zni MH nn339-nfi LCS Neuropharmacology of Cognition
and Mood in Geriatric
Neuropsychiatry Pfi'^
Section on Clinical Pharmacology
Zni MH 00433-07 LCS Role of Neuropeptides and Biogenic
Amines in Neuroendocrine
Regulation ?77
ZOl MH 00447-18 LCS Amine Neurotransmitters and Meta-
bolites in Mental Illness ?85
ZOl MH 01850-10 LCS Clinical Pharmacology of
Antidepressants 7.93
ZOl MH 018B5-03 LCS Central Neurochemistry Service. . .301
ZOl MH 018fi0-01 LCS The Role of Epinephrine in
Rrain 309
Section on Comparative Studies of Rrain and Behavior
ZOl MH 00787-08 LCS Brain Mechanisms of Isolation
Call in Squirrel Monkey (Saimiri
sciureus) 313
ZOl MH 00796-0? LCS Cytochemical Tracing of Thalamic
Connections with Midline Frontal
Cortex 319
ZOl MH 00797-02 LCS Neurobiology of Attachment 323
ZOl MH 00798-01 LCS Studies on the Development of the
Cerebral Cortex 327
ZOl MH 00799-01 LCS Studies on Postnatal
Neuronogenesis 333
ZOl MH 02219-04 LCS Animal Models of Anxiety 337
Section on Histopharmacology
zni MH nn38?-i3 i.cs
Zni MH 00388-11 LCS
ZOl MH 00396-Oq LCS
ZOl MH 00397-09 LCS
ZOl MH 0?377-01 LCS
Localization and Characterization
of Brain Neurochemicals 341
Coexistence of Peptides and
Neurotransmitters 34 B
A Study of Proteins Within the
CNS by Two-Oimensional Gel
Electrophoresis 349
Autoimmune Aspects of nisease. . . .355
A Study of Adenosine Receptors:
Isolation and Characteri zation. . .359
Unit on Research in Behavioral Systems
ZOl MH 0?239-03 LCS Conceptual Analysis of Complex
Biobehavioral Population
Systems 363
Child Psychiatry Branch
ZOl MH 00153-10 CHP Treatment of Obsessional Children
and Adolescents with Clomi-
prami ne 365
ZOl MH 00161-OQ CHP Behavioral Effects of Oietary
Substances in Normal and Hyper-
active Children 369
ZOl MH 00178-06 CHP Rrain Structure and Function in
Oevelopmental Neuropsychiatric
Disorders 373
ZOl MH 00301-05 CHP Diagnosis in Child Psychiatry 377
ZOl MH o??40-03 CHP Neurobiology of Attention Deficit
Disorder 381
VI
CLINICAL NEIIRORFNETICS BRANCH
Section on Clinical Genetics
Office of the Chief
zni MH nnna4-i3 cng
zni MH nona6-ii cng
ZOl MH n2?36-n3 CNG
zni MH a?.?.37-03 CNG
Section on Riochemical Genetics
zni MH nnQ35-?n cng
zni MH nnP4i-n7 cng
Genetic Biologic Studies of
Psychiatric Hisorders .385
Gutpatient Clinic for Genetic and
Pharmacologic Studies of
Affective Disorders 393
Schizophrenia Studies 401
Molecular Genetics of Neuropsy-
chiatric Hisorders 405
Studies of Plasmids and Small
Genomes in Human Cells 413
Riochemical Genetics and Meta-
bol ic Diseases 419
CLINICAL NEIIRnSCIENCE BRANCH
Section on Preclinical Studies
zni MH ni«36-n9 ns
zni MH n2186-05 NS
zni MH n?34n-n? ns
zni MH n?34i-n2 ns
zni MH r)?342-n? ns
GARA/Receptors in the Central
Nervous System: Biochemistry to
Rehavi or 4?9
Rrain Recognition Sites for
Stimulants and Antidepressants:
Relationship to Pharmacological
Activity 437
Riochemical and Clinical Studies
of Gaucher Disease and
nther Neurogenetic Disorders 443
Correction of Inherited Enzyme
Deficiencies by Gene Transfer. .. .447
Gene Regulation Within the Nervous
System 451
VII
Zni MH 0^343-0? NS Molecular Renetics of Inherited
Neurologic and Psychiatric
Disorders 453
zni MH n?344-n? ns Neuropsychiatric nisorders: Protein
Structure Activity Studies 457
Section on Clinical Studies
Zni MH nnil?-in ns Endorphin Research in Mental
II 1 ness 459
Zni MH n?lRl-n5 ns Neurobiology of Schizophrenia 461
zni MH 02184-05 NS Neurobiology of Depression 469
Zni MH n?l«7-n4 ns Diazepam Infusions as a Measure
of Benzodiazepine Receptor
Sensitivity in Hunans 475
Zni MH n?lR8-n3 ns Riological Studies of Borderline
Personal ity Disorder 479
Section on Molecular Pharmacology
Zni MH nni79-n6 ns Morphological Aspects of
Peptides in Mammalian Brain 481
Zni MH n?177-n5 ns Behavioral Functions of Neuro-
peptides 483
Zni MH n?17R-n4 ns Pharmacology of Anxiety 489
Zni MH n?179-05 NS Animal Models of Neuropsychiatric
Disorders 493
Zni MH n?18n-n5 ns Electrophysiological Studies of
Peptidergic and GABAergic Function
in Mammalian Brain 497
Section on Brain Biochemistry
Zni MH n?18?-n5 ns Toward the Visualization of Opiate
Receptors in Living Humans 503
VIII
Zni m 02183-05 NS Is Schizophrenia an Autoimmune
Neuropeptide Receptor nisease?. . .505
ZOl MH 021R9-04 NS Neuropeptides and Their Receptors
are Shared by the Brain and the
Immune System 509
ZOl MH 02190-04 NS Oistribution and Properties of
Opiate and Other Brain
Receptors 517
ZOl MH 021P1-02 NS Brain Receptors for the AIOS Virus
and Other Neurotrophic Vi ruses. . .521
LABORATORY OF DEVELOPMENTAL PSYCHOLOGY
ZOl MH 02152-08 LOP Discipline and Parental Control
in Families with Affective Dis-
orders 525
ZOl MH 02155-08 LDP Children of Depressed and Normal
Parents 529
ZOl MH 02156-08 LDP Personality of Children Reared by
Normal and Depressed Mothers:
Inhibited Children 533
ZOl MH 02164-07 LDP Biological Changes and Physiological
Functioning During Adolescence. . .537
ZOl MH 02169-05 LDP Interactions Between Siblings with
a Depressed Parent 541
ZOl MH 02170-05 LDP Psychiatric Assessment of Infants
and Toddl ers 543
ZOl MH 02171-04 LDP Protective and Risk Factors in
Childrearing: Contributions of
Fathers 547
ZOl MH 02207-04 LDP The Affective Rearing Environment:
A Comparison of Normal and
Depressed Parents 549
ZOl MH 02229-03 LDP Vocalic Analysis of Natural Dis-
course in Well and Depressed
Mothers 553
IX
zni MH n??3i-n3 ldp
zni MH n??3?-n3 inp
zni MH n??33-n? inp
zni MH n??34-n? inp
zni MH n??q7-n? lop
zni MH n?3fii-ni lop
zni MH n?3fi?-ni inp
zni MH n?3fi3-ni lop
zni MH n?3(S4-ni inp
zni MH n?365-ni lrp
zni MH n?3fi6-ni lhp
zni MH n2367-ni inp
zni MH n?3fi«-ni inp
zni MH n?3fiQ-ni lhp
zni MH n?37n-ni lhp
Riological -Behavioral Relations
in Farly Adolescence 557
Pevelopment of Ability to Con-
centrate in Children of Repressed
and Well Mothers 5fil
The Development of Guilt: Lan-
guage, Emotions, and Rehavior. . . .563
Infants of Chronically Repressed,
and Normal Parents 567
Generosity and Sharing in Children
of Normal or Affectively His-
turbed Parents 56P
Relation Between Self- and
Teacher-reports of Social -Emotional
Adjustment 573
Physical /Neurological Hpvelopment
in Children of Healthy and
Repressed Mothers 575
Information-Processing Deficits
in Schizophrenic Children 57P
A Follow-up Investigation of
nffspring of Bipolar Parents 583
The Psychobiological Effects of
Sexual Abuse 587
The Psychophysiology of Multiple
Personal ity Disorder 589
The Clinical Phenomenology of
Multiple Personality Disorder 591
The Dissociative Experiences
Scale (DES) 595
Mutual Interpersonal Influence
in Families With and Without
Affective Disorder 59 q
Caregiving Patterns in Stressed
Fami lies fin3
X
zni MH 02371-ni Lnp
zni MH n?372-ni inp
zni MH n?37P-ni lop
zni MH n?3Rn-ni inp
zni MH n238i-ni inp
LARnRAinpY np NEiiRnpsYCHnLnny
zni MH nn478-31 ln
zni MH n?n3?-ii ln
zni MH n?n33-in ln
zni MH n?n35-n7 ln
zni MH n?n3fi-n7 ln
zni MH n?n37-n6 ln
zni MH n?03«-ns ln
zni MH n?n3q-n5 ln
zni MH n?n4n-n4 ln
Patterns of Alliance in Families
With and Without Parental
Depression 6n7
Psychiatric Status of Children of
Depressed Parents 611
Survivor Children 61B
Stressful Life Events and Child-
hood Adjustment 617
Functioning of Repressed Mothers
Within and Between Episodes 6?1
Neural Mechanisms of Cognitive
Memory and Habit Formation 6?.?,
Neural Coding of Visual Stimuli
in the Awake Monkey 633
Functional Mapping of Sensory and
Memory Systems 639
Anatomy of the Primate Visual
System 643
Neural Representations of Visual
Stimuli in the Extrastriate
Cortex 649
Functional Anatomy of the Somato-
sensory Cortex of the Monkey 653
nntogenetic Development of Cognitive
Memory and Habit Formation 659
Pharmacology of Cognitive Memory
and Habit Formation 667
Functional Analysis of Neuro-
transmitter Systems 675
XI
LABORATORY OF PSYCHOLnGY AND PSYCHnPATHOLnnY
ZOl MH 00471-3? LPP Studies of Heredity and Environ-
ment in Schizophrenia 677
ZOl MH 004R4-?7 LPP Psychophysiological Responsivity
and Behavior in Schizophreni a. . . .6R3
ZOl MH 004R6-1!S LPP Psychophysiological Effects of
Stimulant Orugs in Children 6P1
ZOl MH 004qi-ll LPP Personality Factors and Psycho-
physiological' Responses to Chang-
ing Stimulus Input f^^S
ZOl MH 00503-07 LPP Human Clinical Studies of
Attention Oisorder 6"^^
ZOl MH 00504-07 LPP Models in the Monkey of fieneral-
ized Seizures of the Absence
Type 707
ZOl MH 00508-05 LPP Neuropsychological Evaluation of
Psychiatric and Neurological
Patients 711
ZOl MH 00509-05 LPP Attention Oisorders as Assessed by
Event-Related Brain Potential s. . .721
ZOl MH 0??RR-03 LPP Studies on Etiological Factors in
Schizophrenia 733
ZOl MH 0??Q5-0? LPP Oenetic Factors in Response to
Alcohol 739
ZOl MH 02404-01 LPP Psychophysiological Investigations
of Preattentional and Attentional
Function 743
LABORATORY OF SOCIO-ENVIRONMENTAL STHHIES
ZOl MH 0067?-?? LSES Social Psychological Correlates
of Occupational Position 749
ZOl MH 00679-07 LSES Structural Equation Models in the
Analysis of Data with Measurement
Error 755
XII
Zni MH 00680-05 LSES Work Experiences and the neinsti-
tutionalized Mentally 111 7fiP
Zni MH 00681-01 LSES Reciprocal Effects of Self-Esteem
and Depression 761
ZOl MH 0068^-01 LSES Environmental Oeteminants of
Cognitive Functioning 763
LABORATORY OF CELL RIOLOnv
Office of the Chief
ZOl MH 004?4-l? LCR Biologically Active Peptides in
the Brain 767
ZOl MH 0;'30?-0? LCR Biochemical Studies on Myelin
Rasic Protein 775
Section on Biochemical Pharmacology
ZOl MH 00422-16 LCR Neuropharmacology of Circadian
Rhythms 777
ZOl MH 00429-08 LCR Biochemistry of Membranes 781
Unit on Riochemistry
ZOl MH 00427-10 LCR On the Mechanism of Signal Trans-
duction Through Receptors 783
Unit on Pharmacology
ZOl MH 00434-06 LCR Molecular Mechanisms of Receptor-
Mediated Signal Transduction 785
Unit on Molecular Renetics
ZOl MH 02385-01 LCR Genetic Control of Cell Differen-
tiation, Orowth and Transform-
ation 793
Unit on Molecular and Cellular Neurobiology
ZOl MH 02386-01 LCR Neuropeptide Secretion, Synthesis
and Action in Neural, Endocrine
and Immune Cells 797
XIII
Zni MH n23R7-01 LCR Structural Analysis of the Cn4/HIV
Ligand/Receptor Dyad 805
Unit on Neurobiology
Zni MH 023«^6-01 LCB Mechanical, Thermal and Optical
Signs of Excitation in the
Nervous System 807
LABORATORY OF CFRERRAL METABOLISM
Section on Developmental Neurochemistry
ZOl MH n08Rl-31 LCM Intermediary Energy Metabolism in
Mammal ian Brain 811
ZOl MH 00882-20 LCM Studies on Regional Cerebral Circu-
lation and Metabolism 815
ZOl MH 00887-10 LCM The Extended Visual System of the
Macaque Monkey 823
ZOl MH 0088Q-08 LCM A Method for the Determination of
Local Rates of Protein Synthesis
in Brain 825
ZOl MH 00903-10 LCM Purification and Identification of
Brain Proteinases and their
Cleavage Products 831
ZOl MH 02216-04 LCM Metabolic Mapping of the Brain
During Rewarding Self-
Stimulation «35
ZOl MH 02?17-04 LCM Plasticity in the Developing
Monkey Visual System 83Q
ZOl MH 02220-04 LCM Regional Biochemical Changes in
the Normal Aging Brain 845
ZOl MH 02308-02 LCM Growth and Development of Dopa-
minergic Neurons 849
ZOl MH 02307-02 LCM Role of Proteinases in Production
and Control of Neuropeptides 853
XIV
Section on Clinical Brain Imaging
Zni MH 00507-05 LCM Clinical Rrain Imaging 855
ZOl MH 0?2q6-0? LCM ' In Vivo Tomographic Imaging of
Dopaminergic Systems and their
Turnover 861
LABORATORY OF GENERAL AND COMPARATIVE BIOCHEMISTRY
ZOl MH 00931-14 LRCB Characteristics and Regulation of
S-Adenosyl homocystei ne
Hydrolase 869
ZOl MH 00936-?3 LGCB Homocystinuria: Methionine Metab-
olism in Mammals 875
ZOl MH 00940-06 LGCB Methionine Biosynthesis in Higher
Plants 879
ZOl MH 00942-06 LGCB Biochemical Reactions in Mamma-
lian Cell Chemotaxis 885
ZOl MH 00943-06 LGCB Pathways of Methionine and Thre-
onine Metabolism and Their Control
in Higher Plants 891
ZOl MH 02321-0? LGCB DNA Methyl ation and Gene E 895
LABORATORY OF MOLECULAR BIOLOGY
Section on Biophysical Chemistry
ZOl MH 01037-19 LMB The Role of the Cell Membrane in
Cellular Organization: A Molec-
ular Study 899
Section on Molecular Genetics
ZOl MH 01035-19 LMB The Process of Lysogeny 903
ZOl MH 02228-03 LMB Genetic Neurobiology of Oroso-
phila 907
Section on Regulatory Proteins
ZOl MH 00934-15 LMB The Biochemical Basis of Peptide
Receptor Activity 911
XV
LABORATORY OF NEDROCHEMISTRY
ZOl MH 01031-19 LNC
ZOl MH 0103P-19 LNC
ZOl MH 01038-19 LNC
ZOl MH 01039-19 LNC
ZOl MH 01040-19 LNC
LABORATORY OF NEUROPHYSIOLOGY
ZOl MH 00981-21 LNP
ZOl MH 01092-09 LNP
ZOl MH 01096-03 LNP
ZOl MH 01097-01 LNP
ZOl MH 01098-01 LNP
ZOl MH 01099-01 LNP
The Conversion of Phenylalanine to
Tyrosine 915
Biosynthesis of Catecholamines. . .919
Phenylketonuria and Other Diseases
Caused by Defects in Biopterin-
Dependent Enzymes 921
Pteridine Biosynthesis 925
Molecular Biology of the Pterin-
Dependent Hydroxylases and Ancil-
lary Enzymes 929
Mechanical, Thermal and Optical
Signs of Excitation in the
Nervous System 933
The Frontal Lobe and the Cerebral
Control of Behavior ..935
Spatial Organization of the Primate
Motor Cortex 943
Activity of Corticostriatal Neurons
in Motor Cortex of Primates During
Wrist Movement 949
Anatomical Analysis of Neuronal
Circuits 953
Neurochemical Interactions Between
Cortical and Striatal Dopaminergic
Activity 957
XVI
DIVISION OF INTRAMURAL RESEARCH PROGRAMS
NATIONAL INSTITUTE OF MENTAL HEALTH
RESEARCH PROJECT SERIAL NUMBER LISTING:
Z01MH00070
Z01MH00071
Z01MH00084
Z01MH00086
ZOlMHOOll?
Z01MH00147
Z01MH00153
Z01MH00161
Z01MH00178
Z01MH00179
Z01MH00180
Z01MH00181
Z01MH00182
Z01MH00183
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Z01MH01031
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Z01MH02206
Z01MHO2207
Z01MH02216
Z01MH02217
Z01MH02219
Z01MH02220
Z01MH02222
XVII
RESEARCH PROJECT SERIAL NUMBER LISTING (Cont'd.)
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ZniMHn2231
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Z01MH023n3
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ZniMH02328
ZniMH02340
Z01MH02341
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ZniMH02343
ZniMHn2344
ZniMH02361
ZniMHn2362
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ZniMH024n5
XVIII
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02188-03 OCD
PERIOD COVERED
October 1, 1986 to September 30,. 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Biological Studies of Borderline Personality Disorder
PRINCIPAL INVESTIGATOR (List other pmlessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: D.L. Gardner Staff Psychiatrist OCD, NIMH
Others: R.W. Cowdry Clinical Director NIMH
K.M. O'Leary Social Worker (Research) OCD, DIRP, NIMH
D. Pickar Chief, Sec. on Clinical Studies NS, NIMH
P. Lucas Clinical Fellow, NRSA Fellow NS, NIMH
D.L. Murphy Chief LCS, NIMH
COOPERATING UNITS (If any)
Office of the Director, Division of Intramural Research Programs, NIMH;
Biological Psychiatry Branch, NIMH; Laboratory of Clinical Science, NIMH
LAB/BRANCH
Office of the Clinical Director
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
1.7
PROFESSIONAL:
1.5
0.2
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
PHS 6M0 (Rev. 1/84)
CPO SI4-»II
Summary ^^^ ^^ 02188-03 OCD
Patients with borderline personality disorder and rejection-sensitive
dysphoria participated in a program of clinical and biological evaluation.
In addition to labile moods and behavioral dyscontrol, a high incidence of
discrete major depressive episodes has been observed. Mood ratings recorded
twice daily on visual analogue scales by borderline patients were compared
with ratings completed by patients with major depression, patients with
premenstrual syndrome and normal volunteers and revealed distinguishing
patterns between the four groups with the borderline patients showing low
global mood ratings with high variability.
On standardized psychiatric rating scales, borderline patients scored high
on depression (Beck Depression Inventory), cognitive symptoms of depression
(Dysfunctional Attitude Scale) and hostility (Buss-Durkee Hostility Aggression
Index) even when not in a current depressive episode. Neuropsychological
testing revealed a pattern of poor performance in tests of tonal memory, a
function linked to the right temporal lobe, and in tests measuring visual-
perceptual abilities.
Computerized tomography scans of the brain were studied and compared to scans
of healthy normal volunteers. No significant differences were found between
the two groups on measures of ventricular brain ratio, third ventricular
size or evidence of frontal lobe atrophy. Lumbar puncture procedures are
being performed to measure cerebrospinal fluid metabllites. Naloxone infusions
are being performed to investigate alteration in pain mechanisms. Preliminary
analysis reveals elevated baseline levels of beta-endorphin and ACTH in the
borderline patients when compared to normal volunteers.
The serotonin agonist, m-chlorphenylpiperazine (m-CPP), was associated with an
activated, euphoric response and preliminary results suggest a blunting of the
prolactin response. Noise and learning, a paradigm of performance under stress
modeled after the learned helplessness model, is being administered to study
reactions in stressful situations and associated neuroendrocrlne responses.
ZOl MH 02188-03 OCD
PROJECT DESCRIPTION
Rejection-sensitive or hysteroid dysphoria is a poorly understood syndrome
occurring in many Individuals with a diagnosis of borderline personality
disorder. This syndrome, described by Klein and others, is characterized by
the rapid onset of a dysphoric mood (sometimes characterized more specifically
by depression, anxiety, or rage) following an actual, threatened, or imagined
rejection. Behavioral dyscontrol is not uncommon, involving violence, direct
Injury to self, or overdosage with sedating medications. This disorder accounts
for a significant number of admissions to short-term psychiatric units, and is
one of the more difficult disorders treated in long-term outpatient
psychotherapy.
There are a number of theories about the etiology of the borderline personality
in general, and rejection-sensitive dysphoria in particular, most emphasizing
developmental psychodynamlcs. However, recent phenomenologlcal and family
history studies of the disorder suggest that this disorder may represent a
variant of affective disorder, a neurophyslological dysfunction of limbic system
functioning, or in some cases an adult variant of minimal brain dysfunction.
To date, little biomedical research has been done to explore possible
underpinnings of this disorder.
Previous findings In this project tend to support a role for biological factors
in the various symptoms of this disorder. A high prevalence of neurological soft
sign abnormalities and psychomotor^psychosensory symptoms were found in this
population. Results of medication trials showed that carbamazepine, an
anticonvulsant was effective in reducing episodes of dyscontrol and Impulsivlty,
while tranylcypromine proved to be an effective antidepressant with some
reduction in emotional lability. Alprazolam, an antianxiety agent, lessened
anxiety but was associated with increased impulsivlty and behavioral dyscontrol.
This project continues to explore the relationships among clinical phenomenology,
developmental factors, and family histories; neurophyslological function
(neuropsychological testing); and biochemical measures including cerebrospinal
fluid studies, provocative infusion studies (naloxone, m-chlorphenylplperazlne)
and endocrine challenge tests (TRH stimulation tests and dexamethasone
suppression tests).
MAJOR FINDINGS
Phenomenology
In addition to elaboration of dysphoric episodes and self-Injurious behavior
described in the previous annual report, we have focused on descriptions of
affective symptomatology and perceptual abnormalities. Depression symptoms
generally are characterized by marked mood lability, often unrelated to
environmental or psychological stimuli; however, a high Incidence of discrete
major depressive epidodes has also been observed. Along with a high family
incidence of depressive disorders, this suggests a strong connection between
the borderline disorder and affective disorders.
ZOl MH 02188-03 OCD
Dally Ratings
Mood lability was further explored with self- ratings of mood recorded
twice daily using visual analogue scales. These ratings reflect global
mood as well as the variability of mood during a given day and variability
of raood from day to day. Ratings by borderline patients were compared
with patients with major depression, patients with premenstrual syndrome,
and normal volunteers. Borderline patients showed low global mood ratings
and a high degree of diurnal variation and day to day variability, and the
combination of these two factors differentiated them statistically from
patients seen in the other three groups.
Neuropsychological Testing
Preliminary results of neuropsychological testing completed on 14 borderline
patients and 11 normal volunteers, show a pattern of poor performance by the
borderline patients on the Seashore Test of Musical Talent, a measure of
tonal memory usually linked to functions of the right temporal lobe, and
poor performance on visual perceptual tasks. Patients with borderline
personality disorder scored high on the Beck Depression Inventory and the
Dysfunctional Attitude Scale, measures of depressive symptoms and cognitions
related to depression. Patients also rated themselves high on the Buss-Durkee
Hostility Aggression Index, a measure of anger and hostility. Information
is being gathered on dissociative phenomena using the Dissociative Episode
Scale, and phenomena related to temporal lobe abnormalities using the
Bear-Fedio Inventory and the psychomotor/ psychosensory symptom rating scale.
Biomedical Studies
To examine systematically the various neurotransmitter systems In the border^
line personality disorder, we planned a series of provocative Infusions
which have been perfomed in other psychiatric populations (e.g., schizophrenia,
major depression). Because of the altered pain mechanisms in this disorder,
we administered naloxone, an opiate antagonist, in a dosage of 2 mg/kg,
to nine borderline patients and 11 normal volunteers. Clinically, one
borderline patient reacted with a transient depressive response and one
nonaal volunteer described feeling "out of sorts" and "down" for several
hours. Preliminary results of biochemical studies shows a higher baseline
level of beta-endorphins and ACTH in the borderline patients than in the
normal volunteers. Naloxone appears to have a negligible effect on
endorphin levels.
The serotonin agonist, ra-chlorphenylpiperazine (m-CPP), was administered to
examine the effects of activation of the serotonergic system on symptomatology.
Seven borderline patients received oral doses of .5 mg/kg. Five patients had
activated responses, including 2 euphoric responses, 2 happy/giddy responses,
and one energized/sarcastic response. Two patients had no clinical response.
Preliminary results of biochemical studies suggest a blunting of the prolactin
response when compared with studies in normal volunteers. Rirther studies are
pla nned .
ZOl MH 02188-03 OCD
Cerebrospinal spinal fluid samples have been collected via lumbar puncture
fran nine borderline patients. These are being assayed for neurotransmitter
metabolites and will be compared with samples from normal volunteers.
Noise and learning, a behavioral paradigm based on the learned helplessness
model of depression, examines the performance of patients under stressful
conditions and blood samples measure hormonal responses to stress. Seven
borderline patients have particpated in this procedure thur far. Two
patients terminated the procedure before completion, stating the procedure
was too stressful. Five patients completed the procedure. Preliminary
analysis of hormonal response suggests an exaggerated Cortisol repsonse
when compared with normal controls. Further studies will be pursued.
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
Rejection-sensitive dysphoria and borderline personality disorder are common
disorders, particularly in the young adult population. They account for a
significant number of short-term psychiatric hospitalizations and are
frequently associated with major, often life-threatening overdoses, with
self-mutilation, and with episodes of violence. The etiology of these
disorders is a matter of great controversy and limited data, as is the role
of medication in the treatment of these individuals.
The evaluation phase of this study provides tentative support for a theory of
these disorders which emphasizes an interaction between developmental traumata
and biological predisposition. If further studies confirm the association
between low threshold for dysphoria and dyscontrol on the one hand and procaine
induced high frequency EEG activity over the temporal lobe on the other, the
line between limbic system' abnormalities and labile mood and impulsive behavior
is strengthened. Specific pharmacologic strategies for altering the responsivity
of limbic system structures may ameliorate the dysphorias, may lessen the
likelihood of dyscontrol, and may enhance the usefulness of psychotherapy
in this disorder.
PROPOSED COURSE
Further clinical, developmental, and biological data are needed from a larger
number of patients and normal volunteers. We plan to continue the studies
discussed in the report through the upcoming year. In addition', neuroanatomical
and neurophysiological issues will be addressed by expanding some of these
procedures. We will explore differences in brain structure through VBR
measurements in borderline patients as compared to normals and schizophrenic
patients. Xenon blood flow, SPECT, or PET methodologies wll be coupled with
procaine-activation of dysphoria in an attempt to identify brain structures
involved in dysphoric process.
ZOl MH 02188-03 OCD
PUBLICATIONS
Cowdry RW, Gardner DL: Pharmacotherapy of borderline personality
disorder: alprazolam, carbamazepine, trifluoperazine, and tranylcypromine.
Archives of General Psychiatry, (in press).
Gardner DL, Lucas PB, Cowdry RW: Soft sign neurological abnormalities
in borderline personality disorder and normal control subjects. The Journal
of Nervous and Mental Disease, 175(3): 177-180, 1987.
Lucas PB, Gardner DL, Wolkowitz OM, Tucker EE, Cowdry RW: Methylphenidate-
induced cardiac arrhythymias. New England Journal of Medicine, 315(23):
1485, 1986.
Lucas PB, Gardner DL, Wolkowitz OM, Cowdry RW: Dsyphoria associated with
methylphenidate infusion in borderline personality disorder. American
Journal of Psychiatry, in press.
Cowdry, RW: Psychopharmacology of borderline personality disorders:
a review. J Clin Psychiatry, in press.
Gardner DL, Cowdry RW: Development of melanchoia during carbamazepine
treatment in borderline personality disorder. J Clin Psychopharmacol
6(4):236-239, 1986.
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00183-02 BP
PERIOD COVERED
October 1, 1986
September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line txtween the borders.)
Biology and behavior of aggression and suicide
PRINCIPAL INVESTIGATOR (List other professional personnel tMlow the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Gerald L. Brown, M.D., Medical Officer, BPB, NIMH
F.K. Goodwin, M.D., Director, IRP, NIMH; R.M. Post, M.D., Chief, BPB, NIMH;
M. Linnoila, M.D. , Chief, LCS, NIAAA; J. Kleinman, M.D., St. Elizabeth's Hospital,
NIMH; D.L. Murphy, M.D., Chief, LCS, NIMH; J.L. Rapoport, M.D., Chief, CHP , NIMH
COOPERATING UNITS (if any)
BPB, CHP, LCS, OD, NIMH; St. Elizabeth's Hospital; LCS, NIAAA, National Naval
Medical Center
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.0
PROFESSIONAL
1.6
.4
CHECK APPROPRIATE BOX(ES)
(3 (a) Human subjects
n (a1) Minors
n (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
For several years, studies that relate human aggression (including hyperactivity
and conduct disorder in children) and suicide to various behavioral and biologi-
cal factors have been ongoing. Some of the most significant findings have in-
cluded pharmacokinetic and metabolic studies of amphetamine administered to hyper-
active and conduct disordered children, and a trivariate relationship among a
history of aggressive behavior, a history of suicidal behavior, and lower cerebro-
spinal fluid (CSF) 5-hydroxyindoleacetic acid (5HIAA) . More recently, data indi-
cate that certain aggressive, impulsive, and depressive characteristics in child-
hood are also inversely related to CSF 5HIAA measured during late adolescence;
family instability during childhood also appears to be associated with increased
likelihood of aggressive and/or suicidal behavior in late adolescence. These
data, along with the work of other investigators studying aggressive behavior in
childhood, indicate the possibility of traits associated with disordered serotonin
metabolism; further, the less consistent relationship between lower CSF 5HIAA and
suicidal behaviors vs. aggressive behaviors, may indicate that some suicidal
behaviors are a self-destructive manifestation of a more basic destructive
(aggressive/impulsive) trait.
PHS 6040 (Rev. 1/84)
CPO S14.«ll
ZOl MH 00183-02 BP
Project Description:
Objectives: An objective is new knowledge of the central nervous system
(CNS) of children, adolescents, and adults with special reference to maturational
changes and neuropsychiatric disorders as they relate to aggression and suicide.
Compared to neurobiology known in adult neuropsychiatry, less is known regarding
neuropsychiatric disorders of children. There have been a number of hypotheses
relating catecholamine metabolism and hyperactivity in children. The possibility
of an overly active catecholaminergic system was first advanced. Later, a function-
al deficiency in catecholamines in hyperactive children (now subsumed under DSM-III
diagnoses. Attention Deficit Disorder [ADD] and Conduct Disorder [CD]) was proposed,
with the greater focus on the possibility of a functional dopamine (DA) rather than
norepinephrine (NE) deficiency. Other biochemical alterations, particularly involv-
ing serotonin (5HT) , have also been proposed. More recently, alterations in phenyl-
ethylamine (PEA) have also been proposed. No single neurotransmitter system to date
can be shown to have an etiological role. Several new populations of children and
adolescents are now being studied; i.e., aggressive CD, obsessive-compulsive disor-
der (OCD) , children who have been abused, and those with multiple personality dis-
orders (MPD) .
Considerable indirect pharmacologic evidence has linked amine systems with adult
psychiatric illness (particularly affective illness and schizophrenia) . Search-
ing for interrelationships between central biochemical functioning and repeated
behavioral patterns may be as important as searching for traditional diagnostic
specificity of biochemical findings. Confirmation of relationships between central
biochemistry and behavior could lead to more specific pharmacological treatments.
Direct human data can be valuable in utilizing animal data and in assessing the
differences and similarities between man and animals. Data has begun to be accumu-
lated on central neurochemical function in the various personality disorders; i.e.,
the "aggressive-impulsive" personalities and OCD ' s . Personality disorders involving
criminality show indications of a genetic component; aggressive/impulsive character-
istics have also been linked to a genetic predeterminant of suicidal behavior inde-
pendent of psychiatric diagnosis. Furthermore, other patterns of behavior often
seen within personality disorders - depression, alcoholism, suicide, and obsessive-
ness - also appear to have genetic components. Data from animals strongly suggest
a relationship between aggressive behavior and neurotransmitters. A purpose of this
project is to extend the studies of central amines into larger and more diverse
populations of psychiatric patients, and to assess behavioral-biochemical relation-
ships and whether such findings are diagnostically specific. Dr. Frederick Goodwin
continues to provide overall scientific supervision of this multi-faceted project.
Methods Employed: An inpatient and day-patient program for children and ado-
lescents, involving selected overnight stays, is ongoing on an inpatient nursing
unit. Children and adolescents who are hyperactive, aggressive/impulsive, and ob-
sessive are admitted in order to study a carefully defined sample of ADD/CD and OD.
Specific exclusion and inclusion criteria are employed. All children are
thoroughly evaluated by medical, psychiatric, and psychometric examinations with
all routine and other indicated procedures and clinical laboratory studies.
Several clinical and behavioral rating instruments are utilized. Pharmacologi-
cal study results implemented in this program are briefly summarized below.
Further details of this program can be found in "Cerebrospinal Fluid in Childhood
ZOl MH 00183-02 BP
Behavioral Disorders", Protocol #85-M-115 of Dr. Judith Rapoport. The studies done
in collaboration with Dr. Frank Putnam on children and adolescents with multiple
personalities; often aggressive/impulsive and the victims of child abuse; have been
delayed, but are still anticipated.
Previous NIMH-Navy studies have been described in detail in a previous annual
report (ZOl MH 00092-11 BP) . The results of these studies are briefly summarized
below.
Currently, clinical studies are being carried out in subjects with several kinds of
disorders. Of particular clinical interest is the interrelationship between aggres-
sion and suicide. These subjects will have their indoleamine metabolism assessed
directly and indirectly in several ways; i.e., cerebrospinal fluid (CSF) obtained
by lumbar puncture (LP) , response to glucose tolerance testing (GTT) in alcoholics.
Family studies are under way in these subjects as well (Dr. Linnoila) . LP's are
being performed in children and adolescents who are aggressive/impulsive or compul-
sive (Dr. Kreusi) . Blind clinical evaluations are being performed by Dr. Brown.
Among those individuals incarcerated for murder, responses to GTT and similar arti-
ficial sweetening will be assessed by the Thematic Apperception Test (TAT) along
with baseline LP's in collaboration with Dr. Linnoila and colleagues. This pro-
ject has recently been approved by an HHS Ethics Committee. A further study in-
volves an assessment of serotonin and its metabolite from autopsy material and LP's
in conjunction with their clinical inpatient records in those individuals with a
history of suicide and/or violence (in collaboration with Dr. Kleinman) . Aggres-
sive/impulsive behavior is also being assessed in adult patients with affective
illness, obsessive-compulsive disorder, and normals (Dr. Murphy).
Major Past Findings: Serial plasma pharmacokinetic data indicate that
d-amphetamine (d-AMPH) reaches a peak level in children with ADD/CD within 3-4
hours of an initial dose; however, as much as 70-80% will remain in the serum
at 5-6 h when behavioral effects have largely dissipated. Mean apparent elimina-
tion half-life is 6.8 ± 0.5 h. Test-retest studies of individuals indicate that
both pharmacokinetic data and clinical response data are highly replicable. Sus-
tained release capsules produce a slower rate of absorption and a more plateau-
like, longer-lasting peak level, but do not give a prolonged clinical response.
Socially appropriate behavioral change and motor activity decrease are maximal
at 1-3 h after administration of a single dose (0.5 mg/kg) of d-AMPH. Higher
single doses (1.0 mg/kg) effect earlier similar clinical response, but of less
magnitude. Piribedil is safe but clinically ineffective in ADD /CD. d-AMPH has
been shown to have an anti-aggressive effect in aggressive/impulsive children.
Preliminary results indicate that neither tryptophan (TP) nor valine (a neutral
amino acid which competes with TP and inhibits its crossing the blood-brain
barrier) results in behavioral response or basal temperature change after a
single dose, but attention span increase is similar to that observed following
d-AMPH, while there are clear effects on plasma amino acids in the expected direc-
tions. Another preliminary study indicates that both plasma 3-methoxy-4-hydroxy-
phenylglycol (MHPG) and homovanillic acid (HVA) are affected acutely by single-dose
d-AMPH in a non-pretreated child.
ZOl MH 00183-02 BP
Urine studies in ADD/CD indicate that day and night excretion of MHPG and HVA are
not different; however, d-AMPH after 8 and 14 days is associated with lower MHPG
levels. Behavioral response may be associated with the decrement in MHPG. Urinary
HVA is unchanged. Tyramine (TRM) and parahydroxyphenylacetic acid (PHPA) excretion
are also decreased and phenylethylamine (PEA) excretion is markedly elevated follow-
ing two weeks of d-AMPH. PEA excretion is lower in ADD/CD versus controls; its
significance depends on whether it is expressed in terms of creatinine excretion.
More recent studies indicate a different pattern of metabolite response to methyl-
phenidate (MP) . Both d-AMPH and MP effect no change in DA or its metabolites.
ADD/CD are not different from normals with regard to plasma NE and dopamine-beta-
hydroxylase (DBH) but do have significantly more neurological soft signs by PANESS
examination. New item analysis data indicates the prevalence of varied soft signs
and their rater reliability. Plasma NE correlates with anxiety ratings and changes
both with regard to dose of d-AMPH and time following dose, with higher doses of
d-AMPH (1.0 mg/kg) giving strongest response at 1 hour and lower doses (0.5 mg/kg)
giving strongest response at 3 hours. Elevated plasma NE is also associated with
increases in blood pressure and pulse, and is dose-related. Baseline plasma NE,
measured prior to an early a.m. dose of d-AMPH, does not change after two weeks of
d-AMPH versus two weeks of placebo.
With regard to pharmacological response, d-AMPH is effective and piribedil and
L-DOPA are minimally so; TP produces responses similar to d-AMPH. ADD/CD, with
higher levels of soft signs, have more abnormal EEC's, more minor physical anomal-
ies, lower full-scale I.Q.'s (WISC-R) , and a greater number of errors on the Bender.
Data from psycholinguistic evaluations indicates that ADD/CD have impairments in
certain auditory processing and language skills; furthermore, d-AMPH does not evoke
pronounced effects with regard to language performance in ADD/CD vs. normals; older
and less hyperactive subjects showed the most improvement. Improvement in cognitive
parameters was shown only in normals.
Initial results from late adolescent personality disorders with problems secondary
to poor impulse control, high levels of anger-hostility, and poor judgment indicated
that aggressive behavior is inversely correlated with CSF 5-hydroxyindoleacetic acid
(5HIAA) and positively correlated with CSF MHPG. Personality disorders have shown
no significant difference in CSF cyclic 3 ' ,5-adenosine monophosphate (c-AMP) from
neurological patients with non-CNS disorders or from depressive, manic, and schizo-
phrenic patients. Aggressive behavior was positively correlated with c-AMP and
cyclic-3', 5-guanosine monophosphate (c-GMP) in one group but not in a second.
Those who were administratively discharged from the Service and those with history
of suicidal attempts had lower CSF 5HIAA and higher MHPG, c-AMP, and c-GMP. Border-
line personalities (DSM-III) in a second study showed an inverse relationship be-
tween CSF 5HIAA and the psychopathic deviate (Pd) (MMPI) scale, as well as a history
of aggressive behavior; neither the MHPG relationships nor the cyclic nucleotide re-
lationships were replicated. A trivariant relationship among a history of aggres-
sion, history of suicidal behavior, and lower CSF 5HIAA is apparent.
As experience accumulates from various collaborative studies, the aggressive vari-
able that appears to be most likely associated with lower CSF 5HIAA is that charac-
terized by lability of affect, history of repeated impulsivity, and explosiveness.
Similarly, our experience and that of others appears to indicate that suicidality
10
ZOl MH 00183-02 BP
associated with aggressivity is most likely to be associated with reduced levels of
CSF 5HIAA.
A military male found guilty of violent murder, with a past history of several sui-
cidal attempts, was found to have the lowest level of CSF 5HIAA yet measured by our
group; he also had a hypoglycemic response to a GTT. In that aggressive behavior
has been shown in animals to be associated with lower GABA, new studies of CSF GABA,
both free and bound, have been analyzed in the borderline group of patients; though
CSF GABA is lower in the more aggressive patients and in those with histories of
suicidal behavior, neither difference reaches the <.05 level of significance.
Alcoholics do not differ from personality disorders in CSF HVA. However, mean CSF
5HIAA is higher in the intoxication-withdrawal stage and decreases over time in ab-
stinence to reach a mean level not differing from that of personality disorders.
CSF HVA levels are depressed by disulfiram (Antabuse) , a DBH inhibitor. Disulfiram
use also correlates with an increase in serum NE. Mean serum DBH in alcoholics
versus normal controls was lower, blood pressure was higher, and serum NE was not
different. Disulfiram is also associated with an increase in cholesterol in alco-
holics. Lower CSF DBH is correlated with increasing psychopathology, as measured
by the MMPI, and lower CSF DBH is associated with disulfiram-induced psychoses.
Furthermore, low platelet monoamine oxidase (MAO) , low amine oxidase (AO) , and ele-
vated erythrocyte catechol-0-methyltransferase (COMT) are associated with disulfir-
am-induced psychoses. Neither clinical depression nor aggressive behavior in this
group of early to mid-stage alcoholics can be associated with alcoholism; nor can
improvement in depression or anxiety ratings of hospitalized alcoholics be attribu-
ted to disulfiram.
Further analyses of previous studies indicate that those individuals diagnosed as
antisocial and explosive (DSM-III) have the lowest levels of CSF 5HIAA. Further-
more, the MMPI profile of 42, 28, and 49 with high F scale scores is most closely
associated with low CSF 5HIAA. The only Buss-Durkee Inventory (BDI) category that
has a significant inverse relationship with CSF 5HIAA is "irritability". While
total BDI scores and PD T scores do correlate significantly with a life history of
aggression, the BDI appears to measure a number of aspects of aggressive thoughts
and attitudes as well as behavior, but this scale appears to be a less useful
instrument to relate to CSF 5HIAA levels.
Platelet MAO is not significantly different in medication-free ADD/CD vs. normals;
AO is significantly lower in ADD/CD vs. normals. MAO was not correlated with age
in normal children (groups not different with age as a covariate) ; AO was not cor-
related with age in either group. MAO and AO levels were not related to a low mono-
amine diet platelet number, Hgb and Hct did not differ in the groups, nor was MAO
or AO correlated with either. MAO and AO were determined two times in 20 subjects;
the percentage coefficients of variation (CV) were 18,6 ± 9.4 and 12.0 + 9.2, re-
spectively. Finally, neither MAO nor AO responds significantly to d-AMPH.
Platelet 5HT, though not different in ADD/CD vs. normals is negatively correlated
with both attentional and conduct factors on the Connors Teachers-Rating Scale
(CTRS) , more strongly with conduct. These findings may explain the discrepant
reports of 5HT in ADD/CD when group data is compared to normals.
11
ZOl MH 00183-02 BP
A very low level of CSF 5HIAA was found in a conduct-disordered adolescent, whose
stealing involved a craving for sugar (glucose intake increases brain levels of
5HT) . This individual also had an MMPI profile similar to that reported by Brown
and colleagues for aggressive male adolescents with low levels of CSF 5HIAA.
New Finding: As part of Dr. Brown's collaborative role in making clinical
assessments of aggressivity-impulsivity in children and adolescents, in collabor-
ation with Drs. Kreusi and Rapoport, he remains blind to CSF 5HIAA data that has
now been collected on approximately 20 such subjects. Further refinements in both
the content and reliability of intra-rater assessments are ongoing as the study
continues. Finding that a negative correlation exists between CSF 5HIAA (in the
later group of Navy men) and a childhood history of ADD/CD kinds of behavior has
been furthered. Medical history includes headaches in childhood. These findings
have been further pursued in terms of suicidal behavior and family history of var-
ious kinds of stresses and instability. Data have now been analyzed that show that
both those individuals with a higher score for aggressive/impulsive behaviors, and
those who have a history of suicide attempts, had greater mean scores on items re-
lated to family instability, stress, and loss than those with lower scores for ag-
gressive/impulsive behavior and those without a history of suicidal attempt. Of
interest is that disturbed family history per se is not related to levels of CSF-
5HIAA, possibly indicating that various kinds of disturbed personality and behavior
(present in all of the subjects whom Dr. Brown has studied) not of an aggressive
and/or suicidal character, may also not be associated with serotonin (5HT) metabo-
lism, or at least not in the same way as that seen for aggressive/impulsive/suicidal
individuals. These findings are consistent with other data in the literature that
would support an inverse relationship between aggressive/impulsive behavior or be-
havior thought to indicate dyscontrol and disinhibition as trait characteristics and
CSF 5HIAA or other measures related to 5HT metabolism. The review of histories of
schizophrenics for aggressive/suicidal behavior with LP and autopsy material is
ongoing. Collaborative studies with Dr. Murphy have just begun.
Significance to Mental Health Research: Though childhood neuropsychiatric
disorders have been considerably studied in the 'last few years, many diagnostic,
psychopharmacological , and psychobiological questions are yet to be addressed; an
increased interest in psychopharmacology continues to emerge. Though MP and AMPH
give positive responses in 80% of well diagnosed ADD children, the pharmacokinetic-
s and metabolism of these drugs have been studied only relatively recently. Drug
responsivity in CD is less clear. One avenue to ascertaining possible neuropath-
ology is to understand more clearly the mechanisms of action of pharmacological
compounds which effectively alter the clinical conditions under study. The
relationship between such basic pharmacological knowledge and clinical effects has
been under-studied in children in general. More importantly, for the future, basic
biological factors in childhood neuropsychiatry which might elucidate the psycho-
pharmacological responses are, at this point, only hypotheses. The degree to which
these hypotheses are validated or refuted could be expected to play a significant
role in understanding childhood neuropsychiatry.
CNS functioning appears to have been under-studied in some major groups of psychi-
atric patients; viz., personality disorders, alcoholics, and borderlines. Studies
of animal models, as well as Gilles de la Tourette's syndrome, ADD/CD in children,
and prisoners suggest a relationship between central neurotransmitter systems and
12
ZOl MH 00183-02 BP
aggressive behavior. Human suicidal behavior has an enormous public health and
social significance and, previously, had largely been studied from psychological and
sociological points of view. Both suicidal and aggressive problems are increasing.
These studies lead to the possibility of identifying those at risk for anti-social
and suicidal behaviors and possibly altering those behaviors through neuropharmaco-
logical adjuncts to management of the psychiatric and/or behavioral problems. The
neurobiological aspects of alcoholism, either predisposing, concomitant, or result-
ant, are also of timely significance.
Proposed Course of Project: The principal investigator, Dr. Brown, remains in
the Office of the Chief, BPB, and is no longer administratively a part of the Child
Psychiatry Branch of the Intramural Research Program (IRP) , although active colla-
boration continues with this Branch as well as the Laboratory of Developmental Psy-
chology (Putnam) , and Dr. Brown serves as a special consultant to the Child Psychia-
try Branch in the Extramural Program (ERP) . There is a body of data yet to be
analyzed but some of this is in preparation and in press and will be reported in the
future .
The preparation for the Navy collaborative project began in January 1973. The
approval processes, both in terms of scientific merit and the protection of rights
of patients, were completed in July 1974. Though the active data collection phase
of the collaboration has been terminated, this collaboration continues to be of
mutual benefit to NIMH and NNMC. Some neurochemical, behavioral, and psychological
data are yet to be analyzed and reported from the patients who have participated in
these studies, as well as the attempts at follow-up. Additionally, collaboration,
though delayed by other duties, continues on LP and autopsy studies of schizo-
phrenics within the IRP at St. Elizabeth's Hospital.
PUBLICATIONS :
Brown, G.L. and Goodwin, F.K. : Human aggression and suicide. In Maris, R.
(Ed.): Biological Aspects of Suicide, Vol. 16. New York, Guilford Press, 1986,
pp. 141-161,
Brown, G.L. and Goodwin, F.K.: Cerebrospinal fluid correlates of suicide
attempts and aggression. In Stanley, M. and Mann, J. (Eds.): Psychobiology of
Suicidal Behavior, Ann. NY Acad. Sci. 487: 175-188, 1987.
Brown, G.L. and Goodwin, F.K. : Overview of biological factors in suicide. In
The Task Force on Youth Suicide. Washington, D.C., Department of Health and
Human Services, in press.
Brown, G.L. and Goodwin, F.K.: Risk factors in suicidal behavior. Psychiatry
Lett. , in press.
Brown, G.L. and Goodwin, F.K. : Measurement of human aggression/impulsivity.
In Linnoila, M. and van Praag, H. , (Eds.): Biological Factors in Human
Aggression. ACNP, in press.
13
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
7.ai MH nnn7n-i4 rp
PERIOD COVERED
TITLE OF PROJECT (80 characterz or less, rule must lit on one line between the borders.)
Psychological and Rinlngiral — Tnfpranl-i nn.c; in <-hp Mrmrl anH Anyjpt-y nigorHpr-g
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfiliation)
Robert M. Post, M.D. Chief BP NIMH
Dr. T. Colburn Research Services Branch, NIMH
Dr. R. Cohen Laboratory of Cerebral Metabolism, NIMH
Dr. L. DeLisi Clinical Neurogenetics Branch, NIMH
Dr. T.W. Uhde Biological Psychiatry Branch, NIMH
COOPERATING UNITS (if any)
BPB,CNG,NSB,NPB,CPB,LCM,LCS,LPP,RSB,IRP, NIMH; DEB, NICHD; IRP, NIAAA; PDS , NIH;
USUHS, Dept of Def.; U. of CA; Tufts U.; U. So. Carolina Med. Sch.,- INSERM: St.
Elizabeth's Hospital; St. Michael's Hospital
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Section on Psychobiology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20205
TOTAL MAN- YEARS:
13.0
PROFESSIONAL:
7.0,
6.0
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects D (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Evaluation, study, and treatment of patients with manic-depressive and schizoaffec-
tive illness are the primary goals of the Section. Double-blind, placebo-con-
trolled clinical trials are employed to evaluate routinely used and novel agents
for the treatment of these disorders. Anticonvulsants such as carbamazepine have
been demonstrated to be clinically effective in the acute and prophylactic treat-
ment of manic-depressive illness. We have identified possible clinical and bio-
chemical markers of response to lithium versus carbamazepine and other agents.
For example, antimanic responders to carbamazepine appear to be more severely ill,
more dysphoric, and more rapidly cycling than non-responders, i.e., variables that
tend to be associated with lithium nonresponse. In attempting to elucidate poss-
ible mechanisms of action, we have found that alpha-2 noradrenergic and "peripher-
al-type" benzodiazepine receptor mechanisms may be important to the anticonvulsant
if not the psychotropic effects of carbamazepine. Other neurotransmitter , modula
tor, and peptide substances are being studied which may account for carbamazepine ' s
positive effects on mood and behavior. The Section also seeks to identify regional
alterations in brain electrophysiological and metabolic activity that are related
to changes in behavior and cognition in affective illness. A clinical probe of
limbic system excitability utilizing a novel provocative agent, procaine , is also
being employed. Procaine selectively increases fast activity over the temporal
lobg in association with a variety of behavioral and cognitive alterations and se-
cretion of Cortisol, ACTH, and prolactin. Animal models of electrophysiological
and pharmacological kindling and cocaine-induced behavioral sensitization are stud-
ied and implicate conditioning and learning processes in the progressive behavioral
changes induced. These models may help provide new clinical and biochemical in-
sights into the mechanisms that underlie the progressive and long-term changes in
behavior in a variety of clinical syndromes including cocaine-induced psychopathol-
ogy and affective illness.
15
PHS 6040 (Rev. 1/84) OPO gw»i«
ZOl MH 00070-14 BP
COLLABORATORS :
Dr. M.S. Buchsbaum, Dept. of Psychiatry, U. of California, Irvine
Dr. D.C. Jimerson, Laboratory of Clinical Science, NIMH
Dr. F.K. Goodwin, Office of the Director, IRP, NIMH
Dr. P.W. Gold, Biological Psychiatry Branch, NIMH
Dr. M. Linnoila, Intramural Research Program, NIAAA
Dr. H. Weingartner, Biological Psychiatry Branch, NIMH
Dr. D.R. Rubinow, Biological Psychiatry Branch, NIMH
Dr. C.H. Kellner, Dept of Psychiatry, U. of So. Carolina Medical School
Dr. R. Coppola, Neuropsychiatry Branch, St. Elizabeth's Hospital
Dr. R. Cowdry, Clinical Director, IRP, NIMH
Dr. D. Gardner, Neuroscience Branch, NIMH
Dr. S.R.B. Weiss, Biological Psychiatry Branch, NIMH
Dr. A. Pert, Biological Psychiatry Branch, NIMH
Dr. P. Marangos, Biological Psychiatry Branch, NIMH
Dr. J. Patel, Biological Psychiatry Branch, NIMH
Dr. S. Reichlin, Division of Endocrinology, Tufts University
Dr. J.C. Ballenger, Dept. of Psychiatry, U. of So. Carolina Medical School
Dr. F. Putnam, Neuropsychiatry Branch, NIMH
G. Leverich, Biological Psychiatry Branch, NIMH
Dr. G.L. Brown, Biological Psychiatry Branch, NIMH
Dr. P. Roy-Byrne, Biological Psychiatry Branch, NIMH
Dr. M. Kling, Biological Psychiatry Branch, NIMH
Dr. D. Davis, Biological Psychiatry Branch, NIMH
Dr. K. Kramlinger, Biological Psychiatry Branch, NIMH
Dr. K. Denicoff, Biological Psychiatry Branch, NIMH
Dr. T. Melman, Biological Psychiatry Branch, NIMH
Dr. J-P. Boulenger, French National Institute for Health and Med. Res. (INSERM) ,
Cannes, France
Dr. R. Joffe, Dept of Psychiatry, St. Michael's Hospital, Toronto, Canada
Dr. A.F. Mirsky, Lab. of Psychology and Psychopathology , NIMH
Dr. G. Chrousos, Senior Investigator, Developmental Endocrinology Branch, NICHD
Dr. P. Brouwers, Lab. of Psychology and Psychopathology, NIMH
Dr. CD. Johnson, Lab. of Psychology and Psychopathology, NIMH
Dr. C.R. Lake, Uniformed Services, Univ. of the Health Sciences
16
ZOl MH 00070-14 BP
Dr. A. Doran, Neuroscience Branch, NIMH
Dr. D. Pickar, Neuroscience Branch, NIMH
A. Rosoff, Biological Psychiatry Branch, NIMH
Dr. P. Hauser, Biological Psychiatry Branch, NIMH
Dr. S. Paul, Neuroscience Branch, NIMH
Dr. A. Roy, Intramural Research Program, NIAAA
Dr. P. Hauser, Biological Psychiatry Branch, NIMH
17
ZOl MH 00070-14 BP
I. Project Description
A. Objectives
This project is engaged in the multidisciplinary longitudinal study and
treatment of patients with a spectrum of acute and chronic psychoses, particularly
involving mood and anxiety disorders. Both investigative and treatment approaches
focus on the elucidation of psychological and biological phenomena and their inter-
action.
B. Methods Employed
1. Subjects who meet Research Diagnostic Criteria (RDC) for manic-de-
pressive or schizoaffective illness or the more recent DSM III criteria for a spec-
trum of mood disorders are admitted to the 3-West Clinical Research Unit, Section
on Psychobiology of the Biological Psychiatry Branch. Patients with anxiety and
panic-anxiety are also admitted to the unit under other protocols (see Project ZOl
MH 00071-0 BP) . Normal volunteers are admitted to the unit to provide control data
for specific studies in patients and to assess clinical and biological interrela-
tionships in normal as well as patient populations.
2. Behavioral and Psychological Evaluation
a. Psychological Evaluation: Patients are rated twice daily in a
double-blind fashion and are assessed with a variety of psychological tests (as
previously described) . Life Course of Illness is charted graphically in great de-
tail.
b. Biological Evaluation: EEC sleep, AER, and glucose utilization
on PET are studied during medication-free intervals, as is procaine activation of
EEG, behavior, and cognition. ■ Neurotransmitters, endocrine substances, and pep-
tides are measured in urine, plasma, and CSF before and after acute drug challenges
and longer-term treatment. Endocrine tests are described in project #Z01 MH
00452-12 BP.
3 . Treatment
Drug evaluation is conducted in a double-blind fashion. Routinely
employed drugs include lithium, neuroleptics, tricyclic and MAOI antidepressants.
New and experimental treatments include carbamazepine, valproic acid, phenytoin,
clonidine, sleep deprivation, and T or T potentiation.
4. Animal Models
A rodent behavioral pharmacology laboratory is maintained in colla-
boration with Drs. S.R.B. Weiss and A. Pert to develop new research techniques in
several areas. The longitudinal evolution of behavioral pathology and its underly-
ing biochemical mechanisms are assessed in different paradigms including: 1) elec-
trophysiological kindling; 2) pharmacological kindling; and 3) behavioral sensitiza-
tion to psychomotor stimulants such as cocaine. The role of seizures in the devel-
opment of behavioral pathology is studied utilizing electrical and pharmacological
kindling and CRF. The anticonvulsant mechanism of action of carbamazepine is also
studied in the kindling paradigm.
18
ZOl MH 00070-14 BP
C. Major Findings
1. Carbaroazepine: A New Treatment and an Alternative or Adjunct to
Lithium for Manic-Depressive Illness
a. Acute Antimanic Efficacy: In collaboration with Drs. T, Uhde,
K. Kramlinger, and other physicians in the Branch, we have found that carbamazepine
is effective in the acute treatment of manic patients, including many who were pre-
viously nonresponsive to lithium carbonate. The magnitude and time-course of im-
provement on carbamazepine paralleled that of neuroleptics. Sleep improved signi-
ficantly in the first week of treatment. Twelve of 19 (63%) acutely manic patients
have shown good responses. These responders, compared to nonresponders, were more
severely manic and dysphoric at the onset during their placebo period and they were
also more rapid cyclers. Responders had a negative family history for manic-de-
pressive illness in first degree relatives, while nonresponders were equally divided
among family history positives and negatives. These data suggest an opposite cli-
nical profile of response to lithium and carbamazepine. While manic severity, dys-
phoria, rapid-cycling, and negative family history tend to be associated with poorer
response to lithiiam, these variables are associated with better antimanic response
to carbamazepine .
Potentiation of carbamazepine with the addition of lithium carbonate resulted
in improvement in five of six patients who had previously been inadequately
responsive to the antimanic ef fects* of either drug alone.
b. Acute Antidepressant Efficacy: Fifteen of the first 47 patients
have shown evidence of a marked clinical response to carbamazepine. Patients with
initially more severe depression responded better to carbamazepine than those with
less severe ratings of depression. Those with more rapid cycling (episodes/years
ill) and hospitalizations for mania, but fewer total weeks depressed (i.e., less
chronic depression) , also responded better (see also thyroid correlate of response
below) .
In 15 depressed patients who were inadequately responsive to the acute anti-
depressant effects of carbamazepine alone administered on a double-blind basis,
lithium was added also on a blind basis (with K. Kramlinger) . Eight of the 15
patients showed a marked response to this lithium potentiation. The time-course of
acute antidepressant response was rapid, and was more rapid than previously observed
in responders to lithium treatment alone. These data suggest that lithium potenti-
ates the antidepressant effects of carbamazepine, as has been reported for many
other antidepressant modalities, including heterocyclics and monoamine oxidase in-
hibitors. While the mechanism of action remains to be elucidated, the rapid onset
of lithium potentiation has been suggested by de Montigny to involve the serotonin
system for more traditional antidepressants. Not only was the time course of acute
antidepressant response to lithium potentiation of carbamazepine faster than the
antidepressant response achieved with either agent alone, but it was also faster
than the onset of antimanic response to lithium potentiation. These data further
support the concept that the rapid onset of acute antidepressant effects with lithi-
um potentiation may be achieved by biological mechanisms that are different from
those engaged in the antimanic effects or those attributable to either drug alone.
19
ZOl MH 00070-14 BP
c. Prophylactic Efficacy of Carbamazepine : In a series of lithium-
nonresponsive, rapidly cycling manic-depressive patients, the addition of carbamaze-
pine decreased the mean number of both depressive and manic episodes. The severity
and duration of episodes and percentage of time ill also tend to be reduced.
Potential reasons for loss of efficacy (breakthrough of manic or depressive
episodes) during prophylaxis are being studied with G. Leverich.
d. Side Effects of Carbamazepine-Lithium Combination Treatment:
The side-effects profiles of carbamazepine and lithium tend to be substantially
different, offering the patient the possibility of alternative treatment should one
drug or the other not be well tolerated. When the two drugs are used in
combination, important interactions are observed. Carbamazepine alone decreases
white count in the majority of patients, while lithium increases white count.
During lithium potentiation of carbamazepine treatment of 22 depressed or manic
patients. Dr. Kramlinger observed that lithium reversed the white count suppression
induced by carbamazepine and by the third week of combination treatment, values
were actually increased over those observed during the baseline placebo period.
Lithium induces diabetes insipidus while carbamazepine has been used to treat
the syndrome. However, because the effects of lithium carbonate occur at a level
below the receptor, probably involving adenylate cyclase, carbamazepine is not able
to override the effects of lithium and will not reverse lithium-induced diabetes
insipidus. While carbamazepine induces mild decreases in serum sodium and calcium,
these effects were not significantly reversed by lithium potentiation.
Carbamazepine decreases plasma levels of T , free T , and T without signi-
ficantly increasing TSH. Lithium potentiation results in further decreases in cir-
culating thyroid hormone levels and increases in TSH secretion. These data suggest
that lithium and carbamazepine are impairing thyroid hormone levels at different
steps in the regulation of thyroid function. Other data suggest that carbamazepine
may increase peripheral thyroid metabolism.
In contrast to lithium carbonate, which can induce clinical hypothyroidism that
requires supplemental treatment with thyroid hormone in a small but substantial
percentage of patients, hypothyroidism on carbamazepine is rarely induced and has
not been observed in our series. In fact, those with the greatest decrements in
circulating T and free T have shown the greatest degree of acute antidepressant
response to carbamazepine. These paradoxical data are consistent with studies of
Baumgartner and associates, indicating that patients with greater decrements in
thyroid indices on maprotiline and zimelidine respond better to these treatments.
Rashes have been observed in 13 of 113 patients (11.7%) and carbamazepine was
discontinued in each instance. The rashes were uniformly pruritic in nature and,
in 12 instances, occurred in the second or third week of carbamazepine treatment.
e. Carbamazepine and its -10, 11-Epoxide Metabolite: Levels of
carbamazepine itself in plasma or in CSF do not appear to be related to the degree
of clinical antidepressant or antimanic response. However, preliminary data sug-
gest that the levels of the active metabolite of carbamazepine, carbamazepine-10, 11-
epoxide, measured in CSF, were more closely related to the degree of antidepressant
response. This metabolite has been demonstrated by us to have anticonvulsant
20
ZOl MH 00070-14 BP
effects on amygdala-kindled seizures and by others to have antinociceptive
properties and to be effective in the treatment of trigeminal neuralgia. Based on
these data, we have undertaken a clinical trial of carbamazepine-10, 11-epoxide in
order to establish whether it has psychotropic properties. The first patient
entered in the clinical trial did not respond to the 10, 11-epoxide for the treatment
of acute mania.
f . Selective Responses to Different Anticonvulsant Agents in
Affectively 111 Patients: Response to one anticonvulsant does not appear to
produce response to another. We have observed clearcut response to carbamazepine
but not to valproic acid or phenytoin in an individual patient completing a
double-blind, crossover to these three agents. Conversely, we have observed other
patients who are inadequately responsive to carbamazepine who respond to valproic
acid. These data are not only of clinical import, but suggest the possibility that
differential biochemical or physiological properties of different anticonvulsants
may be related to differential clinical responsivities in different patients. This
may be particularly apparent in the case of carbamazepine compared to clonazepam,
where the two drugs exert differential effects on benzodiazepine receptors, carbama-
zepine likely interacting with the "peripheral-type" benzodiazepine receptor and
clonazepam acting exclusively at the "central-type". We are also beginning to ex-
plore the utility of combination treatment in refractory bipolar patients. We have
just discharged a patient with a 30-year history of ultra-rapid cycling manic-de-
pressive illness who responded well only after T (50 yg) had been added to the com-
bination of carbamazepine, lithium, and valproic acid.
The effectiveness of the anticonvulsants carbamazepine, valproate, clonazepam,
and related drugs raises the paradox of why both anticonvulsants and the induction
of seizures with electroconvulsive therapy (ECT) are useful treatments for acute
manic and depressive illness. We have observed that electroconvulsive seizures in
the rat are paradoxically anticonvulsant to amygdala-kindled seizures. These data
raise the possibility that common biochemical and physiological mechanisms of elec-
troconvulsive therapy and anticonvulsants such as carbamazepine could be related to
their profile of therapeutic efficacy in both phases of affective illness.
g. Studies of Carbamazepine ' s Mechanism of Action:
1) Effects on Classical Neurotransmitters and Modulators:
Evidence of others in laboratory animals suggests that carbamazepine blocks the
reuptake of norepinephrine (NE) , but also inhibits stimulated-induced release; it
increases firing of the locus coeruleus, but decreases NE turnover. Elevated
levels of CSF NE in mania are decreased by carbamazepine.
New evidence from our laboratory suggest that noradrenergic effects of carbama-
zepine are important to its anticonvulsant properties. An alpha-2 mechanism is
likely involved in the anticonvulsant effects, as the alpha-2 antagonist, yohimbine,
reverses the effects of carbamazepine on amygdala kindling. Alpha-2 effects may be
necessary but not sufficient for the anticonvulsant effects, as the alpha-2 agonist
clonidine is not an effective anticonvulsant on this model of kindled seizures.
Although it is as effective as the neuroleptics in the treatment of acute
mania, carbamazepine does not block dopamine receptors or produce other typical
neuroleptic effects. Moreover, it has not been associated with the development of
21
ZOl MH 00070-14 BP
parkinsonian side effects or with the syndrome of tardive dyskinesia as have the
neuroleptic drugs. These data suggest that carbamazepine acts by mechanisms other
than blockade of dopamine receptors.
Alterations in GABA have been postulated in affective illness (see below) as
well as in the seizure disorders. Carbamazepine has been reported to decrease the
turnover of GABA in animal studies (Bernasconi, 1984), although brain levels are
not altered by the drug. This is consistent with our data indicating that CSF GABA
levels are not significantly decreased during treatment with carbamazepine compared
to baseline levels.
GABA-B (baclofen type) mechanisms are implicated in the antinociceptive actions
of carbamazepine based on animal models of trigeminal neuralgia. For example,
Terrence et al (1983) reported that an inactive isomer, d-baclofen, reversed the
antinociceptive effects of carbamazepine and the active isomer 1-baclofen. In con-
trast, we have demonstrated that the anticonvulsant effects of carbamazepine on
amygdala kindling do not appear to involve GABA-B mechanisms, as these effects are
not reversed by d-baclofen.
Thus, it remains to be determined whether the effects of carbamazepine in
manic-depressive illness are more akin to those in trigeminal neuralgia (potential-
ly involving GABA-B mechanisms) or in seizures (such as amygdala kindling that do
not involve GABA-B mechanisms) . In order to assess these differential possibili-
ties, we have undertaken a clinical trial of the active isomer, 1-baclofen, in the
treatment of manic-depressive patients. The first two patients have been entered
in the clinical trial and efficacy has not been demonstrated in these two patients
treated with doses up to 10 mg/day (the initial limit imposed by the FDA) . Further
patients will be tested at higher doses when FDA approval is obtained. Most effec-
tive antidepressants have been reported to increase GABA-B receptors in frontal
cortex (Lloyd et al, 1987), giving further rationale to the baclofen trial.
Effects of carbamazepine on central and "peripheral-type" benzodiazepine re-
ceptors have been studied with biochemical techniques (Marangos et al.), and elec-
trophysiologically in the amygdala kindling model (Weiss et al) . Carbamazepine
binds poorly to the central site (^H-diazepam or ^h-bcCE) , but more potently at the
Ro5-4864 (peripheral) site. Dr. S.R.B. Weiss has found that Ro-15-1788 and BCCM
block the anticonvulsant actions of diazepam, but are ineffective in reversing the
anticonvulsant effects of carbamazepine on amygdala kindling. Conversely, Ro5-4864
does reverse the anticonvulsant effects of carbamazepine and its 10 , 11-epoxide , but
not those of diazepam. PK-11195, which acts selectively at the peripheral site,
blocks the effects of Ro5-4864 on carbamazepine ' s anticonvulsant effects. Taken
together, these biochemical and electrophysiological data suggest that carbama-
zepine may exert its anticonvulsant effects through the "peripheral-type" but not
the "central-type" benzodiazepine receptor, and thus act very differently from
diazepam and clonazepam. It is of interest that "central-type" benzodiazepine re-
ceptors are linked to chloride channels, while the "peripheral-type" may be linked
to calcium channels.
Carbamazepine is potent in displacing binding of adenosine receptor ligands
(Marangos et al.). Contrary to predictions, chronic treatment with carbamazepine
(similar to that with caffeine) increased the number of adenosine receptors, sug-
22
ZOl MH 00070-14 BP
gesting that carbamazepine may possess adenosine antagonist-like properties. It
does not appear that carbamazepine exerts its anticonvulsant effects through the
adenosine receptor, as its effects on kindled seizures are not altered by several
adenosine-active agents (S.R.B. Weiss). The increase in adenosine receptors follow-
ing carbamazepine treatment was long lasting (possibly permanent) , suggesting a
novel mechanism for this effect,
2. Carbamazepine' s Effects on Endocrine and Peptide Systems
Carbamazepine significantly decreased somatostatin measured in CSF of
affectively ill patients (studied in collaboration with Drs. D.R. Rubinow, P.W.
Gold, and S. Reichlin) . These findings, which have recently been replicated in
neurological patients (Steardo et al, 1986) are of interest in relationship to the
reports by others of long-lasting increases in brain somatostatin following
amygdala kindling seizures and observations that depletions of somatostatin exert
anticonvulsant effects on kindled and CRF seizures. Thus, changes in somatostatin
could relate to the anticonvulsant properties of carbamazepine.
Carbamazepine may directly or indirectly potentiate vasopressin effects at the
receptor level. Rubinow and associates have found that carbamazepine induces
escape from dexamethasone suppression and increases urinary free Cortisol excre-
tion. Carbamazepine did not alter CSF opiate binding activity in affectively ill
patients, or affect morphine's antinociceptive effects on tail flick latencies in
the rat. In contrast to lithium, qarbamazepine inhibits rather than potentiates
the TSH response to TRH.
3 . Physiological and Behavioral-Pharmacological Effects of Carbamazepine
The anticonvulsant effects of carbamazepine have been examined in sev-
eral different types of kindling. Remarkable dissociations in the anticonvulsant
efficacy of carbamazepine have been revealed as a function of stage and type of
kindling. For example, carbamazepine is ineffective in blocking the development of
electrical kindling of the amygdala in the rat, even though it is highly potent in
blocking completed amygdala-kindled seizures. Conversely, carbamazepine blocks the
development of lidocaine- and cocaine-kindled seizures, but acutely is ineffective
against completed or high-dose local anesthetic seizures. Pinel (1983) has demon-
strated that spontaneous seizures which occur after many hundreds of kindled sei-
zures are also differentially pharmaco-responsive compared with the early stages of
kindling. Thus, there appears to be a general principle that different stages in
the evolution of kindling — developmental, completed, and spontaneous — may be
differentially responsive and, therefore, involve different neuroanatomical , physio-
logical, and/or biochemical mechanisms. Different types of kindling are also dif-
ferentially responsive.
The bulk of our work has utilized completed kindled seizures in order to eluci-
date possible mechanisms of anticonvulsant effects of carbamazepine. Our data
strongly implicate noradrenergic alpha-2 mechanisms and "peripheral-type" benzodi-
azepine receptor mechanisms in this effect. Data from other investigators also sug-
gest that stabilization of type-2 sodium channels are likely involved in the anti-
convulsant effects of both carbamazepine and phenytoin.
However, our time course analysis of the clinical efficacy of carbamazepine in
epilepsy, pain syndromes, and affective illness suggests that the mechanisms under-
23
ZOl MH 00070-14 BP
lying the anticonvulsant effects of carbamazepine , which occur almost immediately,
may be different from those underlying efficacy in mania and depression, which re-
quire some 2-3 weeks before they become fully manifest. Based on this analysis, one
would want to investigate anticonvulsant mechanisms that require time to develop in
order to have a more parallel model for the time frame of efficacy occurring in
manic-depressive illness. The effects of carbamazepine on cocaine-induced seizures
described below appear to be a useful paradigm in this regard.
In contrast to electrical kindling of the amygdala, which is poorly responsive
to carbamazepine in the early developmental stage, the development of pharmacologi-
cal kindling with local anesthetics is robustly inhibited by carbamazepine. Carba-
mazepine almost completely blocks the development of lidocaine-kindled seizures and
also is highly effective in blocking the development of cocaine-kindled seizures in
three separate studies at doses of 40, 50, and 65 mg/kg, respectively. At these
doses, animals begin to rapidly develop seizures to a dose of drug that was previ-
ously non-convulsive and, in contrast to lidocaine seizures which are well toler-
ated, those associated with cocaine are extremely lethal. Carbamazepine not only
blocks the development of cocaine-kindled seizures, but markedly reduces it associ-
ated lethality. In spite of this robust effect on the development of local-anes-
thetic-induced kindling, carbamazepine is ineffective in blocking completed lido-
caine-kindled seizures or high-dose cocaine seizures following acute administration.
Thus, an interesting dissociation occurs; completed amygdala-kindled seizures
are responsive to the acute anticonvulsant effects of carbamazepine, while local-
anesthetic seizures are unresponsive to this manipulation. However, if carba-
mazepine is administered chronically prior to the local-anesthetic seizures, anti-
convulsant efficacy is observed. Therefore, dissection of the anticonvulsant
mechanisms which require chronic administration of carbamazepine in order to become
manifest against cocaine-induced seizures might provide a suitable first-line stra-
tegy for approximating mechanisms which might also be involved in the psychotropic
effects of carbamazepine, which also require chronic administration before they are
observed.
Lithium carbonate is ineffective in blocking either the development of amygda-
la-kindled seizures or completed kindled seizures, yet appears to have some efficacy
in blocking cocaine-induced behavioral sensitization (described below) . Conversely,
carbamazepine is a highly effective anticonvulsant for some types of kindled
seizures, but is without effect in blocking cocaine-induced behavioral sensitiza-
tion. Thus, comparison and contrast of the effects of lithium and carbamazepine
not only on a biochemical but also on a physiological and behavioral/pharmacological
basis may help to elucidate different mechanisms of action of these two compounds,
which are both effective in manic-depressive patients.
The amygdala-kindling data suggest that different stages in the development of
epilepsy will also show differences in anticonvulsant responsivity. One might ask
whether a similar principle exists for treatment of different stages in the evolu-
tion of affective illness. A considerable body of data suggest that lithium carbon-
ate is less effective in the rapid or continuous cycling type of illness, which
often occurs late in the course of affective illness, while these patients may be
among those who respond best to carbamazepine. Thus, in addition to a variety of
other characteristics that may differentiate lithium responders from carbamazepine
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responders, work with the animal models suggests the utility of considering stages
in the developmental longitudinal course of the illness as a relevant variable,
with the possibility emerging that carbamazepine and related anticonvulsants may be
most useful in the later stages of affective illness, which tend to be less
responsive to treatment with lithium carbonate.
2. Approaches to Classical Neurotransmitter and Peptide Dysfunction in
Affective Illness
a. CSF norepinephrine (NE) is significantly increased in manic pa-
tients compared to either of the other patient or control populations. Indirect
biochemical, pharmacological, and endocrine data continue to suggest a role for
dopamine in some aspects of affective illness. Dopamine and its metabolite HVA and
DOPAC are studied, in collaboration with Drs. D. Rubinow and M. Linnoila, in the
CSF of depressed, manic, and euthymic patients and controls. Studies of the rela-
tionship of plasma HVA to the longitudinal course of affective illness suggest only
weak relationships to mood or anxiety in selected individuals, but no consistent
relationship like those reported for plasma HVA and severity of psychosis in schizo-
phrenics .
b. Dr. Rubinow found that CSF somatostatin is significantly de-
creased in depressed patients compared to those re-studied in the euthymic state or
compared to normal volunteer controls. There have now been seven replications in
other laboratories of the finding of low somatostatin in depressives compared to
normals or other psychiatric comparison groups. These findings are of interest in
relationship to the reports of decreased somatostatin in brain and CSF of patients
with Alzheimer's disease and several other neuropsychiatric syndromes that can pre-
sent with cognitive defects, including multiple sclerosis and parkinsonism. Dr.
Rubinow, in collaboration with Drs. A. Doran, D. Pickar, A. Roy, and S. Paul, has
also found that depressed and schizophrenic patients who were Cortisol hypersecre-
tors, as indicated by escape from dexamethasone suppression, had significantly lower
CSF somatostatin. The causal links in the relationship are unclear, as somatostatin
and Cortisol can each influence the other; for example, glucocorticoid treatment of
normal volunteers decreases CSF somatostatin (Wolkowitz & Rubinow) .
Dr. Gold has completed a series of studies of CRH infusions in affectively ill
patients and controls (as described in Project # ZOl MH 00452-12 BP) and found
evidence for blunted ACTH response in depressive but not manic or improved states.
In contrast to depressed patients, hypercortisolemic patients with Cushing's
disease show ACTH hypersecretion to CRF, providing a possible differential dia-
gnostic test.
3. Course of Affective Illness: Relationship to Biochemical and Clinical
Variables
As described in detail in last year's annual report, we have
characterized the course of more than 100 affectively ill patients' on our clinical
research unit over the past several years. These descriptive data are of consider-
able interest in their own right, but also form a critical substrate for analyzing
the relationship of entire course of illness to subsequent pharmacological response
(see data on carbamazepine above) as well as to various neurobiological alterations
observed in the illness. An example of this usefulness is derived from our recent
study of the relationship of thyroid dysfunctions in manic-depressive illness. It
25
ZOl MH 00070-14 BP
had previously been reported by others that patients with hypothyroidism were overly
represented in a group with rapid cycling.
a. Thyroid Function and Course of Illness: In order to more
systematically examine this question, we assessed thyroid indices at several points
during the NIMH hospitalization in relationship to retrospective and prospective
course-of-illness variables. Values were obtained at the onset and termination of
the medication-free observation interval at NIMH and during treatment with both
lithium and carbamazepine. Thyroid values changed from the first to the second
medication-free period in a highly consistent fashion. T and free T levels in-
creased while T levels decreased, suggesting decreased conversion of T to T con-
sistent with the development of a euthyroid sick syndrome. Depression levels did
not change from the first to the second interval although there was a small in-
crease in psychosis rating indicating that the patients remained ill during this
period of time and did not show spontaneous remissions. The changes in the thyroid
indices were most pronounced in the patients who had been rapid cyclers (demonstrat-
ing more than four episodes per year in the year prior to NIMH admission) and in
those who demonstrated hypercortisolism (increased excretion of urinary free Corti-
sol) during their medication-free evaluation at NIMH. These data are of interest
in relationship to reports that a variety of medical illnesses and glucocorticoid
treatment can induce the euthyroid sick syndrome.
We observed a pattern of increased thyroid indices being positively correlated
with measures reflecting increased rapidity of cycling and a greater severity of
illness. Moreover, rapid cyclers had significantly higher levels of T and free T
than non-rapid cycling patients. There was no relationship of TSH levels to degree
of rapid cycling. Duration of €ime off lithium did not account for the findings and
was unrelated to differences in thyroid indices. In fact, in a subgroup of patients
exposed to lithium, the relationship of rapid cycling to higher levels of thyroid
hormone remained.
Thus, a consistent perspective on the relationship of thyroid dysfunction to
affective illness emerges in our data. Rapid cyclers are characterized by relative
hyperthyroid, rather than hypothyroid, indices (although most values are within the
normal range) and respond to drugs that tend to further reduce thyroid indices, such
as lithium and carbamazepine (see above) . Moreover, we observed that responders
compared to nonresponders to the acute antimanic effects of carbamazepine showed
significantly greater levels of T and free T in the period prior to carbamazepine
treatment. These data are also consistent with the findings of others that some
30-40% of affectively ill patients show blunted TSH responses to TRH and only a
small minority show increased responses. These data from the literature are also
consistent with relative thyroid hyperf unction rather than hypof unction. Moreover,
recent data from Nemeroff et al indicate increased TRH levels in the CSF of
depressed patients, and Reichlin and associates reported that chronic intrathecal
TRH administration for patients with amyotrophic lateral sclerosis produces a pro-
file of thyroid indices similar to that observed in depressed patients with normal
to high hormone levels and a blunted response to TRH.
Joffe et al have suggested that this view of relative thyroid hyperfunction in
affective illness may also be consistent with the data that antidepressant re-
sponses can be potentiated with supplemental thyroid hormone administration with T .
2f)
ZOl MH 00070-14 BP
T , by feedback inhibition, actually suppresses circulating levels of T . Since
CNS uptake is dependent on circulating levels of T and intracellular conversion to
T , this thyroid manipulation may actually induce relative thyroid hypof unction,
like many of the other treatments of affective illness. Joffe would predict, on
the basis of this hypothesis, that depressed patients would respond better to T
potentiation than to T , and preliminary data from his group in Toronto now support
this prediction.
b. Suicidality and Course in Affective Illness: We have found that
49 of 87 of our affectively ill patients (56%) had made suicide attempts. Females
(34/51 or 66%) were more likely than males (15/36 or 41%) to have made an attempt.
While the majority of attempts occur within the first year or two of illness, more
severe attempts (as assessed by a formal "risk" scale) were significantly correla-
ted with duration of illness (r = .52, p < .004, age corrected) and total number of
affective episodes (r = .40, p < .03) . Intensity of suicidal ideation (which was
higher in attempters than non-attempters) was not highly correlated with lethality
of attempt, but was correlated with several variables including number of episodes
of illness. This study provides one of the first attempts to examine suicidality
in affectively ill patients as it relates to the longitudinal course of affective
illness.
4. Depressive Subtypes and Symptoms in Relation to Regional Localization
of Function
a. Psychosensory Phenomena: In collaboration with Dr. P. Hauser,
we have continued to assess signs and symptoms that are usually associated with
psychomotor epilepsy in patients with primary affective illness and panic-anxiety
illness, as well as in patients with temporal lobe epilepsy and in a medical con-
trol group of hypertensive patients. Compared to the medical control group,
patients with affective illness, panic-anxiety disorders, and with epilepsy showed
a highly significant increased incidence in the number of these signs and symptoms.
The qualitative symptom profiles differ slightly among the affective, anxious, and
epileptic patients. Depressed patients with a history of panic attacks have more
symptoms than depressed or anxious patients without panic attacks and show a pro-
file highly characteristic of panic patients. To the extent that psychosensory
distortions and related symptoms usually associated with temporal lobe epilepsy are
occurring with a high incidence in patients with primary affective illness, these
data might suggest that some of the neural substrates involved in complex partial
seizures overlap with affective illness. Contrary to predictions, affective
patients with greater numbers of psychosensory symptoms responded better to lithium
carbonate, and preliminary data suggest that this is not the case for carbamazepine .
b. Psychological, Structural, Metabolic, and Electrophysiological
Approaches to Regional Brain Function in Affective Illness: A variety of psycho-
logical test batteries are employed to assess possible alterations in regional
brain function in patients with affective illness, including the Luria Battery and
the Halstead Categories Test. Impairment in cognitive function has been documented
on these tests during depression. Depressed patients compared to controls are also
deficient in their ability to recognize emotions in pictures of faces presented to
them; recognition of expressions of sad and elated are particularly disturbed
(Rubinow et al) .
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Computerized axial tomography (CAT) scans have been performed on our patients
with affective illness and reveal a similar range of ventricular brain ratios
(VBRs) comparable to those observed in schizophrenic patients. Larger VBR is not
associated with a more chronic or recurrent course of illness in manic-depressive
patients, as has been reported in some studies of schizophrenics. Affectively ill
patients also do not show gross abnormalities of brain structure assessed by
magnetic resonance imaging (MRI) , as studied by Dr. P. Hauser.
Positron emission tomography (PET) scan studies using 2-deoxyglucose indicate
glucose utilization in temporal cortex relative to other areas in the same brain
slice was also lower in depressed patients compared to controls (studied with Drs.
Cohen, DeLisi and Buchsbaum) . These data provide evidence that depressed patients
differ from patients with complex partial seizures who show areas of increased
glucose utilization in the temporal lobe ictally and hypometabolism interictally
(Engel et al, 1982) .
c. Procaine Infusions as a Probe of Limbic System Responsivity :
Graded doses of the local anesthetic procaine were administered to affectively
ill patients (in collaboration with Drs. C. Kellner, F. Putnam and M. Kling) , bor-
derline personality disorders (in collaboration with R. Cowdry and D. Gardner) , and
normal volunteers in an attempt to probe limbic system responsivity. Analysis of
the first 21 subjects by Dr. R. Coppola reveals selective increases in fast EEG
activity, especially 26 to 45 Hz over the temporal cortex, confirming in man the
suggestions from animal studies that local anesthetics activate temporal lobe and
limbic structures. Dose-related alterations in subjective sensory and cognitive
functions were reported as well as a variety of affective responses ranging from
mood elevation to dysphoria. Vivid recall of experientially immediate memories, as
well as hallucinatory-like phenomena, occurred less often. In patients with bor-
derline personality disorder, degree of fast activation of the temporal cortex was
not positively correlated with response to carbamazepine (Cowdry and Gardner) .
Procaine-induced release of ACTH, Cortisol, and prolactin, but not growth hormone,
has also been documented in collaboration with Drs. P. Gold, C. Kellner, and M,
Kling. These data suggest the utility of procaine as a potential pharmacological
probe of the limbic-temporal lobe function.
5. Laboratory Studies of Behavioral Sensitization and Electrophysiologi-
cal Kindling (in collaboration with Drs. S.R.B. Weiss and Agu Pert)
a. Conditioning in Cocaine-induced Behavioral Sensitization: We
have investigated the phenomenology and mechanisms underlying the increased behav-
ioral responsivity to the same dose of the psychomotor stimulant cocaine. Animals
administered cocaine (10 mg/kg i.p.) once-daily show increasing amounts of locomotor
hyperactivity and stereotypy to the same dose over time. An environmental context
or conditioning component has been demonstrated. For example, animals repeatedly
treated with cocaine in the same context (the test cage) showed greater degrees of
hyperactivity and stereotypy than animals receiving identical doses in a different
environment and then injected in the test cage. These findings have been replicated
using drug or saline injections into the nucleus accumbens; cocaine pretreated ani-
mals showed an increased response to intracerebral saline or amphetamine only when
they had been pretreated in the same environment. The similarity of the pretreat-
ment environment (where and in what type of cage animals receive cocaine [40 mg/kg]
28
ZOl MH 00070-14 BP
on day 1) to the test environment where they receive cocaine (10 mg/kg) on day 2 is
also related to the degree of sensitization.
The experience of motor hyperactivity itself in the pretreatment environment
following cocaine challenge (40 mg/kg) appears necessary for cocaine-induced be-
havioral sensitization to occur to a cocaine (10 mg/kg) test dose the next day. If
cocaine-induced activity during the pretreatment is blocked with haloperidol,
diazepam, but not muscimol, sensitization to cocaine (10 mg/kg) does not occur.
(Diazepam and muscimol pretreatments in themselves increase subsequent responsivity
to cocaine, suggesting that GABA mechanisms may facilitate subsequent response to
cocaine . )
b. Neuroleptics Block the Development, But Not Expression of Co-
caine-induced Behavioral Sensitization — A Model of Neuroleptic Nonresponsiveness :
It is also of interest that while neuroleptic blockade of cocaine-induced hyperacti-
vity during the day 1 cocaine pretreatment phase blocks sensitization, neuroleptic
administration prior to the day 2 testing phase does not block the sensitizing ef-
fect of cocaine. Two doses of haloperidol (0.2 mg/kg and 0.5 mg/kg) that were suf-
ficient to block the development of sensitization when administered on day 1, were
both unable to block the expression of sensitization when administered on day 2.
To the extent that behavioral sensitization accounts for some of the progressive
development of psychopathology to cocaine in man, these data suggest that neurolep-
tic treatment, once sensitization has already developed, will be ineffective.
These findings may also represent an animal model for neuroleptic nonresponsiveness
in some psychotic conditions.
c. Anatomical Substrates for Cocaine-induced Behavioral
Sensitization: Using lesion strategies, we have attempted to dissect possible
neural substrates mediating the conditioned component of cocaine-induced behavioral
sensitization. We found that selective dopaminergic lesions of the nucleus accum-
bens that were insufficient to block day 1 high-dose cocaine-induced hyperactivity
did block the expression of cocaine-induced behavioral sensitization. Similarly,
electrolytic lesions of the amygdala as well as selective dopaminergic lesions of
the amygdala, blocked cocaine-induced behavioral sensitization. This effect was not
achieved by lesions of the dorsal or ventral hippocampus or midline cerebellar
structures. These data suggest that nucleus accumbens and amygdala and, in partic-
ular, the dopaminergic components of these pathways, may be involved in the media-
tion of cocaine-induced behavioral sensitization.
d. Pharmacological Kindling: Repeated, intermittent electrical
stimulation of the brain results in increasing duration, spread, and complexity of
electrical after-discharges culminating in the appearance of major motor seizures
to a previously subthreshold stimulation (Goddard et al , 1969). We have employed
this procedure in order to study long-lasting changes in neural and behavioral ex-
citability that accompany this process. Repeated daily injections of the same dose
of lidocaine (65 mg/kg, i.p.) also lead to an increasing incidence, severity, and
duration of seizures to the same dose over time, a phenomenon we have called pharm-
acological kindling. Cocaine (65 mg/kg) also produces pharmacological kindling,
but with a much higher seizure incidence and lethality.
Carbamazepine is a potent inhibitor of the development phase of lidocaine-
kindled seizures, but is ineffective against completed lidocaine seizures.
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ZOl MH 00070-14 BP
Carbamazepine also slows the development of cocaine-kindled seizures and their
accompanying lethality, but is ineffective in preventing high-dose cocaine-induced
seizures and may even increase the cocaine-induced lethality (S.R.B. Weiss).
However, chronic carbamazepine does decrease cocaine-induced seizures and
lethality.
e. CRF Seizures and Behavior: Interaction with Amygdala Kindling:
Dr. S.R.B. Weiss, in collaboration with Dr. A. Pert, has conducted a series of stud-
ies on the behavioral and convulsive effects of corticotropin releasing hormone
(CRF) administered intracerebroventricularly . CRF induces the late onset (i.e.,
following a lag of approximately 4-8 hours post injection) of seizures that behav-
iorally and electrophysiologically resemble those produced from electrical stimula-
tion of the amygdala. Following five repeated once-daily administrations, toler-
ance develops to the seizure inducing effects of CRF. Despite this, CRF seizures
enhanced the development of amygdala-kindled seizures such that animals pretreated
with CRF develop electrically kindled seizures twice as fast as vehicle-injected
controls. CRF-treated animals also show increases in aggressive behavior toward
other rats, an effect that was markedly enhanced in the electrically kindled rats.
Conversely, electrically kindled rats showed a decreased convulsive response to CRF
similar to that seen with repeated CRF injections.
The convulsive response to CRF was not reliably reproduced by local intracere-
bral injection into amygdala, hippocampus, septum, hypothalamus, periaqueductal
gray (PAG) or the pre-pyriform area identified by K. Gale as a highly sensitive
trigger zone for other seizures. However, the aggressive behavior could be elici-
ted by CRF injections into PAG. Moreover, small lesions of the amygdala decreased
the CRF-induced aggression following i.e. v. administration, but small or large amyg-
dala lesions did not affect the development of seizures. Lesions of the hippo-
campus, pre-pyriform area, and olfactory tubercle similarly did not block the devel-
opment of seizures produced by i.e. v. CRF.
These data suggest that CRF is inducing seizures behaviorally similar to those
produced by electrical stimulation of the amygdala, but they are not dependent on
an amygdala substrate for their occurrence. Further, these data suggest the poss-
ibility that an endogenously produced, stress-related peptide such as CRF may,
under pathological conditions, be associated with alterations in convulsive and
aggressive responsivity . No discrete brain focus of this effect has so far been
found and since 50-100 yg of CRF into the CSF appears to be required, this effect
may be pharmacological rather than physiological.
D. Proposed Course of Project
We have helped to introduce and document carbamazepine as an effective
treatment modality for manic-depressive and schizoaffective illness. Preliminary
predictors of clinical response have been elucidated. We propose to further delin-
eate clinical and biological markers of carbamazepine response. Preliminary evi-
dence suggests that many patients who clearly do not respond to lithium carbonate
will respond to carbamazepine. It will be increasingly important to establish
whether response to carbamazepine, compared to lithium carbonate, delineates se-
parate subgroups of patients with affective illness. As such, carbamazepine re-
sponders might be distinguished on the basis of: 1) severity; 2) pattern (rapid
30
ZOl MH 00070-14 BP
cycling) ; 3) genetics (family history negative) ; 4) course of illness (late) ; or 5)
biological markers.
The degree of generalization of carbamazepine response to other anticonvulsant
agents such as phenytoin or valproic acid will be another area of both clinical and
theoretical import. This is also particularly the case in light of our recent
findings that electroconvulsive shock exerts potent anticonvulsant effects on
limbic system seizures. Are anticonvulsant effects of a variety of treatment
modalities (including ECT) linked to therapeutic response in affective illness?
Carbamazepine is clearly useful in pain syndromes that do not involve a convulsive
process, and effectiveness of anticonvulsant agents in a subgroup of patients with
affective illness does not imply an underlying ictal process.
The possible mechanisms of action of carbamazepine in our patients, as well as
in behavioral pharmacological models, will also be pursued. A clinical trial of
baclofen will help elucidate the role of GABA-B mechanisms in carbamazepine ' s effi-
cacy. We will investigate whether carbamazepine ' s anticonvulsant metabolite,
carbamazepine-10,ll-epoxide, also has important psychotropic properties in manic-
depressive patients.
Alterations in somatostatin as they relate to affective and seizure mechanisms
will also be systematically explored, especially in light of growing evidence of
alterations in somatostatin in depression and in a variety of neuropsychiatric
disorders (D.R. Rubinow) .
As described in detail in Project #Z01 MH 00071-07 BP, Dr. T.W. Uhde will con-
tinue to explore the similarities and differences in patients with panic anxiety
syndromes and those with affective illness in terms of acute symptomatology, longi-
tudinal course of illness, and response to pharmacological agents. Catecholamines
appear to be altered in both the mood disorders and in panic anxiety disorders.
Response to treatments which act on catecholamine systems such as clonidine will be
compared and contrasted in both patient populations. Since caffeine has been shown
to increase plasma Cortisol and induce escape from dexamethasone suppression, the
clinical, mechanistic, and theoretical implications of this important observation
will be systematically followed up by Dr. Uhde and his associates.
Dr. D.R. Rubinow is continuing to study and treat patients with menstrually-
related exacerbation of mood and behavior disorders. He will be examining this
problem from a clinical and endocrinological point of view, and as a model for
studying the acute onset and offset of affective dysfunction.
Work in animal models will continue to focus on possible mechanisms underlying
behavioral sensitization and electrophysiological kindling. In collaboration with
Drs. S.R.B. Weiss, P. Marangos, and J. Patel, neurotransmitter receptors, protein
phosphorylation, and ion channels will be examined as possible mediators or modula-
tors of the electrophysiological kindling paradigm. The mechanisms of anticon-
vulsant action of carbamazepine on amygdala-kindled seizures will also be further
studied. The role of environmental context and conditioning will also be examined
in these paradigms; anatomical and biochemical substrates will be studied.
The mechanisms of cocaine-induced kindled seizure lethality will be explored.
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E. Significance to Biomedical Research and the Program of the Institute
Based in part on work in this Branch, carbamazepine has emerged as a new
treatment for manic-depressive illness. The anticonvulsants have emerged as a -
class of new treatments as valproic acid and clonazepam also appear effective in
mania. Thus, a whole new range of treatment options has evolved from the work with
carbamazepine .
Carbamazepine 's clinical and theoretical importance is further highlighted by
the fact that it is effective in some patients who do not respond to lithium carbon-
ate. Studies of the mechanism of action of carbamazepine may provide new leads to
the understanding of mechanisms of action of other effective antimanic and antide-
pressant drugs as well as basic mechanisms underlying affective dysregulation.
Mechanisms can now be compared and contrasted with lithium and also with a range of
other anticonvulsants that are effective in manic-depressive illness. Thus, basic
and clinical research has led to important findings in neurobiology and the develop-
ment of a new treatment for affective illness with carbamazepine.
Study of endocrine and peptide substances in man and animals may also provide
new conceptual and practical treatment approaches to the relationship between manic
and depressive symptoms and biochemistry. Examination of the interaction between
classical neurotransmitters and the peptides should prove fruitful in understanding
normal and pathological functioning. The multi-disciplinary assessment of our pa-
tients' mood, behavior, cognition, physiology, and biochemistry should allow more
precise characterization of important biobehavioral relationships and their un-
derlying neural substrates.
Elucidating the mechanisms underlying behavioral sensitization and kindling,
which appear to involve processes akin to memory, may provide important information
regarding the coding of behaviorally relevant long-term changes in the CNS . Kind-
ling and behavioral sensitization have aided in the conceptualization of a variety
of psychiatric disorders that show progressive increases in behavioral pathology
over time, and have led to the introduction of new treatment strategies as well as
the novel conceptualization of the efficacy of pharmacotherapy as a function of
state of syndrome development. This is clearly documented with neuroleptics, which
block the development, but not expression, of cocaine-induced behavioral sensitiza-
tion, and with carbamazepine, which blocks completed amygdala-kindled seizures, but
not their development.
Studies of chronic cocaine indicate a potent conditioned component to
behavioral sensitization. Conditioning is now also being recognized as a major
factor in the clinical treatment of cocaine-addicted patients. If subjects are
exposed to cocaine-related paraphernalia or similar cues even months after they
have been withdrawn from the drug, powerful craving and/or withdrawal symptoms can
be re-introduced. Thus, elucidation of the mechanisms and anatomical and
biochemical pathways involved in conditioned components of cocaine-induced
behavioral sensitization in the preclinical animal models should lead to a better
understanding of related phenomena and possible new treatment alternatives in man.
Similarly, understanding the mechanisms involved in the high lethality of
cocaine-induced kindled seizures may lead to better treatment interventions for
this potentially catastrophic reaction. Both the behavioral sensitization and
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kindling perspective, to the extent that they can be extrapolated to the human
cocaine use condition (and all of the data so far suggest that they can be) , imply
that there may be additional hidden liabilities to cocaine use beyond those that
are widely known. That is, that both behavioral and convulsant toxicity may become
an increasing problem with repeated use and that a given dose of cocaine, which was
previously well tolerated, upon sufficient repetition, may not only lead to in-
creasing pathological effects on behavior, but, in some instances, may produce
lethal seizures as observed in pharmacological kindling. Thus, these preclinical
findings which are of interest in their own right and as potential models for manic
and other psychotic symptomatology and psychiatric disorders, may also be of con-
siderable public health interest and pave the way for reconceptualization of the
pathophysiology of cocaine-related syndromes and new treatment interventions.
PUBLICATIONS
Joffe, R.T., Post, R.M., Ballenger, J.C., Rebar, R. , Rakita, R. , and Gold, P.W. :
Neuroendocrine effects of the dopamine agonist, piribedil, in depressed patients.
Clin. Neuropharmacol. 9: 448-455, 1986.
Joffe, R.T., Post, R.M., Ballenger, J.C, Rebar, R. and Gold, P.W.: The effects
of lithium on neuroendocrine function in affectively ill patients. Acta Psychiatr.
Scand. 73: 524-528, 1986.
Joffe, R.T., Post, R.M., Rubinow, D.R., Berrettini, W.H., Hare, T.A. , Ballenger,
J.C, and Roy-Byrne, P.P.: CSF GABA in affective illness. In Shagass, C,
Josiassen, R.C., Bridger, W.H., Weiss, K.J., Stoff, D. and Simpson, G.M. (Eds.):
Biological Psychiatry, 1985 (Developments in Psychiatry, Vol. 7). Amsterdam,
Elsevier, 1986, pp. 1553-1555.
Kellner, C.H., Rubinow, D.R., and Post, R.M. : Cerebral ventricular size and cogni-
tive impairment in depression. J. Affective Pis. 10: 215-219, 1986.
Post, R.M. and Rubinow, D.R.: Somatostatin and heat sensitivity in multiple sclero-
sis [Letter to the Editor]. Lancet 2: 810, 1986.
Post, R.M., Rubinow, D.R., and Ballenger, J.C: Conditioning and sensitization in
the longitudinal course of affective illness. Br. J. Psychiatry 149: 191-201, 1986.
Post, R.M. , Rubinow, D.R., Gold, P.W., Jimerson, D.C and Linnoila, M. : Neurochemi-
cal correlates of mania. In Shagass, C, Josiassen, R.C , Bridger, W.H., Weiss,
K.J., Stoff, D. and Simpson, G.M. (Eds.): Biological Psychiatry, 1985 (Developments
in Psychiatry, Vol. 7). Amsterdam, Elsevier, 1986, pp. 832-834.
Post, R.M. and Uhde, T.W. : Anticonvulsants in non-epileptic psychosis. In Bolwig,
T.G. and Trimble, M.R. (Eds.): Epilepsy in Psychiatry. Chichester, England, John
Wiley and Sons, 1986, pp. 177-212.
Post, R.M., Uhde, T.W., Rubinow, D.R., Gold, P.W. , Joffe, R.T. and Weiss, S.R.B.:
Efficacy and mechanisms of action of carbamazepine in affective disorder. In
Shagass, C, Josiassen, R.C, Bridger, W.H., Weiss, K.J., Stoff, D. and Simpson,
G.M. (Eds.): Biological Psychiatry, 1985 (Developments in Psychiatry, Vol. 7).
Amsterdam, Elsevier, 1986, pp. 886-888.
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Post, R.M., Uhde, T.W,, Rubinow, D.R., Joffe, R.T. and Gold, P.W.: Antidepressant
effects of carbamazepine: possible biochemical mechanisms. In Shagass, C,
Josiassen, R.C., Bridger, W.H., Weiss, K.J., Stoff, D. and Simpson, G.M. (Eds.):
Biological Psychiatry, 1985 (Developments in Psychiatry, Vol. 7). Amsterdam,
Elsevier, 1986, pp. 909-911.
Post, R.M., Uhde, T.W., Rubinow, D.R., Joffe, R.T., Weiss, S.R.B., Patel, J.,
Bierer, L. and Marangos, P.: Possible interactions of the anticonvulsant carbamaz-
epine with calcium. In Shagass, C. , Josiassen, R.C., Bridger, W.H., Weiss, K.J.,
Stoff, D. and Simpson, G.M, (Eds.): Biological Psychiatry, 1985 (Developments in
Psychiatry, Vol. 7) . Amsterdam, Elsevier, 1986, pp. 326-328.
Post, R.M., Weiss, S.R.B., Pert, A., and Uhde, T.W. : Chronic cocaine administra-
tion: sensitization and kindling effects. In Raskin, A. and Fisher, S. (Eds.):
Cocaine: Clinical and Biobehavioral Aspects. New York, Oxford University Press,
1986, pp. 109-173.
Weiss, S.R.B., Post, R.M., Gold, P.W., Chrousos, G. , Sullivan, T.L., Walker, D.
and Pert, A, : CRF-induced seizures and behavior: interaction with amygdala
kindling. Brain Res. , 372: 345-351, 1986.
Weiss, S.R.B., Post, R.M., Marangos, P.J., and Patel, J.: Peripheral-type benzo-
diazepines: behavioral effects and interactions with the anticonvulsant effects
of carbamazepine. In Wada, J. (Ed.): Kindling III. New York, Raven Press, 1986,
pp. 375-391.
Joffe, R.T., Uhde, T.W., Post, R.M. and Minichiello, M.D.: Motor activity in de-
pressed patients treated with carbamazepine. Biol. Psychiatry 22: 941-946, 1987.
Post, R.M. : Mechanisms of action of carbamazepine and related anticonvulsants in
affective illness. In Meltzer, H. and Bunney, W.E. , Jr. (Eds.): Psychopharma-
cology; A Generation of Progress. New York, Raven Press, 1987, pp"] 567-576.
Post, R.M., DeLisi, L.E., Holcomb, H.H., Uhde, T.W., Cohen, R. , and Buchsbaum, M.S.
Glucose utilization in the temporal cortex of affectively ill patients: positron
emission tomography. Biol. Psychiatry 22: 545-553, 1987.
Post, R.M., Kramlinger, K.G. and Uhde, T.W. : Carbamazepine-lithium combination:
perspective on clinical efficacy and side effects. Int. Drug Therapy Newslett. 22:
5-8, 1987.
Post, R.M., Uhde, T.W., Roy-Byrne, P.P. and Joffe, R.T.: Correlates of antimanic
response to carbamazepine. Psychiatry Res., 21: 71-83, 1987.
Post, R.M., Uhde, T.W., Rubinow, D.R. and Weiss, S.R.B.: Antimanic effects of
carbamazepine: mechanisms of action and implications for the biochemistry of
manic-depressive illness. In Swann, A. (Ed.): Mania: New Research and Treatment.
Washington, D.C., APA Press, 1987, pp. 96-176.
Weiss, S.R.B., Nguyen, T. , Rubinow, D.R., Helke, C.J., Narang, P.K., Post, R.M.
and Jacobowitz, D.M. : Lack of effect of chronic carbamazepine on brain somatostat-
in in the rat. J. Neural Transm. 68: 325-333, 1987.
34
ZOl MH 00070-14 BP
Weiss, S.R.B. and Post, R.M. : Carbamazepine and carbainazepine-10,ll-epoxide inhib-
it amygdala kindled seizures in the rat but do not block their development. Clin.
Neurophannacol. 10: 272-279, 1987.
Joffe, R.T. and Post, R.M. : Experimental treatment for affective disorder. In
Berger, P. A. and Brodie, K.H. (Eds.): American Handbook of Psychiatry, Vol. VIII.
New York, Basic Books, in press.
Joffe, R.T., Post, R.M., Roy-Byrne, P.P., and Uhde, T.W. : Hematological effects
of carbamazepine in patients with affective illness. Am. J. Psychiatry, in press.
Kellner, C.H., Post, R.M. , Putnam, F.K., Cowdry, R.W., Gardner, D. , Kling, M.A. ,
Minichiello, M. and Coppola, R. : Intravenous procaine as a probe of limbic system
activity in psychiatric patients and normal controls. Biol. Psychiatry, in press.
Post, R.M. : Animal models: clinical relevance. In Koob, G. and Ehlers, C.
(Eds.): Animal Models of affective Disorders. Chicago, University of Chicago
Press, in press.
Post, R.M. : Approaches to treatment-resistant bipolar affectively ill patients.
Psychiatric Annals, in press.
Post, R.M.: Affective disorders: ^somatic treatment (pharmacotherapy). In Kaplan,
H.I. and Sadock, B.J. (Eds.): Comprehensive Textbook of Psychiatry. Baltimore,
Williams & Wilkins, in press.
Post, R.M. : Combination treatment: antiepileptic agents. In Johnson, F.N. (Ed.):
Modern Lithium Therapy. Oxford, IRL Press, in press.
Post, R.M. , Rubinow, D.R., and Gold, P.W.: Neuropeptides in manic-depressive ill-
ness. In Nemeroff, C.B. (Ed.): Neuropeptides in Psychiatric and Neurological
Disease . Baltimore, Johns Hopkins University Press, in press.
Post, R.M, and Uhde, T.W. : Psychotropic effects of carbamazepine and its mechan-
isms of action in affective illness. In Burrows, G.D. and Werry, J.S. (Eds.):
Advances in Human Psychopharmacology, Vol. iv. Greenwich, Connecticut, JAI Press,
Inc. , in press.
Post, R.M. and Uhde, T.W.: The use of carbamazepine in mania. In Burrows, G.D.,
Norman, T.R., and Davies, D. (Eds.): Antimanics, Anticonvulsants, and Other Drugs
in Psychiatry (Drugs in Psychiatry, Vol. 4) . Amsterdam, Elsevier/North Holland Bio-
medical Press, in press.
Post, R.M. and Uhde, T.W.: Clinical approaches to treatment-resistant bipolar
illness. In Hales, R.E. and Frances, A.J. (Eds.): APA Annual Review, Vol. 6.
Washington, D.C., APA Press, in press.
Post, R.M. and Uhde, T.W.: Refractory manias and alternatives to lithium treatment.
In Georgotas, A. and Cancro, R. (Eds.): Textbook of Depression and Mania. Amster-
dam, Elsevier Science Publ . Co., in press.
35
ZOl MH 00070-14 BP
Post, R.M., Uhde, T.W., Rubinow, D.R. and Huggins, T.: Differential onset of anti-
depressant efficacy following sleep deprivation, ECT, and carbamazepine: clinical
and theoretical implications. Psychiatry Res. , in press.
Post, R.M. and Weiss, S.R.B.: Behavioral pharmacology of carbamazepine: differen-
tial effects on kindling. Int. J. Clin. Psychopharmacol . , in press.
Post, R.M., Weiss, S.R.B. and Rubinow, D.R.: Recurrent affective disorders:
lesions from limbic kindling. In Ganten, D. and Fuxe , S. (Eds.): Current Topics
in Neuroendocrinology . New York, Springer Verlag, in press.
36
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00071-07 BP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Psychobiological Correlates and Treatment of Panic and Related Mood Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
T.W. Uhde, M.D., Chief, Unit on Anxiety and Affective Disorders, BPB, NIMH
R.M. Post, M.D.
J. -P. Boulenger, M.D.
P.P. Roy-Byrne, M.D.
B.J. vittone, M.D.
B. Scupi, M.S.W^.
BPB, NIMH
French National Institute for Health and
Medical Research, Cannes, France
Univ. of Washington, Seattle, Washington
BPB, NIMH
BPB, NIMH
COOPERATING UNITS (rf any) CNB, CRB , LCS , LPP , NSB, OD , NIMH; Outpatient Dept . , NIMH; LCS,
NIAAA; French Nat'l Inst, of Hlth. & Med. Res., Cannes; UC, Irvine & San Diego;
VA Med. Ctr., Bronx; Med. Univ. of So. Carolina, Charleston; Univ. Pittsburgh,
Pittsburgh, Univ. of Washington, Seattle; Catholic Univ., Washington, D.C.
HB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Anxiety and Affective Disorders
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
5
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
El (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Patients with pathological degrees of anxiety who meet DSM III-R criteria for
panic disorder, with or without agoraphobia; generalized anxiety disorder; or
social phobia are evaluated using psychological, physiological, and biochemical
methodologies. Patients with major affective illness, particularly those with a
significant anxiety component, are also eligible for participation in the program.
Particular attention is given to the role of the noradrenergic , dopaminergic, ad-
renergic, and serotonergic neurotransmitter systems as assessed by: 1) measure-
ment of the metabolites MHPG and HVA in plasma; 2) adrenergic receptor number and
function in platelets; and 3) neuroendocrine and behavioral responses to the
alpha-2 adrenergic agonist clonidine and antagonist yohimbine and the serotonin
agonist m-chlorophenylpiperazine (MCPP) . Research investigating the relationship
of noradrenergic and adenosinergic function to other neurotransmitter systems and
hypothalamic-pituitary-adrenal axis also has been initiated. Caffeine and nifedi-
1;he
pine challenges are administered to assess their behavioral and biochemical ef-
fects. Other approaches to understanding the pathophysiology of anxiety and its
potential treatment with alprazolam, carbamazepine , clonidine, imipramine and
verapamil will be explored.
An animal model using genetically "nervous" and "normal" pointer dogs has been
developed and studied in relation to noradrenergic and adenosinergic function.
37
PHS 6040 (Rev. 1/84)
CPO >I4>SI«
COLLABORATORS :
ZOl MH 00071-07 BP
M. Geraci
M.S. Buchsbaum, M.D.
T.P. Zahn, Ph.D.
M. Kafka, Ph.D.
L. Siever, M.D.
D.C. Jimerson, M.D.
N. Salem, M.D.
M. Albus, M.D.
W. Potter, M.D.
M. Linnoila, M.D.
L. Bierer, M.D.
C. Kellner, M.D.
E. Klein, M.D.
J. Maser, Ph.D.
T. Mellman, M.D.
G. Leverich
K. Kramlinger, M.D.
P. Marangos, Ph.D.
S. Risch
S. Sinclair
W. Kaye, M.D.
S. Steinberg
S.R.B. Weiss, Ph.D.
M. Stein, M.D.
G. Gurguis, M.D.
C. Shea, M.A.
D. Arnkoff, Ph.D.
Psychiatric Nursing Department, NIMH
University of California at Irvine
LPP, NIMH
NSB, NIMH
VA Medical Center, Bronx, New York
LCS, NIMH
LCS, NIAAA
CNB, NIMH
LCS, NIMH
LCS, NIAAA
VA Medical Center, Bronx, New York
Medical University South Carolina, Charleston, South
Carolina
BPB, NIMH
CRB, NIMH
BPB, NIMH
BPB,., NIMH
BPB, NIMH
BPB, NIMH
Univ. of California at San Diego
BPB, NIMH
Univ. of Pittsburgh, Pittsburgh, Pennsylvania
Veterinary Medicine, Univ. of Pennsylvania
BPB, NIMH
BPB, NIMH
BPB, NIMH
BPB, NIMH
Catholic University, Washington, D.C.
38
ZOl MH 00071-07 BP
I. Project Description
A. Objectives
This project employs a multidisciplinary team in the study and treatment of
pathological anxiety, major affective and related mood disorders.
B. Methods Employed
1. Subjects
a. Patients who meet Research Diagnostic Criteria for panic, pho-
bic, and generalized anxiety disorders, as well as patients who meet DSM III cri-
teria for major affective illness, are candidates for participation in the pro-
ject. Inpatients are studied and treated on the 3-West Clinical Research Unit and
outpatients are followed through the Ambulatory Care Research Facility. A number
of previously validated scales to measure state and trait anxiety are utilized and
an analogue anxiety scale and panic anxiety scale have been developed to more ade-
quately assess the relationship among state anxiety, phobic anxiety, avoidance be-
havior, and depressive symptomatology.
b. Normal volunteers are also accepted into the project to provide
control data, as well as to assess the relationship between normal state anxiety
and selected psychological and biological variables.
2. Psychological and Biological Evaluation
a. Baseline Evaluation. During an initial evaluative period pa-
tients undergo extensive neurological, psychological, biochemical, and neurophys-
iological evaluation. This initial evaluation is indicated due to the heterogen-
eous nature of the panic and phobic disorders. Anecdotal reports suggest that
many medical illnesses may present as or exacerbate pre-existing conditions of
pathological anxiety.
b. Life Chart Methodology. A life chart technique has been devel-
oped to plot the frequency, intensity, and interval between panic attacks. The
character and the change in the quality of panic attacks is assessed as a function
of duration and longitudinal course of illness. This approach allows the Unit to
document the development, recurrence, and progression of the panic and phobic dis-
orders. Life charting is an important aspect of the overall project because few
systematic studies have been conducted on the natural progression of these disor-
ders.
As part of this assessment, life events and their impact on the course of
illness are investigated with the PERI-M life events inventory. Moreover, the
influence of personality (DSM III, Axis II diagnosis) on the phenomenology and
course of illness is systematically evaluated with the Structured Interview for
DSM III Personality Disorders (SIDP) . These studies are conducted in
collaboration with Drs. K. Kramlinger, T. Mellman, P. Roy-Byrne, and with M.
Geraci, G. Leverich, and B. Scupi.
39
ZOl MH 00071-07 BP
c. Sleep and Sleep Deprivation. Electroencephalographic sleep re-
cordings are obtained for three consecutive nights. Although many panic anxious
patients, like endogenously depressed individuals, have improved sleep following
treatment with tricyclic and monoamine oxidase inhibitors, little is known about
the sleep architecture of panic and phobic anxious patients. The effects of one
night's sleep deprivation on mood and behavior are investigated in patients with
panic disorder and major depressive disorder. Sleep studies are conducted in col-
laboration with Drs. T. Mellman and P. Roy-Byrne.
d. Galvanic Skin Response. The effects of yohimbine on
physiological measures of galvanic skin response, reaction time to auditory tones,
pulse, and respiratory rate are studied in panic and phobic anxious patients and
age-matched normal volunteers. This investigation is performed in collaboration
with Drs. M. Albus, B. vittone, and T. Zahn.
e. Computerized Axial Tomography. Cerebral CAT Scans are
obtained, and, in collaboration with Dr. C. Kellner, cerebral ventricular size is
determined in patients with panic disorder. Scans are performed on a GE 8800 or
9800 Scanner.
f. Psychomotor Activity. Twenty-four hour motor activity is ass-
essed with a miniaturized activity monitor worn on the wrist of patients with pri-
mary anxiety disorders under a variety of experimental conditions.
g. Caffeine. Caffeine is administered to panic patients and nor-
mal controls to assess behavioral and biochemical responses to this agent whose
effects are thought to be mediated through the adenosine, GABA-benzodiazepine, and
noradrenergic systems.
To assess the effects of caffeine on plasma adenosine, an HPLC assay for
adenosine and caffeine has been developed in collaboration with Drs. N. Salem and
P. Marangos. The clinical studies are conducted in collaboration with Drs. L.
Bierer, J. -P. Boulenger, T. Mellman, R. Post, and M. Geraci and S. Sinclair.
h. Clonidine — An Alpha-Adrenergic Agonist. Clonidine is admin-
istered intravenously to anxious and affectively ill patients and normal
volunteers to assess clinical, physiological, and neuroendocrine responses to this
noradrenergic drug. These studies are conducted in collaboration with Drs. G.
Gurguis, W. Kaye, R. Post, L. Siever, and B. Vittone.
i. Yohimbine — An Alpha-Adrenergic Antagonist. Yohimbine is ad-
ministered in an oral challenge to panic anxious and affectively ill patients and
normal controls to assess the clinical and biochemical effects of this noradrener-
gic antagonist which is known to potently increase noradrenergic function in the
animal. These studies are conducted in collaboration with Drs. M. Albus, G.
Gurguis, B. vittone, T. Zahn, and with M. Geraci.
j . Nifedipine -- A Calcium Channel Blocker. Nifedipine is
administered orally to agoraphobic patients exposed to nonphobic and phobic
situations to determine the antianxiety effects, if any, of calcium channel
blockers. This study is conducted in collaboration with Dr. E. Klein and M.
Geraci.
40
ZOl MH 00071-07 BP
k. Plasma MHPG, HVA, and Urinary Free Cortisol. Amine metabolites
and urinary free Cortisol are systematically evaluated using daily 24-hour urine
collections across clinical state changes on and off medication. These studies
are conducted in collaboration with Drs. G. Gurguis, D. Jimerson, M. Linnoila, and
W. Potter.
1. Dexamethasone Suppression Test. Dexamethasone is administered
to patients to evaluate the hypothalamic-pituitary-adrenal axis. Basal values are
performed at baseline and at 4:00 pm, following dexamethasone administration.
m. Urine and Plasma Studies. Amine metabolites, electrolytes, and
peptides are also measured in the urine and blood.
n. Alpha-Adrenergic Receptors. In collaboration with Dr. M.
Kafka, platelet alpha receptor function as well as prostaglandin-stimulated
increases in cyclic-AMP are assessed in patients and age-matched normal
volunteers.
o. Platelet Imipramine Binding. In collaboration with Dr. W.
Berrettini, [^H] imipramine binding to platelets is measured in patients and normal
controls .
3. Treatment
a. Psychotherapeutic . Treatment and evaluation are conducted in
individual and/or group supportive sessions. In addition, ongoing clinical case
conferences are utilized. Collaborators in these studies include Drs. E. Klein,
T. Mellman, B. Vittone, G. Gurguis, M. Stein, and M. Geraci and B. Scupi.
b. Routine Somatic Treatment. Both routine and experimental com-
pounds are evaluated during double-blind clinical trials. Ongoing clinical trials
include calcium channel blockers, tricyclic antidepressants, monoamine oxidase in-
hibitors, and minor tranquilizers. These studies are performed in collaboration
with Drs. E. Klein, T. Mellman, B. vittone, M. Stein, G. Gurguis, and M. Geraci
and B. Scupi.
C. Major Findings (Studies in Humans) :
1 . Medical Illnesses and Anxiety
Detailed physical, neuropsychiatric, and laboratory evaluations
continue to be performed in patients admitted to our program. As reported in
previous years (ZOl MH 00071-04/05/06 BP) , 60% of our panic patients had
previously undiagnosed medical illnesses. Although these illnesses appeared to be
unrelated to the direct pathogenesis of the panic attacks themselves, these data
extend previous research suggesting that psychological (major life events) and
physiological (medical illnesses) stressors may trigger panic attacks in
biologically vulnerable individuals.
In an attempt to further define the characteristics of brain structure in
panic disorder, we continue to investigate the cerebral ventricular size (VBR) in
agoraphobic patients with panic attacks. The mean VBR in our patients was 3.4 ±
41
ZOl MH 00071-07 BP
2.4 SD (range 1.0-9.0). Male (M) and female (F) patients had similar VBR (M: 4.0
± 2,8 versus F: 2.9 ± 1.8; t = 1.21, df = 23, p = NS) and were of similar age at
the time of the scan (M: 34.1 ± 6.5 versus F: 36.2 ± 7.0; t = 0.77, df = 23, p =
NS) . Of the 25 scans, one was read as clinically abnormal in a patient with
abnormally small ventricles for age. VBR for patients who had a history of major
depression (n = 7, 28%) (2.9 ± 1.5) or severe agoraphobia (n = 7, 28%) (3.2 ± 1.5)
did not differ from the patients without a history of depression (3.6 + 2.6, t =
0.65, df = 23, p = NS) or severe agoraphobia (2.6 ± 3.5, t = 0,28, df = 23, p =
NS) . There was a significant inverse relationhip between vBR and duration of
illness (r = -0.55, p < 0.01). There was no significant association between VBR
and age (r = -0.06, p = NS) , panic attacks within the past month (r = 0.04, p =
NS) , or state anxiety (r = 0.19, p = NS) .
There was a significant association between VBR and duration of
benzodiazepine use (r = 0.51, p < 0.02) and percent of time ill treated with
benzodiazepines (r = 0.67, p < 0.001) , although the mean VBR (3.8 ± 2.5) of the
panic disorder patients who had received benzodiazepine treatment was similar to
the patients without previous benzodiazepine exposure (2.5 ± 1.6; t = 1,26, df =
23, p = NS) .
The findings, published in the J. Affective Disord., suggest that the
ventricular size of panic disorder patients falls well within the normal range
compared with reported values of mean VBR in normal control groups in the
literature.
The nature of the relationship between VBR and duration of benzodiazepine
exposure (r = 0.51, p < 0.02) in our study remains unclear but might be related
either to a direct or indirect drug effect or merely be an artifact reflecting a
tendency towards greater drug use in a subpopulation of patients with more severe
illness.
2. Psychosensory Symptoms
The Unit continues to investigate the role of psychosensory symp-
toms in the phenomenology and longitudinal course of panic disorder. As noted in
ZOl MH 00071-05/06 BP , panic disorder patients experience increased psychosensory
symptoms during episodes of illness. During well intervals, the number of
psychosensory symptoms in panic patients is similar to normal controls, although
both nondepressed panic disorder patients and affectively ill patients report
comparable increases in total number of psychosensory symptoms during clinical
relapses. Since psychosensory phenomena occur spontaneously and after stimulation
of the amygdala and hippocampus in patients with psychomotor epilepsy, these
findings support an involvement of tempore- limbic structures in these psychiatric
conditions. Of interest, preliminary findings suggest that the number and type of
psychosensory symptoms does not predict clinical response to a wide variety of
psychotropic medications.
3. Life Events and Onset of Panic Disorder
The life course of panic disorder continues to be assessed retro-
spectively in all patients with panic attacks. As reviewed in greater detail in
42
ZOl MH 00071-07 BP
previous reports (ZOl MH 00071-04/05/06) , several conclusions can be made regard-
ing the phenomenology and longitudinal course of panic disorder. First, the onset
of panic attacks generally begins in adolescence or early adulthood and, if
untreated, frequently leads to an impaired life style characterized by patho-
logical degrees of anticipatory or free-floating anxiety and agoraphobia. Second,
lifetime symptoms of major depression occur in approximately 50% of the patients,
although only 25% of all panic disorder patients develop longstanding endogeno-
morphic symptoms of depression. Third, tricyclic antidepressants appear to have
antipanic effects independent of the presence of concomitant depressive symptoma-
tology.
During the past year, the Unit has focused on the role of life stressors and
life events on the onset and course of illness of panic disorder. In order to
evaluate the role of life events in the onset of panic disorder, we explored the
number, type, and effect of life events occurring prior to the first panic attack
in patients with panic disorder compared with similar data obtained in normal con-
trol subjects. Results of this study, reported in the American Journal of Psychi-
atry, indicate that panic disorder patients experienced more life events directly
involving them. Comparisons by category, however, yielded differences only for
events related to work and health. In particular, patients did not have more
"exit" events than controls. Although there were no differences in objective
degree to life change or stress, the patients did report greater subjective "dis-
tress" .
These results suggest that patients with panic disorder, prior to the onset
of their illness, personally experience more life events "happening to them" than
controls. More importantly, life events seem to have a more adverse subjective
effect on patients.
The major difficulty with this type of retrospective analysis involves the
possible distorting effect of time on recall. Our analysis failed, however, to
find an interaction between recall time and subject group by ANOVA. Using linear
statistical analyses, the effects of time on recall was assessed in panic disorder
patients and normal controls.
Not unexpectedly, time affected the number of events reported. However,
there was no difference, in terms of the influence of time on the recall of each
type of event, between the groups. The theoretical rationale for this statistical
analysis and methodological implications of these findings were published in the
Journal of Affective Disorders.
4. Life events and Course of Panic Disorder
The previous study indicated that an increased frequency of life
events precedes the onset of panic disorder. Although there was no difference be-
tween panic disorder patients and normal controls in the total number of exit
events, it remained unclear whether those patients who did experience a major
separation or loss (e.g., death of spouse) prior to the onset of their illness
would have a higher prevalence of subsequent major depressive episodes, panic
attack frequency, or onset of agoraphobia.
43
ZOl MH 00071-07 BP
Thirty-three patients with panic disorder participated in the study. They
ranged in age from 21 to 45 years (meanlSD; age = 31.4 ± 6.5 years), and their
duration of illness ranged from 1 to 16 years (meantSD = 6.1 ± 4.8 years) . In all
cases, panic attacks were the first psychiatric symptom these patients had experi-
enced and constituted the complaint that brought them to psychiatric attention.
Life events were determined using the Peri-M Events Scale. In this study we
were only interested in the presence or absence of a major loss or separation, a
type of event unlikely to be forgotten. Before we examined our data, we decided
to define this type of event as including the death of a parent, sibling, or
spouse, or the permanent dissolution of the patient's major attachment bond
through divorce or another action (e.g., the breakup of a longstanding romantic
relationship) . There were 11 patients in the "major loss" subgroup, five having
experienced the death of their father, two having been divorced, and four having
faced the dissolution of a longstanding (longer than a year) romantic
relationship. The remaining 22 patients had experienced other types of life
events. Four patients who had undergone similar but less severe separation events
(the death of an aunt, a transient separation from a mate, etc.) were excluded
from the analysis.
Life course of illness was characterized with our life charting method.
Variables extracted for this analysis included number of panic attacks in the year
after onset, longest time free of panic attacks, rapidity of onset of agoraphobia,
and presence or absence of a subsequent major depressive episode. In patients
with a major depressive episode, we also recorded the time elapsed from the first
panic attack to the episode, the severity of the episode, and the total number of
episodes.
Parametric data were analyzed using a two-tailed Student's t test, and
dichotomous data were analyzed using chi-square analysis. Patients with a severe
loss preceding the onset of their illness had a significantly greater prevalence
of subsequent major depression than did the no-loss group (85% versus 36%; x^ =
6.08, df = 1 , p < .02). There were no significant differences between groups in
number of panic attacks, longest time free of panic attacks, or rapidity of onset
of agoraphobia (45% within 3 months in the severe-loss group versus 36% in the
no-loss group) . For the majority of patients suffering a subsequent depression,
these depressions were severe (78% in the severe-loss group and 78% in the no-loss
group) and produced functional impairments regardless of whether or not there had
been a severe loss preceding the onset of the illness. The time from first panic
attack to the depression and the total number of lifetime depressions were similar
whether or not there had been a prior loss.
These data, published in the American Journal of Psychiatry, suggest that the
occurrence of major loss in patients with panic disorder confers an increased risk
for a "secondary" depression without influencing the course of the primary anxiety
symptomatology .
5. Panic Disorder: Relation with Obsessive Compulsive Symptoms
There has been much recent interest in the clinical and biological
overlap of both panic and obsessive-compulsive disorders with major affective dis-
orders. Although there is less known about the relationship between panic
44
ZOl MH 00071-07 BP
disorder and obsessive-compulsive disorder, some evidence suggests a partial
overlap in clinical phenomenology.
We determined the prevalence of obsessive-compulsive symptoms in our panic
disorder population. We also compared clinical variables and treatment outcome in
panic disorder patients with obsessive-compulsive symptoms to panic disorder pa-
tients without obsessive-compulsive features. Nineteen of 70 (27%) patients who
met Research Diagnostic Criteria (RDC) for panic disorder reported obsessive-com-
pulsive symptoms during a diagnostic interview utilizing the Schedule for
Affective Disorders and Schizophrenia (SADS) , anxiety disorders section. Of this
group, 10 (53%) reported obsessional symptoms only, two (10%) reported compulsive
symptoms only, and the remaining seven (37%) reported obsessional plus compulsive
symptoms. These 19 patients with panic disorder (PD) plus obsessive-compulsive
symptoms (OCS) comprised the study group (PD + OCS) .
A comparison group without OCS was selected to form a homogeneous population
with certain classic features of panic disorder. Followup data were obtained by a
structured telephone interview of patients who had completed the NIMH treatment
program a mean of 16.3 ± 13.4 months (range 3-46) for the PD + OCS group, and 13.4
± 10.0 months (range 7-32) for the PD - OCS group prior to phone contact. Sixteen
of 19 of the PD + OCS group (84%) and 14 of 25 of the PD - OCS group (56%) were
available for the phone interview (p = NS) . The interview included information on
the three-month prevalence and self-rated change in the following items: panic,
generalized anxiety, avoidance, obsessions, and compulsions. Patients made a glo-
bal judgment as to whether each of these five symptoms had improved, were
unchanged, or had worsened since their discharge from the program. The interview
also included social disability scale.
Compared to the PD - OCS group, a lifetime history of major depression by
DSM-III criteria was more frequent in the PD + OCS group (p < .001) as was a past
history of alcohol or drug abuse (p < .05) . The PD + OCS group patients had a
significantly earlier onset of illness (20.3 ± 4.7 years) compared to patients
without obsessive-compulsive symptoms (27.1 ± 7.7; X^ = 3.43, p < .01). The two
groups did not differ significantly with regard to the presence or severity of
agoraphobia. There was no difference noted in the two groups for frequency of
panic attacks, total years ill, or percentage of time in remission prior to the
NIMH evaluation.
There was a significantly greater incidence of primary affective disorders
(63% versus 20%) and alcoholism or substance abuse (47% versus 8%) in the first
degree relatives of panic patients with obsessive-compulsive symptoms compared to
the first degree relatives of patients without obsessive-compulsive symptoms.
There was no difference in the incidence of panic or phobic disorders.
While both groups reported improvement in panic attacks, persistent attacks
were more common at follow-up in the PD + OCS patients, even though 89% and 77% of
the PD + OCS and PD - OCS groups, respectively, had greater than one panic attack
per month at the time of initial evaluation. Fewer PD + OCS patients compared to
PD - OCS patients reported "improvement" in generalized anxiety (6/16 versus
12/14, X2 = 5.36, p < .02) and a greater number reported the persistence of
moderate to severe generalized anxiety (13/16 versus 4/14, X^ = 6.3, p < .01).
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These data suggest that the presence of obsessive-compulsive symptoms in
panic disorder may be a clinical predictor of a subgroup of panic disorder
patients with distinct features and substantially less optimal treatment outcome,
particularly in relation to symptom interference with functioning. The greater
impairment in this subgroup cannot be attributed solely to the additional problems
associated with a second separate neuropsychiatric disorder (i.e.,
obsessive-compulsive disorder) , since these patients also reported a greater rate
of panic attacks and more severe generalized anxiety at follow up.
6. Sleep, Sleep-related Panic, and Sleep Deprivation
In reports ZOl MH 00071-04/05/06 BP we reported in detail the find-
ings on the sleep EEG of panic disorder patients. Since a major focus of the
Unit's ongoing research is the investigation of the relation between panic and
major depressive disorders, we were particularly interested in the nature of
rapid-eye-movement (REM) parameters in patients with panic disorder compared to
normal controls. Preliminary data from our laboratory, published in Psychiatry
Research, suggested that panic disorder patients did not have marked reductions in
REM latency typical of patients with melancholic depression. In fact, in this
initial study, our panic disorder patients had a significantly lower REM density
and a normal progression in the length of each successive REM period. These
findings have been both confirmed and extended by our laboratory in a second
separate study.
In our second study, we investigated the sleep EEG of patients with panic and
major depressive disorders and normal controls. The sleep of the panic disorder
patients was generally disturbed, as manifested by significant decreases in sleep
time and sleep efficiency and increased sleep latency. These disturbances were
more prominent in the panic disorder patients compared with both the depressed
patients and normal controls'. Preliminary findings also suggest that REM
latencies are reduced in depressed patients compared to the panic disorder
patients and normal controls.
Six of 13 patients experienced sleep panic attacks. The sleep panics were
all characterized as sudden awakening with fear or apprehension, without recall of
any specific dream content. The symptoms most commonly reported for these
episodes included palpitations occurring during 100% of the episodes, sweating
(67%) , hot or cold flushes (50%) , choking or smothering sensation (50%) , feelings
of unreality (50%), and chest pain or discomfort (50%). The attacks occurred
between 24 and 225 minutes from sleep onset and between 65 minutes before and 48
minutes after the first REM period. The epoch preceding the awakenings with panic
were scored as stage 2 for two of the six sleep panics and stage 3 for the
remaining four attacks. Some EEG slowing preceded the awakenings from stage 2,
and the awakenings from stage 3 were preceded by a maximum of only two minutes of
stage 3 sleep. The amount of time from the awakening to resuming stage 1 or stage
2 sleep ranged from two to seven minutes.
Nights of sleep panic featured increased REM latencies (101.2 minutes ± 40.4
versus 69.9 ± 18.9, p < .05) and increased minutes of stage 3 sleep (26.2 ± 26.5
versus 22.5 + 25.0, t = 2.98, p < .05) in comparison to non-panic nights. There
was a trend for less movement time to occur on panic nights. These data suggest
that panic attacks occurring from sleep are not an infrequent feature of panic
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disorder and provide a potentially useful model for elucidating mechanisms of
panic.
All six of the attacks recorded in this study occurred from non-REM sleep
and, in comparison with other spontaneous awakenings, there appears to be some
specificity for stage 3 sleep. In fact, all of the panic awakenings could be
characterized as being preceded by a transition from a lighter to deeper stage of
non-REM sleep; i.e., proceeding from stage 2 toward delta sleep.
That panic can occur in association with the progession toward a "deeper"
stage of sleep is of interest with regard to the observation that increased basal
arousal is often predictive of subsequent panic in many panic induction studies,
such as those utilizing sodium lactate infusions. Our findings suggest that
increased basal arousal is not a requirement for panic and that panic may actually
occur in the context of diminishing arousal.
Several studies have documented that one night's total sleep deprivation is
associated with a clinically robust but transient improvement in mood in depressed
patients. In fact, patients with the more classic symptoms of melancholia appear
to be the best responders to sleep deprivation, while more atypical depressives
tend to be poor responders to sleep deprivation. On the basis of these observa-
tions, we hypothesized that nondepressed panic disorder patients would fail to re-
spond positively to one night's total sleep deprivation.
The effects of one night's total sleep deprivation was studied, therefore, in
panic disorder patients and compared with results in depressed patients and normal
controls previously studied in our laboratory. As a group, the depressed patients
demonstrated significantly more improvement in nurse-rated measures of anxiety
than both the panic disorder patients and controls. Depressed patients showed a
decrease, while panic patients had an increase in measures of anxiety, including
some patients who experienced panic attacks. These findings, published in the
Archives of General Psychiatry, suggest that both the sleep EEG and behavioral re-
sponse to sleep deprivation are reliable markers in distinguishing between nonde-
pressed panic disorder patients and patients with major depressive disorder,
melancholic sub type .
7. Urinary Free Cortisol and Plasma MHPG in Panic Disorder
Alterations in noradrenergic function have been postulated to play
an important role in the modulation of fear and anxiety. Moreover, the
noradrenergic system appears to be functionally related to
hypothalamic-pituitary-adrenal (HPA) axis function. The Unit on Anxiety and
Affective Disorders, therefore, studied urinary free Cortisol and plasma MHPG in
12 panic disorder patients and 12 normal controls.
There was no significant difference in either MUFC or plasma MHPG levels be-
tween panic patients and normal controls. Two of 12 patients versus none of 12
controls had a MUFC greater than the normal range of 9 to 95 ijg/24 hrs . There was
no significant correlation between plasma MHPG and MUFC in the total sample (r =
0.27, df = 22, p = NS) or in the panic patients (r = 0.17, df = 10, p = NS) or
controls (r = 0. 52 , df = 10, p = NS) as separate groups.
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Six patients reported a mean of 2.3 ± 1.6 (range 1-5) panic attacks during
the three day period of the study (panic attack-positive patients [PA+] ) . The
mean Spielberger anxiety ratings for the total patients group were 46.3 ± 6.3.
There was no significant difference in mean Spielberger anxiety ratings in
PA-positive (49.2 ± 1.7) versus PA-negative (43.4 + 8.1) patients. The
PA-positive patients did not have significantly different plasma MHPG (3.2 + 0.8
SD) or MUFC (62.4 pg/24 hours + 31.4 SD) values compared with the PA-negative
patients (MHPG: 4.0 ± 0.8 SD, t = 1.66, df = 10, p = NS; MUFC: 51.6 pg/24 hours ±
26.2 SD, t = 0.64, df = 10, p = NS) , normal controls (MHPG: 3.7 ± 0.9, t = 1.0, df
= 16, p = NS; MUFC: 52.7 ± 18.8, t = 0.82, df = 16, p = NS) , or the combined group
of PA-negative plus normal controls (MHPG: 3.8 ± 0.9 SD, t = 1.39, df = 22, p =
NS; MUFC: 52.4 Mg/24 hours ± 20.8 SD , t = 0.90, df = 22, p = NS) .
There were no significant correlations between MHPG and frequency of panic
attacks (r = -0.46, df = 10, p = NS) or measures of state anxiety (r = -0.40,
df = 10, p = NS) , global anxiety (r = -.32, df = 10, p = NS) , agoraphobia
(r = -0.32, df = 10, p = NS) or depression (r = 0.11, df = 10, p = NS) in the pa-
tients. There was also no significant correlation between MUFC and frequency of
panic attacks (r = 0.35, df = 10, p = NS) or measures of state anxiety (r = -0.31,
df = 10, p = NS) , global anxiety (r = -0.06, df = 10, p = NS) , agoraphobia
(r = 0.49, df = 10, p = NS) or depression (r = 0.27, df = 10, p = NS)
in the panic disorder patients.
Our MUFC data suggest that increased HPA axis function is not a prominent
feature of panic disorder. Our Unit has reported, however, that patients with
panic disorder have elevated evening plasma Cortisol levels and reduced ACTH and
Cortisol responses to corticotropin releasing hormone (CRH) (see 15C) . Similar
findings with the CRH test have been reported in depressed patients. These
observations suggest that while panic patients may not have persistently elevated
indices of increased HPA function, there may be discrete periods of
hypercortisolemia associated with the illness. Mean plasma MHPG values did not
differ between panic patients and controls.
We also failed to find a relationship between MHPG and frequency of panic
attacks or ratings of global anxiety or agoraphobia. These observations and our
finding that plasma MHPG did not distinguish PA-positive from PA-negative patients
are consistent with the suggestion that noradrenergic overactivity is not a
biological pre-requisite for all panic attacks or other types of pathological
anxiety. Moreover, although an association between adrenergic activation and
plasma Cortisol have been reported in depressed patients, we found no correlation
between plasma MHPG and urinary free Cortisol in either panic disorder patients or
normal controls. These observations, to be published in Biological Psychiatry,
further suggest that the mechanisms underlying central adrenergic and peripheral
HPA activation may have different dimensions of functional relatedness, depending
on the nature and state of the psychiatric syndrome.
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8. Plasma HVA in Panic Disorder
To assess dopamine function in panic disorder, plasma HVA and MHPG
was investigated in 15 panic disorder patients and nine normal controls. As
reported in ZOl MH 00071-06 BP, we found no difference in plasma HVA between panic
disorder patients and normal controls. However, plasma HVA values showed a
bimodal distribution. Comparisons of high and low HVA subgroups were tabulated.
Patients in the "high" HVA subgroup, compared with those in the "low" subgroup,
had significantly higher Spielberger State Anxiety scores, more panic attacks in
the previous year, and shorter maximal time free of panic attacks.
These findings, published in Biological Psychiatry, suggest that panic disor-
der patients with higher concentrations of plasma HVA are more anxious, have in-
creased panic attacks, and have shorter symptom-free remissions. Although the
panic disorder patients did not as a group have higher HVA compared to normal con-
trols, these preliminary findings suggest a possible role for dopamine systems in
the neurobiology of panic disorder.
9. Effects of Diazepam on Mood, Memory, and Pain
The effects of clonidine on the somatosensory pain threshold have
been discussed in previous annual reports (ZOl MH 00071-04 and ZOl MH 00071-05) .
The Unit has expanded these studies to include the assessment of the effects of
diazepam on mood, memory, and pain.
Using previously described methods (ZOl MH 00071-04/05) , the Unit recently
found that 10 mg of diazepam produces a significant impairment in effortful memory
and attention, but had no effect on automatic memory, semantic memory, or judgment
memory. These findings were published in Psychopharmacology . Data also suggest
that diazepam has a subtle analgesic effect due to its ability to prevent the nor-
mal improvement in discriminability associated with somatosensory retesting. Al-
though diazepam produced mild-moderate subjective effects such as the induction of
"lethargic", "dreamy", "drowsy", and "muggy-headed" states, none of these subjec-
tive feelings was related to diazepam's effects on free-recall and attention.
Overall, the effects of diazepam on pain and memory appear to be a separate
phenomenon. These findings, together with previous data (ZOl MH 00071-04/05)
showing that pain and anxiety may have opposite relationships in panic disorder
patients compared to normal controls, suggest that diazepam may have differential
effects on pain sensitivity in patients with anxiety disorders.
10. Effects of Diazepam on Cortisol and Beta-endorphin
Benzodiazepines have been shown to have neuroendocrine effects in
both animals and humans. The most consistently observed endocrine changes in re-
sponse to benzodiazepines have been decreases in ACTH and Cortisol. Increases in
growth hormone (GH) have been observed by some but not all investigators.
Numerous studies have demonstrated that various kinds of stress can activate the
HPA axis, causing increases in both Cortisol and ACTH. Benzodiazepines have also
been shown to antagonize stress-induced increases in both ACTH and Cortisol in
animals and humans.
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The neuroendocrine effects of 5 and 10 mg of orally administered diazepam
were, therefore, assessed in ten normal subjects under baseline (pre-stress) and
laboratory-controlled stressful conditions. Although the 10 mg diazepam dose had
no effect on Cortisol at baseline, it significantly reduced the increase in
Cortisol produced by 15 minutes of exposure to a painful electrical stimulus.
There were no significant effects on growth hormone, beta-endorphin, or ACTH
either at baseline or following painful stimulation.
Our results, to be published in the Journal of Clinical Psychopharmacology,
document the ability of diazepam to blunt stress-induced increases in plasma
Cortisol in normal subjects. These results suggest the possibility of employing
the cortisol-response to diazepam in assessing the function of the benzodiazepine
system in different psychiatric conditions.
11 . Caffeine: Behavioral and Biochemical Effects
Several studies have been conducted by the Unit on Anxiety and
Affective Disorders to investigate the behavioral and biochemical effects of
caffeine in panic disorder patients and normal controls. The following section
reflects the scientific rationale and chronological sequence of our research with
caffeine.
a. Caffeine: Retrospective Survey. As previously reported
(ZOl MH 00071-04/05/05) , a caffeine-consumption survey was designed and adminis-
tered to patients with panic and major depressive disorders and compared to normal
controls matched for age, sex, and socioeconomic status. Data from this survey,
published in Psychopharmacology Bulletin and the Archives of General Psychiatry,
indicated an increased sensitivity to the psychostimulant and anxiogenic effects
of caffeine in panic disorder patients compared to their normal controls. This
relationship was not found in patients with major affective disorders. The
findings of this survey, suggesting an increased vulnerability to the anxiogenic
effects of caffeine in patients with panic disorder, led us to directly test the
single-dose behavioral and biochemical effects of caffeine in panic disorder
patients and normal controls. To pursue this goal, our Unit first investigated
the effects of three separate doses of caffeine in normal controls.
b. Caffeine: Effects on Anxiety, Blood Pressure, Lactate, and
Cortisol in Normal Controls. Using double-blind, placebo-controlled conditions,
three doses of oral caffeine (240, 480, and 720 mg) were administered to 14 normal
controls. Caffeine produced dose-related increases in state anxiety, mean
arterial pressure, plasma lactate, and plasma Cortisol. Plasma NE and its
principal metabolite, MHPG, failed to increase. Two of 14 normal controls devel-
oped unequivocal panic attacks following the 720 mg dose of caffeine. This
research demonstrated that caffeine in sufficient doses may induce anxiety,
including panic attacks, in normal subjects. The lack of caffeine's effects on
MHPG further suggested that noradrenergic systems might not be responsible for the
major psychostimulant effects of caffeine in euthymic humans.
c. Caffeine: Effects on Plasma Adenosine Levels. Using the
beforementioned design (Section C, 10b) , plasma adenosine was measured in normal
volunteers. The measurement of plasma adenosine after oral caffeine in humans was
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investigated since caffeine-induced behavioral changes in animals are thought to
be mediated by blockade of adenosine receptors. In a subgroup of eight normal
volunteers presented in Section C, 10b of this report, three oral doses of
caffeine (240, 480, and 720 mg) and placebo were administered on four separate
occasions. Adenosine levels were determined as described in Section B, 2g.
Despite dose-related increases in anxiety and plasma caffeine levels (up to
73.3 uM) , no significant change in plasma adenosine concentrations was observed
after caffeine administration. Although plasma adenosine levels did not change,
these data support a role for adenosine receptor systems in caffeine-induced
anxiety states since the caffeine levels reached after administration of 720 mg,
the only dose which in this small sample produced significant anxiogenesis, are in
a range (44-73 uM) known to compete with the binding of various ligands to the
adenosine receptors in human brain (Ki-35-115 uM) .
d. Increased Sensitivity to Caffeine in Panic Disorder Patients.
To directly test our hypothesis that panic patients have an increased
vulnerability to the anxiogenic effects of caffeine, a caffeine dose (480 mg)
which failed to elicit panic attacks or severe degrees of generalized anxiety in
the normal controls, was administered under double-blind, placebo-controlled
conditions, to 24 panic disorder patients and compared to the 14 normal controls
reported in lib. The results of this study support our hypothesis of increased
sensitivity to caffeine in panic patients, as indicated by a significantly greater
increase in measures of anxiety on the Zung Anxiety Scale in the patients compared
to normal controls. Moreover, pine of 24 panic patients, but none of the 14
normal controls experienced panic attacks by DSM III criteria. Compared to normal
controls, the panic patients also had significantly higher levels of Cortisol,
lactate, and glucose following caffeine, although only increased levels of lactate
distinguished between panicking and nonpanicking patients. It should be
underscored that the normal controls did have significant increases in both
measures of anxiety and plasma Cortisol, compared to their placebo control
condition. Thus, while panic patients appear more sensitive to the anxiogenic
effects of caffeine, normal subjects are not insentient to the psychostimulant
properties of caffeine.
e. Alprazolam Blocks Anxiogenic Effects of Caffeine. We have con-
ducted preliminary studies investigating the effects of alprazolam, a
triazolabenzodiazepine with antipanic properties in humans, on caffeine-induced
anxiety. Blinded caffeine 480 mg was administered to patients participating in a
double-blind, alprazolam-placebo crossover study. While six of 16 (37.5%) on the
placebo phase of the study had panic attacks following single dose caffeine (480
mg) , none of 11 (0%) of the alprazolam- treated patients had panic attacks
following this same acute oral dose of caffeine (p = .027, Fisher's exact test).
Of interest, alprazolam blocked the usual caffeine-induced increment in lactate
but had no effect on plasma Cortisol levels. These behavioral and biochemical
effects suggest that the benzodiazepine receptor system may play an important role
in blocking some of caffeine's psychostimulant and biochemical effects. The role
of the GABA-benzodiazepine receptor system in mediating caffeine's principal
panicogenic effects remains to be elucidated.
f. Caffeine-induced Escape from Dexamethasone Suppression. The
dexamethasone suppression test (DST) has been suggested as a sensitive and
specific tool for the diagnosis of major depressive disorder, melancholic subtype.
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Because psychiatric patients have been reported to consume excessive amounts of
caffeine, because caffeine produces dose-related increases in plasma Cortisol
(refer to Sections C, 10b and 10c of this report) , and because the effects of
caffeine (probably the most widely consumed psychotropic agent the world) on the
DST had not been previously reported in the literature, the single-dose effects of
caffeine 480 mg on the standard dexamethasone suppression test was investigated in
23 normal volunteers, 13 depressed and two panic disorder patients. Using a
single-blind design, an oral dose of caffeine 480 mg or placebo was administered
randomly on two separate days at 2:00-2:30 pm the day following the 11:00 pm
administration of dexamethasone 1 mg. Test days were separated by at least 48
hours. Blood samples were obtained at 4:00 pm.
Caffeine significantly increased the post-dexamethasone Cortisol values.
Whereas the 4:00 pm Cortisol values after placebo averaged 2.3 ± 2.3 (mean + SD) ,
the comparable mean value after caffeine was 5.3 ± 5.8 (paired t = 3.7, p < .001).
A plasma Cortisol level of > 5 pg/dl has been used most commonly to signify non-
suppression. Of the 38 subjects, five (13%) were found to be nonsuppressors on
placebo and 12 (31%) were nonsuppressors on caffeine. Caffeine-induced
nonsuppression was observed in both depressed patients and normal volunteers.
This study is the first investigation to our knowledge demonstrating that escape
from dexamethasone suppression can be induced by caffeine. Of interest, the 480
mg single dose of caffeine given to subjects in this study is roughly comparable
to four to five cups of coffee and within the range typically consumed on a daily
basis by 20%-40% of the population. Since several lines of evidence suggest that
psychiatric patients, particularly depressed and schizophrenic patients, may
consume excessive amounts of caffeine, our findings may explain in part the wide
variability and discrepant findings in the literature on the DST in psychiatric
patients.
12. GH-response to Clonidine
Studies using clonidine to assess noradrenergic function continue
to be investigated by the Unit on Anxiety and Affective Disorders. Several lines
of evidence, reviewed in previous reports (ZOl MH 00071-04 BP and
ZOl MH 00071-05 BP) , suggest that the GH-response to clonidine may provide an
index of postsynaptic alpha -adrenergic function. Since noradrenergic
dysfunction, particularly noradrenergic overactivity, represents one of the major
current theories of anxiety, the GH-response to clonidine was studied in
nondepressed panic disorder patients compared to depressed patients and normal
controls triple-matched for age, sex, and menstrual cycle status.
The mean peak growth hormone response to clonidine was significantly
decreased in the panic disorder patients (n = 11, x = 1.8 ± 1.2 SD) and depressed
patients (n = 11, 3.8 ± 4.3) compared to normal controls (n = 11, 12.7 + 11.7)
(F = 7.91, p < 0.002). This finding remained significant when men (n = 4, F =
20.48, p < 0.0004), women (n = 7, F = 4.51, p < 0.03), and the subgroup of
patients (four men and three women) without elevated baselines (F = 6.31, p <
0.008) were analyzed as separate groups. There was no significant difference in
baseline GH levels among the three groups.
Our findings, published in Biol. Psychiatry, suggest that panic disorder and
depressed patients demonstrated a similar blunted growth hormone response to
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clonidine compared to age- and sex-matched normal controls. These findings are
consistent with an emerging body of data suggesting a partial, but incomplete,
overlap in the phenomenology, epidemiology, and neurobiology of panic and major
depressive disorders.
13. Cortisol-response to Clonidine
Abnormalities in regulation of noradrenergic function have been
proposed as part of the pathology of depressive and panic anxiety disorders.
However, abnormalities in HPA axis function have largely been limited to patients
with depressive disorders. Using the Cortisol response to clonidine, an
alpha -adrenergic receptor agonist, this study examined the relationship between
the noradrenergic system and the HPA axis in ten patients with major depression
(4 unipolar, 6 bipolar) , ten patients with panic disorder, and ten normal
controls.
There was a trend for the three diagnostic groups to differ in baseline
Cortisol values prior to infusion (panic: 6.1 ± 4.7 jjg/dl; depressed: 12.6 ± 6.2
pg/dl; controls: 9.6 ± 6.9 yg/dl; F = 2.98, p < .07), The significant difference
was between the depressed and panic groups (p < .02) .
The mean fall in plasma Cortisol after clonidine was 1.7 ± 2.4 pg/dl, 5.2 ±
4.9 yg/dl, and 2.8 ± 2.8 yg/dl in the panic disorder (p < .06), depressed (p <
.01) , and normal controls (p < .02) , respectively. There was a trend for the
three groups to differ in the fall in plasma Cortisol in response to clonidine (F
= 2.56, p < .10) , with the trend seen between the depressed and panic groups
(p < .06) . However, when the fall in plasma Cortisol was expressed as a
percentage change from baseline, there was no significant difference among the
three groups (panic: 26.2 + 38.6%; depressed: 38.8 ± 17.7%; controls: 31.3 ±
18.3%; F = .563, p > .10) .
The pre-clonidine plasma Cortisol level was significantly negatively
correlated with the absolute clonidine-induced change in Cortisol level in the
depressed patients (r = -0.87, df = 8, p = < .005), in the controls (r = -.75,
df = 8, p = < .02) , and in the panic patients (r = -0.77, df = 8, p = < .001).
The composite correlation for the group of 30 patients was: r = -.81, df = 28, p <
.0001. However, the pre-clonidine plasma Cortisol level did not correlate
significantly (p > .10) with the percentage of change in plasma Cortisol level in
any of the three groups.
This is the first study to compare the Cortisol response to clonidine across
subjects with panic disorder, major depression, and noiniial controls. While the
enhanced Cortisol drop following clonidine might suggest a difference in
noradrenergic modulation of the HPA axis in depression compared to panic disorder,
we believe that the inhibitory effects of clonidine on Cortisol secretion may be a
less than satisfactory probe of this relationship.
The apparent greater drop in Cortisol may simply be a function of higher
baseline Cortisol levels in the depressed group. We found that the drop in
Cortisol was highly correlated with the baseline Cortisol level; when a
percentage, rather than absolute, drop was measured, this correlation was not
seen. Accordingly, the percentage drop in Cortisol, a measure independent of
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baseline Cortisol level, did not differ across diagnostic groups. Because of the
dependence on baseline Cortisol levels, the absolute drop in Cortisol in response
to clonidine may provide little information about alpha -adrenoreceptor
sensitivity. These findings, and other data to be published in Biological
Psychiatry, have led us to conclude that "stimulatory" rather than "inhibitory"
behavioral and neuroendocrine responses may be more reliable paradigms for the
study of neurochemical-neurotransmitter function in panic disorder.
14. Panic Disorder: Platelet Imipramine Binding
The nature of the relationship between panic and major affective
disorders is currently a subject of scientific controversy. The favorable res-
ponse of patients with panic disorder to tricyclic and monoamine oxidase inhibitor
antidepressants, the high prevalence of major depressive episodes in panic
disorder patients, and the greater frequency of depression in relatives of de-
pressed patients with concomitant panic disorder compared to relatives of de-
pressed patients without panic disorder, suggest an overlap between panic disorder
and major depression. Similar neuroendocrine responses to clonidine also suggest
a neurobiological relationship between the two disorders. Several lines of
evidence, however, suggest important differences between the two disorders. Since
most studies have reported that the number of binding sites on blood platelets are
reduced in depressed patients compared to normal controls, we investigated
platelet imipramine binding in panic disorder patients.
[3H] Imipramine binding to platelets was measured in 17 drug-free panic disor-
der patients and 14 healthy controls. No difference in Bmax or Kd values was
found between the two groups. Patients with a past history of major melancholic
depression or severe agoraphobia had binding parameters similar to those of panic
disorder patients without a history of depression or severe agoraphobia. Thus,
our findings, published in Biological Psychiatry, suggest that nondepressed panic
disorder patients, with or without a past history of endogenous depression or
agoraphobia, may not display a lower density of platelet [ ^H] imipramine binding
sites compared to normal controls, as has been reported in many depressed
patients.
15. Panic Disorder and Neuroendocrine Function
The Unit on Anxiety and Affective Disorders has investigated
several neuroendocrine tests in panic disorder patients. With the exception of
new data presented in 15a, the results of these ongoing investigations were
presented in more detail in last year's report (ZOl MH 00071-06 BP) and are only
briefly summarized here.
a. T , T , and TSH Levels. Anxiety is a common manifestation of
hyperthyroidism and thyroid disorders may mimic the clinical presentation of panic
disorder. What is less clear from the literature is whether or not panic
disorder, in the absence of clinically overt thyroid disease, is routinely
associated with physiologic disturbances in thyroid function. Using a closely
age- and sex-matched normal control group, we were able to explore the hypothesis
that patients with panic disorder might exhibit subtle abnormalities in routine
indices of thyroid function. The samples studied consisted of 26 panic disorder
patients, and 26 closely age- and sex-matched normal volunteers. There were no
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significant differences in measurements of T , T , FT , TSH, or TBG between
patients with panic disorder and normal controls. When these values were compared
separately by sex, no differences emerged. Furthermore, our findings do not rule
out the possibility that hypothalamic-pituitary modulation of peripheral thyroid
function may be altered in panic disorder.
This study, to be published in the American Journal of Psychiatry, suggests
that abnormalities in baseline and peripheral indices of thyroid function are not
a requisite biological correlate of panic disorder. That abnormal T , T , FT ,
and TSH levels were not found in our panic disorder patients does not preclude the
possibility that patients with primary thyroid dysfunction might be predisposed
toward the secondary development of anxiety syndromes, even after correction of an
underlying thyroid disorder.
Our Unit had previously speculated that the blunted TSH response to TRH might
reflect a condition of subclinical hyperthyroidism in panic disorder. However,
given our new findings presented here, it seems less likely that the blunted TSH
response is a consequence of subclinical hyperthyroidism, since we found no
tendency for panic disorder patients to exhibit evidence of excess peripheral
thyroid hormone levels.
b. Thyroid Releasing Hormone (TSH) Test. A TRH test was adminis-
tered to 12 patients with panic disorder and ten normal volunteers. A Bmax TSH of
less than 7 U/ml was used as the criterion for a blunted TSH response. Four of 12
panic disorder patients and none of the ten controls demonstrated a reduced TSH
response to TRH (p = .06) . In addition, the panic patients had a significantly
lower Bmax TSH value compared to the normal controls. These findings, published
in the American Journal of Psychiatry, suggest that the blunted TSH response to
TRH may be common to both panic disorder and major depressive disorders and
represent inappropriate hyporesponsiveness of the thyrotrope.
c. Corticotropin Releasing (CRH) Test. A CRH test was performed
on eight panic disorder patients and compared with 27 normal controls previously
studied by the Biological Psychiatry Branch. Compared with normal controls, panic
disorder patients had decreased ACTH responses (p < .01) and reduced Cortisol re-
sponses (p < .05) to CRH. These findings, published in the Am. J. Psychiatry,
suggest that panic disorder patients may have an element of chronic
hypercortisolemia and an abnormality in CRH secretion similar to that proposed for
depressed patients. In addition, of the two baseline ACTH values obtained prior
to the CRH challenge, the mean initial ACTH value was significantly higher than
the second value. In control subjects, the two basal ACTH values were not
significantly different. This initial elevated basal ACTH level in the panic
disorder patients suggests a more acute perturbation in CRH secretion, one not
seen thus far in any other group of hypercortisolemic psychiatric patients tested.
The possibility that patients with panic disorder might more readily release CRH
in response to environmental perturbation is intriguing in light of the animal
data documenting that intracerebroventricular administration of CRH produces a
variety of behavioral and physiological changes classically associated with the
stress response. Finally, the ability of CRH to increase both locus coeruleus
activity and plasma norepinephrine levels is provocative in light of theories
implicating increased central noradrenergic activity in the etiopathology of panic
disorder.
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d. Dexamethasone Suppression Test. A standard dexamethasone sup-
pression test was administered to 16 panic patients and 22 normal controls. Using
a standard Cortisol value of greater than 5 yg/dl to indicate nonsuppression,
there was no significant difference between the proportion of panic patients (25%)
and normals (14%) with an abnormal test. Moreover, an internal standard for the
dexamethasone suppression test was determined using our normal control data.
Using a 95% confidence interval (mean + 2 SD) as the criterion for abnormal
response (7.1 yg/dl), only 6% and 4% of the panic patients and normal controls,
respectively, demonstrated Cortisol escape from dexamethasone suppression. Our
results, published in Biological Psychiatry, indicate that panic patients do not
respond abnormally to dexamethasone testing when a control group is used to
determine the range of normality for a given assay and testing condition.
16. Alprazolam Withdrawal
Alprazolam is an effective and widely-used benzodiazepine in the
treatment of panic disorder. A potential disadvantage of alprazolam, however, is
the development of withdrawal symptoms following the abrupt discontinuation of
relatively high doses. Therefore, the Unit investigated both the clinical and
biological correlates of gradual alprazolam withdrawal and the utility of
carbamazepine in the treatment of alprazolam withdrawal.
a. Behavioral and Biological Correlates. In the first study, ten
patients (five men and five women, mean age 31.0 ± 8.7) maintained on an average
of 4.95 mg ± 3.22 S.D. alprazolam (range 1.0 to 12.0 mg) for 4 to 22 months (x =
11.7 ± 5.8) were studied during alprazolam withdrawal on the 3-West inpatient
unit. For the purposes of this study, the behavioral and biological indices of
the "withdrawal" period were compared to a stable "post-withdrawal" period.
Spielberger anxiety ratings were found to be significantly elevated during
the "withdrawal" compared to the "post-withdrawal" period. The Cortisol values
were consistently more elevated during alprazolam withdrawal than when
medication-free. During "withdrawal" as compared to "post-withdrawal", there was
also a trend for increased pulse rates and systolic blood pressure. Significant
differences in diastolic blood pressure, temperature, and hours of sleep were not
demonstrated. There was a significant correlation between changes in (withdrawal
minus post-withdrawal) Cortisol and pulse values.
These findings, published in the American Journal of Psychiatry, suggest that
increased measures of anxiety and plasma Cortisol are commonly associated with
gradual tapering of relatively low doses of alprazolam.
b. Treatment. In an attempt to explore withdrawal modifying
strategies, a preliminary study investigating the utility of carbamazepine in the
treatment of alprazolam withdrawal has been initiated by the Unit on Anxiety and
Affective Disorders. Three patients who had extreme difficulties during blind
withdrawal from alprazolam demonstrated during a second withdrawal phase that they
were able to tolerate a comfortable and more rapid alprazolam withdrawal when
treated with carbamazepine than without this agent. These preliminary findings,
published in the American Journal of Psychiatry, suggest that carbamazepine might
provide a potentially useful therapeutic tool in the treatment of benzodiazepine
withdrawal .
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17. Panic Disorder: Treatment
a. Verapamil. Calcium channel blocking agents are widely used in
the treatment of cardiovascular disorders. Recent evidence also suggests that
these drugs might have positive therapeutic effects in patients with major
affective disorders. In a double-blind, placebo-controlled study, we investigated
the effects of verapamil, a calcium channel blocker, in the treatment of panic
disorder. The study was designed as a double-blind, crossover study and each
patient received both verapamil and placebo.
Each patient first received four weeks of placebo treatment. After the first
placebo period, verapamil was initiated at a dose of 160 mg/day and increased by
160 mg at weekly intervals to a maximum dose of 480 mg/day. This dose was
maintained for five weeks. The dose was then decreased to 240 mg/day for one week
prior to discontinuation and placebo substitution for an additional four weeks.
Thus, the study was an off-on-off design lasting a total of 16 weeks. Analysis of
variance with repeated measures was employed comparing the pretreatment placebo
period versus treatment period versus post-treatment placebo period with the
corrected Huynhfeldt probability; posthoc analyses were performed employing the
Tukey test.
Eleven patients completed the study. A one-way repeated measure analysis of
variance (ANOVA) (first placebo versus active drug versus second placebo periods)
revealed a significant drug effect on anxiety as measured by the Zung anxiety
scale (mean ratings: 58 ± 10.5, 51.9 ± 11.1, 51.5 ± 10,6, respectively, F = 4.19,
df 1.6/16.1, p < 0.04) . Posthoc analysis revealed a significant difference
between Zung ratings during the placebo pretreatment period and the ratings in the
post-treatment period (p < 0.05) with a similar trend between the "pretreatment"
and the "on-treatment" values (p < 0.06). However, there were no significant
changes on the Spielberger state anxiety, the NIMH agoraphobia, and the Beck
depression scales. Nine of 11 patients (82%) had a decrease in the number of
panic attacks during the last four weeks of verapamil treatment compared to the
four weeks on placebo preceding verapamil (p < 0.02, Signed Rank test).
These findings, to be published in the Am. J. Psychiatry, suggest that
verapamil has modest anxiolytic and antipanic effects. Additional studies are
required to substantiate this finding and compare the efficacy of verapamil to
that of drugs such as imipramine, phenelzine, and alprazolam which have a
well-established role in the treatment of panic disorder.
b. Carbamazepine. Our findings of a high frequency of
psychosensory symptoms (see C2) and electroencephalographic abnormalities in
patients with panic disorder led our unit to investigate the efficacy of
carbamazepine in panic disorder patients. While ten of 14 completers demonstrated
some improvement on carbamazepine, the overall clinical response was judged to be
minimal as reflected by only a small decrement (-4.5, p < .02) on the Zung Anxiety
Scale and a nonsignificant change (-1.6, p = NS) on the Spielberger State Anxiety
Scale. Forty percent of the patients had a decrease in frequency of panic attacks
on carbamazepine, while 50% had an increase and 10% showed no change. Neither the
presence of EEG abnormalities nor prominent psychosensory symptoms predicted
response to carbamazepine .
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This is the first study, to our knowledge, to systematically examine the
efficacy of carbamazepine in the treatment of panic disorder using a controlled,
double-blind design. Our findings suggest that carbamazepine is of limited value
in the treatment of most patients suffering from panic disorder with or without
agoraphobia. On most outcome measures, carbamazepine failed to show any benefit
over the preceding time period on placebo. While a small decrement was noted on
the Zung Anxiety Scale, this was of minimal clinical significance.
d. Clonidine. In previous reports (ZOl MH 00071-06 BP) , we
presented the rationale for investigating the potential antianxiety effects
following both an acute intravenous challenge (2 yg/kg) and chronic treatment with
clonidine. The following summarizes our experience to date with this paradigm.
1. Acute Effects. Fourteen patients agreed to participate
in the acute clonidine study. Of these 14, two did not complete both a placebo
and a clonidine challenge. Only the 12 patients who completed both parts of the
challenge (i.e., clonidine and placebo) are reported in the analysis. Seven women
and five men made up this patient group. The mean age (± SD) was 36.2 ± 7.0 years
(range 26-49 years) .
Ten healthy subjects completed both the placebo and clonidine challenge.
Nine women and one man made up this control group. The mean age (± SD) was 27.9 ±
13.6 years (range 19-56 years) . The mean ages of the patient and control groups
did not differ significantly (Student's t-test, p = NS) . All patients were
medication-free for at least three weeks prior to the start of the study.
Placebo failed to produce significant changes in state anxiety in the
patients or normal controls (p = NS) . While intravenous clonidine also failed to
produce changes in state an:?iety in the healthy subjects (p = NS) , clonidine did
produce significant decreases in Spielberger anxiety from a mean baseline value of
60.2 ± 11.5 to 47.1 ± 12.9 sixty minutes after clonidine (paired t = 8.12, df =
11, p < .0001) in the patients.
The placebo-corrected changes in anxiety with clonidine (i.e., change in
anxiety with clonidine minus change in anxiety with placebo) were significantly
greater in the patients than in the healthy subjects (-8.5 + 8.5 versus -0.7 ±
6.0, t = -2.35, df = 19, p < .05).
Healthy subjects showed a significant mean drop in systolic blood pressure
with clonidine (-12.9 + 7.3 mm Hg) as compared to placebo (0.7 ± 3.5 mm Hg, paired
t = -6.16, df = 9, p < .0005). Patients also showed a significant mean drop in
supine systolic blood pressure with clonidine (-14.6 + 10.3 mm Hg) as compared to
placebo (2.4 + 9.7 mm Hg) (paired t = -5.03, df = 6, p < .005). Healthy subjects
and patients did not, however, differ significantly in their supine systolic blood
pressure response to clonidine (t = -0.42, df = 16, p = NS) . The variance of the
systolic blood pressure response to clonidine was not significantly different
between healthy subjects and patients (Levene's F-test = 1.9, p = NS) .
Neither group showed a significant change in diastolic blood pressure with
placebo (p = NS) . Healthy subjects showed a significant drop in supine diastolic
blood pressure with clonidine (-8.9 ± 4.8 mm Hg, paired t = -5.93, df = 9, p <
.0005), as did the patients (-10.4 ± 5.2 mm Hg, paired t = -5.60, df = 7, p
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.001) , but the two groups did not differ significantly in this response (t =
-0,63, df = 16, p = NS) . The variance of the diastolic blood pressure response to
clonidine was not significantly different between healthy subjects and patients
(Levene's F-test = 0.2, p = NS) .
Neither group showed a significant change in heart rate with placebo (p =
NS) . Healthy subjects showed a significant drop in supine heart rate with
clonidine (-6.7 ± 6.1 BPM, paired t = -3,48, df = 9, p < .01), with a similar
trend seen for the patients (-4.0 ± 5.8 BPM, paired t = -1.95, df = 7, p < .10).
Healthy subjects and patients did not differ significantly in their supine heart
rate response to clonidine (t = 0.95, df = 16, p = NS) . The variance of the heart
rate response to clonidine was not significantly different between healthy
subjects and patients (Levene's F-test = 0.1, p = NS) ,
There were no significant correlations between changes in anxiety and changes
in systolic blood pressure or diastolic blood pressure. Decreases in anxiety with
clonidine were not significantly correlated with increases in sleepiness in the
healthy subjects (r = ,04, df = 6, p = NS) or in the panic disorder patients (r =
.30, df = 10, p = NS) . Changes in heart rate with clonidine also were not
significantly correlated with changes in anxiety in healthy subjects (r = .43,
df = 8, p = NS) nor in patients (r = .40, df = 6, p = NS) ,
2. Chronic Effects. Eighteen patients agreed to participate
in the chronic study. Five men and 13 women, with a mean age of (± SD) 34 ± 8.6
years (range 23-58 years) , participated in this trial.
Clonidine failed to exhibit anxiolytic effects on any of the rating scales.
All comparisons were made between pretreatment scores (while on placebo) and
scores during the last week of treatment at the highest daily dosage administered.
No statistically significant changes were noted on the Zung Anxiety Scale (-1.5 ±
10.3, p = NS) , Global Rating of Anxiety (-1.0 ± 2.0, p = NS) , or the HSCL-90
global severity index (-0.2 ± 0.5, p = NS) , anxiety subscale (-0.3 ± 0.9, p = NS) ,
phobic subscale (-0.3 ± 0.7, p = NS) , or panic subscale (-0.3 ± 0.8, p = NS) . A
trend toward a decrement in anxiety was noted on the Spielberger State Anxiety
(-4.4 + 10.3, p < .10), but the magnitude of this change was of minimal clinical
significance.
The lack of apparent efficacy for the group as a whole notwithstanding, it is
noteworthy that two patients demonstrated remarkable responses to treatment,
showing a drop in Spielberger State Anxiety score of 22.7 and 21.3 points,
respectively. The first patient, a 25-year-old woman, has not responded to any
other treatment agent to date, including blind trials of imipramine, phenelzine,
and carbamazepine , Moreover, the primary clinicians treating these patients rated
ten of 14 patients as demonstrating some clinical benefit from clonidine
pharmacotherapy. Of interest, the area of symptomatic improvement was
inconsistent across patients. Thus, some patients demonstrated a marked reduction
in panic attacks, but not generalized anxiety or agoraphobia. Other patients had
a clinically significant improvement in generalized anxiety but experienced no
blockade of panic attacks. As a result, a greater number of individual patients
received clinical benefits from chronic clonidine pharmacotherapy than is readily
evident from statistical analysis of separate rating scales.
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D. Major Findings (Animal Research)
During the past three years, the Unit on Anxiety and Affective Disorders has
established a viable colony of "normal" and "nervous" pure-bred pointer dogs.
These dogs offer the advantage of investigating both "normal" behavior and "spon-
taneously-occurring" (rather than laboratory-conditioned) fear behaviors. The
'"nervous" line may be particularly useful in the study of several behaviors and
characteristics relevant to human psychopathology , including genetically-transmit-
ted inheritance with phenotypic expression of "nervous" behaviors at eight to 12
months of age. This delayed manifestation of pathology in dogs parallels in a
similar, temporal fashion, the emergence of agoraphobia in humans during
adolescence and early adulthood.
While the colony was being established the development of observation
chambers with one-way mirrors and rating scales were developed. Careful and
precise techniques for the surgical removal of the whole brain under general
anesthesia were also developed and can be reliably performed under suitable
conditions. A brain mold for the purebred pointer dog has been developed which
allows the Unit to prepare regional brain tissue immediately after surgical
removal for later measurement of transmitter levels and ligand binding to various
receptors such as alpha -adrenergic, benzodiazepine, imipramine, opiate, and other
binding studies.
Research with this animal model has been conducted in collaboration with
Drs. E. Klein, S. Steinberg, and S. Weiss. The following sections (D1-D4) report
on preliminary findings with this model.
1. Heritability of Fear Behaviors
During the past year, the Unit has systematically evaluated the
validity of the inheritance of nervous behaviors in the A- and E-lines of the
Arkansas pure-bred pointer dogs. After breeding dogs from each line at our own
facilities, the Unit blindly assessed the behaviors at nine months of age or
older. We evaluated "nervous" behaviors using previously validated scales of fear
or fear-related behaviors: weighted activity (WA) , weighted nervous score (WNS) ,
and new morbidity score (NMS) .
The Unit developed several additional scores of fear based on 21 behaviors
(i.e., tremor, circling, salivate, etc.) observed under four conditions (dog
alone, dog exposed to human sitting on chair, human calling dog, human approaching
dog) .
On all measures, the offspring of E ("nervous") -line parents had
significantly higher nervous scores compared to the offspring of A ("normal") -
line parents. There were no sex differences in relation to fear or fear- related
behaviors. These data confirm and extend previous observations demonstrating the
heritability of fear behaviors in "nervous" pure-bred pointers.
i
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2. Hearing and Non-hearing Pointer Dogs
During the past year, our unit was impressed by a behavioral
pattern which was not mentioned in earlier work; namely, that nervous dogs seemed
to be less responsive to the presence of a human if the human was not within their
field of vision, suggesting a possibility of a hearing deficit. Such a deficit
could potentially contribute to or largely determine the aforementioned abnormal
behavior in the nervous dogs. Since a hearing deficit has not been previously
described in these dogs, we decided to evaluate the hearing status of both nervous
and normal dogs in our colony and further assess the relationship between a
possible hearing deficit and the abnormal behavior.
The standard brainstem auditory-evoked response (BAER) , which has been
recently applied to dogs, was used for the assessment of hearing status. Of 16
normal dogs tested, all but one showed normal responses in both ears. One dog (a
4-year-old male) showed a normal response in one ear but no response in the other
ear. Direct otoscopic examination gave no clue to the cause of this unilateral
deficit. In contrast, the testing in the "nervous" dogs revealed that 20 of 27
dogs had no brainstem evoked response that could be detected in either ear. These
dogs were thus considered to be deaf, while the remaining seven dogs had normal
responses. However, behavioral ratings revealed that hearing and deaf dogs did
not differ in their pathological response to the characteristic fear-provoking
stimuli (e.g., human interaction), whereas both hearing and deaf nervous dogs
markedly differed from normal dogs on this measure. Thus, regardless of the
hearing status, there was a robust difference between nervous and control dogs.
That is, these results support a conclusion that hearing status does not effect
the behavioral outcome in these dogs and that these traits are not causatively
related, although genetic linkage between the behavioral abnormality and the
deafness might be expected.
3. Response to Diazepam and RO 15-1788
The Unit has investigated the effects of diazepam, RO 15-1788, and
placebo in ten "nervous" and seven "normal" pointer dogs. Although RO 15-1788
reversed diazepam-induced hind leg ataxia in both lines, there were no significant
drug group effects by ANOVA on the previously validated WA, WNS, NMS scales or the
ten NIMH subscales of fear behaviors. These data suggest that most abnormal
behaviors in this animal model of "anxiety" are unlikely to be mediated by an
endogenously-produced, central type, benzodiazepine anxiogenic ligand. Also, the
limited effects of diazepam somewhat parallel the relatively poor effects of this
agent in the treatment of panic and agoraphobic syndromes in humans.
4. Yohimbine Binding
We studied alpha -adrenergic receptor binding as determined by
[3H] -yohimbine in platelets and brains of the nervous and normal dogs in our
colony. Our findings indicate that alpha -adrenergic receptor density and
affinity are similar in platelets and frontal cortex, but we did not observe
significant differences in binding between the two groups of dogs.
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5. Adenosine Binding
Since benzodiazepines and adenosine derivatives have marked effects
on behavioral arousal, these systems were studied in the brains of both nervous
and normal dogs. Adenosine receptors were found to be increased in the
hippocampus, and adenosine reuptake sites were found to be increased in the
cerebellum of the nervous dogs. No changes were observed in benzodiazepine
binding. These findings suggest that adenosine neuromodulatory function might be
impaired in the nervous pointer dogs. The report of increased sensitivity to
caffeine in panic disorder patients compared to normal controls (see lib and lid) ,
and the findings here of increased adenosine binding in the hippocampus in the
nervous pointer dogs, provide indirect evidence for altered adenosinergic
modulation in stress and anxiety syndromes.
6. Motor Activity
Nervous pointer dogs are characterized by a typical and highly
reproducible pattern of fear-related behaviors which become manifest at the age of
3-9 months. These fear- related behaviors are most pronounced when these dogs are
exposed to humans or novel environments. Upon such exposure these dogs frequently
respond with reduced exploratory activity, hyperstartle, marked avoidance of the
human observer, frequent catatonic freezing, cardiovascular changes, urination and
defecation. In contrast, normal dogs do not demonstrate these abnormal behaviors
under similar conditions. The marked motoric components of the fear response in
the nervous dogs (reduced exploratory activity and catatonic freezing) prompted us
to investigate spontaneous motor activity in these dogs to determine whether
differences in motor activity between the two lines can be demonstrated under more
"naturalistic", nonstressful, conditions over a 24-hour period of time. Our unit
studied spontaneous motor activity, using nontelemetric activity monitors, in both
nervous and normal pointer dogs.
Data from eight normal dogs (two males and six females, mean age 10.5 ± 1
month) and ten nervous dogs (five males and five females, mean age 11.2 ± 2.2
months) were obtained. There was no significant difference in motor activity
between the nervous and normal pointer dogs. Of interest, however, was the
observation that the rest-activity cycle was not evident in either dog line.
II . Proposed Course
Efforts will be expanded to document the phenomenology, natural course,
family dynamics, and personality structure of patients with panic disorder. Dr.
T. Mellman will initiate a study exploring the impact of personality structure on
the phenomenology and treatment of panic disorder. In collaboration with B.
Scupi, a Panic Disorder Questionnaire, assessing current and past life
experiences, has been developed. This Panic Disorder Questionnaire plus a number
of standardized scales including the Parental Bonding Instrument, Retrospective
Childhood and Current Fear Scale, Locke-Wallace Marital Satisfaction Inventory,
Family Assessment Device, and the Childhood and Adult History Questionnaire will
be administered. This study will assess whether retrospective perspectives of
early childhood experience and family structure are associated with specific types
of symptomatology or predict treatment outcome measures. In collaboration with
Dr. J. Maser and B. Scupi, an inventory to assess phenomenology and natural course
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of panic and the prevalence of co-morbidity of panic disorder with other
psychiatric illnesses has been designed and will be administered to a large sample
(> 2000) of subjects worldwide.
Human research conduced by the Unit on Anxiety and Affective Disorders has
demonstrated an alteration in neuroendocrine and noradrenergic function in panic
disorder patients compared to age- and sex-matched controls. These abnormalities
include blunted GH, TSH, and ACTH to clonidine, TRH, and CRH, respectively. Dis-
turbances in noradrenergic function are also suggested by increased DHE binding to
platelets and increased sensitivity to the alpha -adrenergic antagonist,
yohimbine. Other lines of evidence reviewed in previous reports
(ZOl MH 00071-04/05/06 BP) also suggest a similarity in neuroendocrine and
noradrenergic dysfunction in panic and major depressive disorders. As a result of
these findings, the Unit will continue to investigate neuroendocrine and
noradrenergic systems across a spectrum of mood and anxiety disorders.
We also intend to expand our research with caffeine. Further delineation of
the clinical response to caffeine is indicated because caffeine consumption is
correlated with symptoms of generalized anxiety in patients with panic attacks,
but not in normal volunteers. Caffeine derivatives also activate noradrenergic
activity in animals when iontophoretically applied to the locus coeruleus. Fur-
thermore, caffeine has been shown to antagonize the biochemical and phar-
macological effects of benzodiazepines, and alprazolam, a triazolabenzodiazepine,
blocks the typical time course of caffeine-induced arousal, panic attacks, and
generalized anxiety in humans (see Section C9) . Other lines of evidence suggest a
major role for adenosine-regulated systems in the mediation of caffeine's
psychostimulant properties. All three of these systems have been independently
implicated in the neurobiology of anxiety and stress. Moreover, caffeine was
found by our Unit to significantly elevate measures of the stress-related hormone,
Cortisol, and induce Cortisol escape from dexamethasone suppression. Thus, we
intend to extend and expand our ongoing research with caffeine by investigating
the behavioral, physiological, neuroendocrine, and biochemical effects of this and
other methylxanthines in both animals and humans.
Drug trials with carbamazepine, clonidine, and verapamil have been concluded
during the past year. While preliminary data suggest that each of these agents
has significant antianxiety effects in a subgroup of panic disorder patients, none
appears to demonstrate the same potency as standard antipanic agents such as
imipramine and alprazolam. A new study investigating the effects of dipyridamole,
compared to placebo and imipramine, will be initiated under the direction of
Dr. M. Stein. The study of dipyridamole, an agent which blocks the reuptake of
adenosine, reflects the Unit's interest in the role of adenosine systems in the
neurobiology of anxiety disorders.
In addition to these drug trials in panic disorder, the Unit will expand its
clinical studies to include patients with social phobias and obsessive-compulsive
disorder. Since no controlled studies of the drug responsiveness of a pure social
phobic sample have been completed, the value of pharmacological interventions with
this disorder is unknown. A number of clinical studies have suggested that
cognitive restructuring and exposure interventions are effective in the treatment
of social phobia. During the next year, the Unit will complete a study to
investigate the relative efficacy of alprazolam, phenelzine, and
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cognitive-behavioral treatment of social phobia. This study will be conducted in
collaboration with C. Shea, a doctoral candidate in clinical psychology at
Catholic University. The inclusion of social phobics and obsessive-compulsive
patients in the anxiety clinic will also provide a patient resource for
investigating the specificity of several biological "markers" of panic disorder
compared to patients with other severe forms of anxiety.
In the pointer dog model of "anxiety", the Unit will focus on the behavioral
pharmacology of adenosine and adenosine derivatives and xanthine compounds. These
experiments in animals will parallel our studies in humans investigating the
neurobiology of fear-related behaviors and the mechanisms of antianxiety drugs.
Drug trials with novel antianxiety agents in humans and in "nervous" pointer
dogs, in conjunction with concomitant measurements of their neurotransmitter ef-
fects, should enhance our understanding of alterations in neuroendocrine and
neurotransmitter pathways associated with pathological human anxiety and animal
fear, and lead to the development of more potent and specific pharmacotherapies.
III. Significance to Biomedical Research and the Program of the Institute
Several epidemiological surveys have suggested that pathological degrees of
anxiety may adversely influence a large segment of our population. Panic
disorder, an anxiety syndrome associated with agoraphobia, results each year in
the impairment of individuals previously well-functioning and productive. Patho-
logical anxiety has been recently found to be one of the most prevalent mental
health problem in this country. The role of anxiety and stress in coronary heart
disease and other medical illnesses has been suggested by a number of studies.
Moreover, emerging epidemiological and familial data suggest that a subgroup of
patients with major depressive illness plus panic attacks may represent an
important and distinct subtype of major affective illness. We intend to
investigate biological correlates in the plasma and cerebrospinal fluid of this
subtype, who may be a greater risk for alcoholism and suicide, compared to
patients with major depressive illness without panic attacks. An improved un-
derstanding of the clinical and biological aspects of both normal and pathological
anxiety is thus critically needed. It is hoped that the developing battery of
clinical and biological tests in patients with anxiety and related mood disorders
will ultimately provide a clinical and biological profile of these illnesses and
lead to more refined subcategorizations, as well as to more selective and
efficacious treatment approaches.
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PUBLICATIONS
Lewy, A.J., Siever, L. J. , Uhde, T.W. and Markey, S.P.: Clonidine reduces plasma
melatonin levels. J. Pharm. Pharmacol. 38: 555-556, 1986.
Roy-Byrne, P.P., Geraci, M. , and Uhde, T.W. : Life events and the course of illness
in panic disorder. Am. J. Psychiatry, 134: 1033-1035, 1986.
Roy-Byrne, P.P., Geraci, M. , and Uhde, T.W.: Life events and the onset of panic
disorder. Am. J. Psychiatry, 143: 1424-1427, 1986.
Roy-Byrne, P.P., Uhde, T.W., and Post, R.M.: Effects of one night's sleep
deprivation on mood and behavior in patients with panic disorder: comparison with
depressed patients and normal controls. Arch. Gen. Psychiaty, 43: 895-899, 1986.
Roy-Byrne, P.P., Uhde, T.W., Post, R.M. , Gallucci, W.T. , Chrousos, G.P. and Gold,
P.W.: The CRH stimulation test in patients with panic disorder. Am. J. Psychia-
try, 143: 896-899, 1986.
Roy-Byrne, P.P., Uhde, T.W., Sack, D.A., Linnoila, M. , and Post, R.M. : Plasma HVA
and anxiety in patients with panic disorder. Biol. Psychiatry, 21: 849-853, 1986.
Uhde, T.W.: Treating panic and anxiety. Psychiatric Annals 16: 536-541, 1986.
Uhde, T.W., vittone, B.J., Siever, L.J., Kaye, W.H., and Post, R.M. : Blunted
growt^i hormone response to clonidine in panic disorder patients. Biol.
Psychiatry, 21: 1081-1085, 1986.
Boulenger, J. -P. and Uhde, T.W.: Crises aigues d'angoisse et phobies. Aspects
historiques et menifestations cliniques du syndrome d'agoraphobie . In Pichot, P.
(Ed.): L'Anxiete. Paris, Masson Publ. Co., 1987, pp. 115-137.
Mellman, T.A. and Uhde, T.W. : Alprazolam: withdrawal syndrome with gradual
taper. Am. J. Psychiatry, 143: 1464-1466, 1987.
Roy-Byrne, P.P., Geraci, M. and Uhde, T.W. : Life events obtained via interview:
the effect of time of recall on data obtained in controls and patients with panic
disorder. J. Affective Disord. 12: 57-62, 1987.
Roy-Byrne, P.P., Uhde, T.W., Holcomb, H. , Thompson, K. , King, A.K., and
Weingartner, H.: Effects of diazepam on cognitive processes in normal subjects.
Psychopharmacology , 91: 30-33, 1987.
Uhde, T.W.: Treatment of panic disorder. In Rakel, R.E. (Ed.): Conn's Current
Therapy. Philadelphia, W.B. Saunders, 1987, pp. 949-953.
Uhde, T.W., Berrettini, W.H., Roy-Byrne, P.P., Boulenger, J.-P. , and Post, R.M. :
Platelet ^H-imipramine binding in patients with panic disorder. Biol. Psychiatry,
22: 52-58, 1987.
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Uhde, T.W. and Kellner, C.H.: Cerebral ventricular size in panic disorder. J.
Affective Disord. 12: 175-178, 1987.
Boulenger, J. -P. and Uhde, T.W.: Les aspects biologiques de I'anxiete. In
Mendlewicz, J. (Ed.): Manuel de Psychiatrie Biologique. Paris, Masson, in press.
Boulenger, J. -P., Salem, N. , Jr., Marangos , P.J., and Uhde, T.W. : Plasma
adenosine levels: Measurement in humans and relationship to the anxiogenic
effects of caffeine. Psychiatry Res. 21: 247-255, 1987.
Klein, E.H. and Uhde, T.W. : Verapamil in the treatment of panic disorder. Am. J.
Psychiatry, in press.
Mellman, T.A. and Uhde, T.W. : Obsessive-compulsive symptoms in patients with
panic disorder. Am. J. Psychiatry, in press.
Nemiah, J.C. and Uhde, T.W. : Obsessive-Compulsive Disorder. In Kaplan, H.I. and
Sadock, B.J. (Eds.): Comprehensive Textbook of Psychiatry, Fifth Edition.
Baltimore, Williams & Wilkins, in press.
Nemiah, J.C. and Uhde, T.W. : Phobic disorders: Simple phobia, social phobia, and
agoraphobia without panic attacks. In Kaplan, H.I. and Sadock, B.J. (Eds.):
Comprehensive Textbook of Psychiatry, Fifth Edition. Baltimore, Williams &,
Wilkins, in press.
Roy-Byrne, P.P., Mellman, T.A. and Uhde, T.W. : Biologic findings in panic
disorder: neuroendocrine and sleep-related abnormalities. J. Anxiety Disord., in
press.
Roy-Byrne, P.P. and Uhde, T.W. : Exogenous factors in panic disorder: clinical and
research implications. J. Clin. Psychiatry, in press.
Siever, L.J., Uhde, T.W. , Insel, T.R. , Kaye, W.H., Jimerson, D.C., Lake, C.R.,
Kafka, M. , Targum, S. and Murphy, D.L.: Abnormalities in the primary affective
disorders compared to other tricyclic-responsive disorders. Psychopharmacol .
Bull. , in press.
Siever, L.J. , Uhde, T.W. , Potter, W.Z. and Murphy, D.L. : Norepinephrine in the
affective disorders and their treatment. II. Receptor assessment strategies. In
Lake, C.R. and Stirba, A.L. (Eds.): Norepinephrine: Clinical Aspects, in press.
Stein, M.B. and Uhde, T.W. : Thyroid indices in panic disorder. Am. J.
Psychiatry, in press.
Uhde, T.W. : The noradrenergic system in anxiety disorders. In Sen, A.K. and Lee,
T. (Eds.): Receptors and Ligands in Psychiatry and Neurology. London, Bath
Press, in press.
Uhde, T.W., Joffe, R.T., Jimerson, D.E. and Post, R.M. : Normal urinary free
Cortisol and plasma MHPG in panic disorder: clinical and theoretical implications.
Biol. Psychiatry, in press.
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Uhde, T.W., Liebowitz, M. and Rainey, J.: Panic attacks and lactate metabolism: a
biochemical clue revisited. Ann. Intern. Med., in press.
Uhde, T.W. and Mellman, T.W. : Commentary on "Relaxation Induced Panic (RIP): When
resting isn't peaceful" by Adler, C.A., Craske, M.G. and Barlow, D.H. Integrative
Psychiatry, in press.
Uhde, T.W. and Nemiah, J.C: Panic and generalized anxiety disorders. In Kaplan,
H.I. and Sadock, B.J. (Eds.): Comprehensive Textbook of Psychiatry, Fifth
Edition. Baltimore, Williams & Wilkins, in press.
Weiss, S.R.B. and Uhde, T.W.: Animal models of anxiety. In Meltzer, H. and
Bunney, W.E., Jr. (Eds.): Psychopharmacology : The Third Generation of Progress.
New York, Raven Press, in press.
Stein, M.B. and Uhde, T.W. : Cortisol response to clonidine in panic disorder:
comparison with depressed patients and normal controls. Biol. Psychiatry, in
press.
Klein, E., Marangos, P.J., Montgomery, P., Bacher, J., and Uhde, T.W.: Adenosine
receptor alterations in nervous pointer dogs: A preliminary report. Clin.
Neuropharmacol. , in press.
Uhde, T.W. and Boulenger, J.-P.:v Caffeine model of anxiety. In Lerer, B. and
Gershon, S. (Eds.): New Directions in Affective Disorders. New York, N.Y.,
Springer-Verlag, in press.
Uhde, T.W.: Caffeine: A chemical model of anxiety. In Ballenger, J.C. (Ed.):
Neurobiological Aspects of Panic Disorder (Frontiers of Clinical Neuroscience) .
New York, N.Y., Alan R. Liss, in press.
Stein, M.B. and Uhde, T.W. : Panic disorder and major depression: Lifetime
relationship and biological markers.. In Ballenger, J.C. (Ed.): Neurobiological
Aspects of Panic Disorder (Frontiers of Clinical Neuroscience). New York, N.Y. ,
Alan R. Liss, in press.
Mellman, T.A. and Uhde, T.W. : Sleep studies in panic disorder. In Ballenger,
J.C. (Ed.): Neurobiological Aspects of Panic Disorder (Frontiers of Clinical
Neuroscience). New York, N.Y., Alan R. Liss, in press.
Uhde, T.W.: What the clinician should know about caffeine. In Roy-Byrne, P.
(Ed.): Biology of Panic. Washington, D.C., American Psychiatric Association
Press, in press.
Uhde, T.W. and Gorman, G. : Chemical models of anxiety. In Silverstone, T. (Ed.):
Psychopharmacology of Anxiety. London, Oxford University Press, in press.
Gurguis, G. and Uhde, T.W. : Panic disorder: New research directions. In Norton,
G.R., Walker, J.R. , and Ross, C. (Eds.): Panic Disorder and Agoraphobia: A Guide
for the Practitioner. New York, N.Y. , MacMillan, in press.
67
; PROJECT .■.bi-'BER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
ZOl MH 00452-12 BP
PERIOD COVERED
October 1, 1986 to September ,30. 1987
TITLE Of PROJECT (BO chtrtcltn or Itss. Titlt must tit on on» lin* tntwatn thi bofdarj.j
Neuroendocrine Studies of Major Psychiatric Disorders
PRINCIPAL INVESTIGATOR (LiMt oth»r prolissionti ptrtonnal btlow iht Principal InnttigaUy.) (Ntma, titl*, Itborttory, »nd «»t/futi tmiltlion)
Philip W. Gold, M.D. , Chief, Section on Clinical Neuroendocrinology, BPB, NIMH
Dr. D.L. Loriaux, Chief, Developmental Endocrinology Branch, NICHD
Dr. E.H. Oldfield, Senior Investigator, Surgical Neurology Branch, NINCDS
Dr. G.P. Chrousos, Senior Investigator, Developmental Endocrinology Branch, NICHD
Dr. R.M. Post, Chief, Biological Psychiatry Branch, NIMH
Dr. D.R. Rubinow, Chief, Unit on Peptide Studies, BPB, NIMH
COOPERATING UNITS (If any)
BPB, CNB, LCS, LNP, NIMH: DEB, EB, NICHD; SNB, NINCDS; SOB, NCI; LCS, NIAAA;
UCLA School of Medicine; University of Pittsburgh School, of Medicine
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Section on Clinical Neuroendocrinology
INSTITUTE AND LOCATION
NIMH, Bethesda, MD 20892
TOTAL MAN- YEARS:
11.0
PROFESSIONAL-
12.0
OTHER: , „
4.0
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects G (b) Human tissues D (c) Neither
D (al) Minors
D (a2) Interviews
SUMMARY OF WORK ft/je ifandvtf unrtductd fyp* Do nol txcttd tfi« spact provided.)
A primary focus of our work has been on mechanisms of physical and emotional
stress and their relevance to major psychiatric and endocrine disorders. Our
recent work with corticotropin releasing hormone (CRH) illustrates our compre-
hensive approach to this area of inquiry. For instance, we have advanced data
which indicate that CRH is of physiological relevance to human pituitary-adrenal
adrenal function, demonstrated its role in the pathophysiology of hypercortisol-
ism in depression and anorexia nervosa, and administered it as a clinically use-
ful means of determining the differential diagnosis between depression and
Gushing 's disease. Our data regarding interactions between the CRH system and
the locus ceruleus-norepinephrine system suggest that a positive feedback loop
between these two major effectors of the stress response may account for many
of the clinical and biochemical manifestations of melancholic depression. As
indirect support for a role of CRH in the mood component of depression, we have
shown that procaine produces does-dependent activation of pituitary-adrenal func-
tion in association with mood changes in patients with affective illness. More-
over, in vitro studies show procaine-induced dose-dependent activation of the
CRH neuron which is prevented by carbamazeplne. In clinical studies with volun-
teers and patients with panic disorder, we have implicated CRH in exercise and
lactate-lnduced panic and have advanced in vivo and in vitro data that alprazo-
lam may exert therapeutic effects by suppressing the CRH neuron. In studies
exploring the mechanisms by which the Immune system may stimulate adrenal' corti-
costeroid counterregulation of the Immune response, we have shown that both
Interleukln-I and interleukin-II produce dose-dependent activation of the CRH
neuron.
69
PHS 6040 (R«v. \l%*) e*o*i4.«ii
ZOl MH 00452-12 BP
COLLABORATORS:
Dr. G. Merriam, Senior Investigator, Developmental Endocrinology Branch, NICHD
Dr. S.R.B. Weiss, Staff Fellow, BPB, NIMH
Dr. L. Laue, Medical Staff Fellow, Developmental Endocrinology Branch, NICHD
Dr. T. Laughlin, Medical Staff Fellow, Developmental Endocrinology Branch,
NICHD
Dr. G. Cutler, Sr., Investigator, Developmental Endocrinology Branch, NICHD
Dr. A. Roy, Medical Staff Fellow, Clinical Neuroscience Branch, NIMH
Dr. J. Rock, Medical Staff Fellow, Surgical Neurology Branch, NINCDS
Dr. D. Pickar, Clinical Neuroscience Branch, NIMH
Dr. S. Paul, Chief, Clinical Neuroscience Branch, NIMH
Dr. H. Gwirtsman, UCLA School of Medicine
Dr. D. Jacobowitz, Chief, Section on Histopharmacology, LCS, NIMH
Dr. H. Brandt, Medical Staff Fellow, Clinical Neuropharmacology Branch, NIMH
Dr. K. Kalogeras, Visiting Fellow, Biological Psychiatry Branch, NIMH
Dr. T. Luger, Visiting Fellow, Developmental Endocrinology Branch, NICHD
Dr. A. Markwick, Guest Worker, Biological Psychiatry Branch, NIMH
Dr. R. Rittmaster, Medical Staff Fellow, Developmental Endocrinology Branch,
NICHD
Dr. L. Nieman, Medical Staff Fellow, Developmental Endocrinology Branch, NICHD
Dr. H. Schulte, Visiting Fellow, Developmental Endocrinology Branch, NICHD
Dr. B. Adinoff, Medical Staff Fellow, Laboratory of Clinical Science, NIAAA
Dr. M. Linnoila, Chief, Laboratory of Clinical Science, NIAAA
Dr. M. Herkenham, Senior Investigator, Laboratory of Neurophysiology, NIMH
Dr. W. Kaye, University of Pittsburgh School of Medicine
70
ZOl MH 00452-12 BP
Dr. A. Calogero, Guest Worker, Section on Clinical Neuroendocrinology, BPB,
NIMH
Dr. C. Liapi, Guest Worker, Section on Clinical Neuroendocrinology, BPB, NIMH
Dr. A. Margioris, Guest Worker, Section on Clinical Neuroendocrinology, BPB,
NIMH
Dr. D. Rabin, Guest Worker, Section on Clinical Neuroendocrinology, BPB, NIMH
Dr. A. Chiarenza, Guest Worker, Section on Clinical Neuroendocrinology, BPB,
NIMH
Dr. C. Liapi, Guest Worker, Section on Clinical Neuroendocrinology, BPB, NIMH
Dr. I. Khan, Guest Worker, Section on Clinical Neuroendocrinology, BPB, NIMH
Dr. J. Sivo, Guest Worker, Section on Clinical Neuroendocrinology, BPB, NIMH
Dr. J. R. Calabrese, Research Psychiatrist, Cleveland Clinic Foundation
71
ZOl MH 00452-12 BP
Project Description
A. Objectives
The fundamental strategy of the Section on Clinical Neuroendocrinol-
ogy has been to extend current concepts regarding the physiology of neuroendo-
crine regulation in animal models and healthy volunteers, and to apply this
understanding in an effort to unravel the pathophysiology of neuroendocrine
disturbances in a variety of patient populations. Moreover, because neuroendo-
crine abnormalities constitute the cardinal manifestations of major psychiat-
ric illness, such as depression and anorexia nervosa (vida infra), it is hoped
that this work may unravel fundamental mechanisms of disease. Our clinical
populations span multiple disciplines including patients with several major
psychiatric diagnoses (e.g., affective illness, anorexia nervosa, panic anxi-
ety disorder, schizophrenia, alcoholism), as well as a range of major neuroen-
docrine abnormalities (e.g.. Gushing 's disease, ectopic ACTH secretion, ACTH-
independent hypercortisolism, primary and secondary adrenal insufficiency
Nelson's syndrome, and diabetes insipidus).
Our work on neuroendocrine regulation in patients with major psychiatric
illness reflects a rapidly enlarging body of literature which suggests an
intimate linkage between neurohormonal functional activity and major compon-
ents of the symptom complexes of illnesses such as depression and anorexia
nervosa. For instance, several aspects of the syndromes of primary affective
disorder and anorexia nervosa suggest hypothalamic dysfunction. Thus, patients
with depression often manifest disturbances in appetite, libido, reproductive
function (i.e., amenorrhea), water metabolism, Cortisol secretion, and in the
temporal organization of a variety of phenomena whose circadian periodicity is
thought to be governed by hypothalamic pacemakers. Patients with anorexia
nervosa show not only profound alterations in eating behavior, but also marked
changes in hypothalamic-pituitary regulation, gonadotropin secretion, and in
plasma levels of Cortisol. In addition to efforts to explore the mechanism of
these abnormalities in psychiatric populations, our interest in neuroendocrine
systems also relates to the fact that the monoaminergic neurotransmitters
long thought to play a dominant role in major psychiatric illness modulate the
synthesis and release of a number of hypothalamic peptides and pituitary hor-
mones; thus, examination of pituitary hormones in plasma can shed light on
the functional activity of central biogenic amine systems. Moreover, the
hypothalamic hormones themselves have been shown to be widely distributed
within brain, to exert specific receptor-mediated biological actions, and to
influence the functional activity of brain neurotransmitter systems. Several
hypothalamic hormones have also been shown to have profound effects on coordi-
nating complex behaviors and physiological processes of relevance to adaptation
and the maintenance of internal homeostasis.
Our interest in neuroendocrine regulation in patients with medical ill-
nesses such as Gushing 's disease reflects our commitment to the clinical study
of neuroendocrine regulation relevant to health and disease. We have focused
our work on patients with abnormalities of the hypothalamic-pituitary-adrenal
(HPA) function, because it is this axis which is most consistently disturbed in
a variety of psychiatric illnesses; moreover, the hypercortisolism of depres-
sion can be of sufficient magnitude that it has been called a "pseudo-
Gushing 's state and can be difficult or impossible to distinguish from mild or
72
ZOl MH 00452-12 BP
early Gushing 's disease. In this regard, one of our goals was to comprehen-
sively compare and contrast the pathophysiology of the hypercortisolism in
depression and Gushing 's disease, and to develop a means of assisting in their
often difficult diagnosis. We also hoped to explore possible mechanisms for
elucidating the differential diagnosis between Gushing 's disease and ectopic
AGTH secretion, and among various causes of adrenal insufficiency. Although
our work in both psychiatric and medical populations focused on HPA regulation,
we have also attempted to integrate data concerning the regulation of other
neurohormones in these patients, including arginine vasopressin, oxytocin,
somatostatin, the endogenous opiates, and growth hormone-releasing hormone.
In our clinical studies, several strategies have been routinely utilized:
1) direct measurement in the CSF and in the plasma of behaviorally active pep-
tides during the basal state and/or following stimulation according to verified
stimulation paradigms; 2) administration of hypothalamic releasing factors to
test responses of the pituitary axis and to elucidate patterns of endogenous
monoaminergic disturbance and neuroendocrine dysfunction; 3) elucidation of the
effect of psychoactive agents on hypothalamic-pituitary axis function and on
levels of behaviorally active peptides; 4) assessment of the temporal organiza-
tion of neuroendocrine function; 5) administration of hormone antagonists to
explore the functional relevance of various agents to normal physiology and to
pathophysiological processes; 6) screening for restriction-fragment length
polymorphism utilizing probes for genes whose functional regulation may be
abnormal, based on our studies of neuropeptide dysregulation in major psychiat-
ric illness.
As an adjunct to our clinical studies, we maintain an active pre-clinical
studies program whose principal interests include the mechanisms of physical and
emotional stress and the endocrine functions of the brain. In this regard, our
pre-clinical program is closely allied with our clinical studies program, which
we anticipate will focus on a coordinated series of studies to compare and con-
trast pathophysiological features in depression and anorexia nervosa. In this
regard, both depression and anorexia nervosa can be precipitated by stress, and
each is clearly influenced by the principal biologic effectors of the stress
response. Moreover, neuroendocrine abnormalities constitute the cardinal bio-
chemical manifestations of both depression and anorexia nervosa, and each con-
stitutes the kind of syndromal process likely to be linked to alterations in
in one or more neurohormones known to function as integrative neuromodulators.
The specific pre-clinical components of our group which currently interface
with our clinical program include studies utilizing immunocytochemistry, high
resolution autoradiography, molecular biology (including JLn situ hybridiza-
tion), and behavioral pharmacology.
Studies of Neuropeptide Regulation in Health and Disease:
A. Studies of relevance in the comparison of pathophysiological mechan-
isms in depression and anorexia nervosa;
73
ZOl MH 00452-12 BP
1. Corticotropin Releasing Hormone (CRH)
The stress responsiveness of anorexia nervosa and depression and
the sustained hypercortisollsm which is a consistent concomitant of these ill-
nesses made the discovery of corticotropin releasing hormone an exciting event
for endocrinologists and psychiatrists. Shortly after the sequencing of CRH,
our group commenced a series of studies in healthy volunteers which validated
the physiologic relevance of CRH to normal human pituitary-adrenal function,
led to the development of strategies for studying pulsatile ACTH secretion,
elucidated the biological and pharmacokinetic properties of ovine and human
CRH, and demonstrated a circadlan rhythm in the cortlcotroph's response to CRH
and its dependence upon its exposure to pulsatile CRH for normal functioning.
Our studies of the normal physiology of the HPA axis have proven helpful
in the establishment of a clinically applicable ovine corticotropin releasing
hormone stimulation test which we have applied in a systematic way to tease
apart pathophysiological mechanisms in psychiatric and neuroendocrine dis-
eases. In patients with anorexia nervosa and depression, we showed that the
response of the corticotroph cell to exogenous synthetic CRH was appropriately
restrained by glucocorticoid negative feedback, indicating that hypercortisol-
lsm in these disorders reflected an abnormality at or above the hypothalamus,
resulting in the hypersecretion of endogenous CRH. In support of this hypothe-
sis was our data that healthy volunteers responded to a continuous infusion of
CRH with a pattern and magnitude of Cortisol secretion similar to that seen in
anorexia nervosa and depression. Moreover, CSF CRH was frankly elevated in
anorexia nervosa, and correlated positively with post-dexamethasone Cortisol
levels in depression.
In addition to our elucidation of the mechanism of hypercortisollsm in
depression and anorexia nervosa, we also hypothesized that CRH may play an
important role in the overall symptom complexes of these two Illnesses. Thus,
the ICV administration of CRH to experimental animals produced many of the
physiological and behavioral concomitants of these disorders, including not
only hypercortisollsm but also hypothalamic hypogonadism, decreased libido,
anorexia and increased motor activity.
Although we have no definitive information regarding the cause of the dys-
regulation in the CRH system in anorexia nervosa and depression, our in vitro
studies show that norepinephrine and serotonin are potent stimuli to the CRH
neuron, while GABA is Inhibitory. Moreover, we noted a positive correlation
between CSF CRH and CSF NE in depression, suggesting that activation of the
locus ceruleus-norepinephrine (LC-NE) system may play a particularly important
role in the dysregulation of CRH in this disorder. In this regard, the recent
report that CRH markedly increases the firing rate of the LC-NE system suggests
that a positive reverberatory feedback loop between the CRH and the LC-NE sys-
tem (e.g., the two major effectors of the stress response) may account for many
of the clinical and biochemical manifestations of depression. In contrast to
depression, Kaye et al. have shown that indices of NE-LC system function are
generally diminished in patients with anorexia nervosa, regardless of clinical
state.
74
ZOl MH 00452-12 BP
2. Peripheral and Central Vasopressin Regulation
In studies of vasopressin regulation in anorexia nervosa, we have
shown that underweight subjects exhibit a previously undescribed abnormality
in which plasma vasopressin secretion proceeds entirely dissociated from osmo-
tic control. This defect in plasma vasopressin secretion was manifested
clinically as an intermittent inappropriate vasopressin secretion syndrome,
which could contribute to hemodilution and to the life-threatening hypokalemia
seen in underweight anorexic subjects. An additional finding was that the
abnormal osmoregulation of plasma vasopressin secretion was almost always
associated with indices of the hypersecretion of centrally directed vasopressin
into the CSF, a defect which persisted not only in patients re-studied follow-
ing the short-term correction of the weight loss, but in many after the long-
term correction of weight loss as well. We have previously postulated that
the increase in centrally directed vasopressin secretion might influence the
symptom complex of anorexia nervosa. Hence, extensive experimental evidence
shows that not only does AVP potentiate the actions of CRH, but that AVP also
delays the extinction of learned behaviors acquired during aversive condition-
ing - an effect analogous to the anorexic person's perseverative preoccupation
with the adverse consequences of eating. Synergy between AVP and CRH could
also contribute to the substantially higher magnitude of hypercortisolism in
anorexia nervosa.
In contrast to patients with anorexia nervosa, patients with depression
showed intact osmoregulation of plasma vasopressin secretion, though the quan-
titative vasopressin response to osmotic stimulation (i.e., the sensitivity of
the osmoreceptor) is significantly reduced in depression. This reduction in
osmotically mediated vasopressin secretion was associated with a significant
decrease in the secretion of vasopressin into the CSF of depressed patients.
Thus, as with indices of LC-NE function, the functional activity of centrally
directed vasopressin seems abnormal in opposite directions in depression and
anorexia nervosa.
3. Growth Hormone Regulation
Integrated basal growth hormone secretion is increased in both
depression and anorexia nervosa. To explore the mechanism of the hypersecre-
tion of growth hormone in these disorders, we administered synthetic growth
hormone releasing hormone (GH-RH 1-44) to drug-free anorexic and depressed
patients. Surprisingly, the GH responses in these populations were in the
opposite direction. Hence, in patients with anorexia nervosa, GH responses
to GH-RH were exaggerated at reduced weight and returned to normal after the
short-term correction of the weight loss, while in depression the GH responses
to GH-RH were blunted, returning to normal after clinical recovery. Our data
indicate that in the underweight anorexics, both the more severe hypercortisol-
ism and the weight loss per se result in a decrease in somatomedin C production
and/or biologic efficacy, so that the reduced restraint of somatomedin C upon
the somatotroph allows an exaggerated GH response to GH-RH. In depression,
on the other hand, the hypercortisolism is not of a sufficient magnitude to
inhibit somatomedin C production and/or actions, but of subsequent magnitude to
inhibit the GH response to GH-RH at the level of the pituitary corticotroph
cell.
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B. Additional Studies of CRH Regulation of Potential Relevance to Depres-
sion: In vivo and in vitro Studies of Procaine Effects on Mood and
Neuroendocrine Function
Anatomic and physiologic considerations suggest that alterations in the
functional activity of certain limbic structures, including amygdala and hippo-
campus, may play a role in both the symptom complex and neuroendocrine distur-
bances seen in affective disorders. Hence, direct stimulation of these struc-
tures in humans has been shown to produce sensory experiences with often vivid
emotional recall, and to reproduce many of the mood states characteristic of
clinical depression. Moreover, studies utilizing electrical stimulation of
limbic structures, or their spontaneous activation through seizure activity,
suggest that these brain areas can exert a potent influence on the secretion of
stress-associated hormones. Because the use of these methods in clinical stu-
dies of depression is severely limited by ethical considerations, we felt that
availability of a safe pharmacologic probe of limbic activity would greatly
enhance our understanding of the role of these structures in the pathophysiol-
ogy of affective disturbances.
A number of experimental observations suggested that local anesthetics
(e.g., procaine, lidocaine, cocaine) might be candidates for such a pharmaco-
logic probe of limbic activity. Hence, local anesthetics given chronically
to rats can produce kindled seizures which cross-sensitize with amygdala-
kindled seizures and which are associated with marked increases in metabolic
activity in specific limbic areas ,j including amygdala and hippocampus. More-
over, previous studies had suggested that intravenous procaine administration
in humans could produce many of the same psychosensory and mood effects seen
with electrical stimulation of limbic structures. We were particularly inter-
ested in the potential involvement of CRH in these effects, as we have shown
that intracerebroventricular CRH can produce amygdaloid seizures in rats which
sensitize animals to the development of lidocaine-kindles seizures.
To explore the potential relationship between CRH and limbic seizures of
potential relevance to affective disorder, we gave IV procaine to healthy vol-
unteers, patients with affective disorder (who tend to have sustained changes
in mood) and patients with borderline personality disorder (who tend to have
paroxysmal mood shifts which have been previously postulated to reflect lim-
bic system instability). We have previously shown that procaine is capable of
inducing psychosensory and EEC effects suggestive of action at temporal lobe
and limbic structures. Procaine stimulated ACTH, Cortisol, and prolactin, but
not growth hormone secretion in our subjects in all diagnostic groups.
Although the magnitude of the ACTH responses were similar, there was signifi-
cantly greater variability of the response in the borderline group compared to
controls. As yet, the group of affective disorder patients is too heterogene-
ous with respect to state to allow meaningful comparisons.
We have attempted to clarify the mechanism of the ACTH response to pro-
caine using an in vitro short-term rat hypothalamic organ culture system
(1 hypothalamus per well) in which we have previously demonstrated robust CRH
responses to predicted provocative stimuli. In this system, procaine, lido-
caine, and cocaine all stimulated CRH release in a dose-dependent fashion in
the micromolar concentration range; these concentrations are attainable in the
plasma after IV administration to humans at the dose range used in our clini-
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cal studies.
In a subsequent study exploring the effects of carbamazepine procaine-
induced In vitro CRH secretion, we showed that this antl-selzure , antl-
klndllng agent significantly reduced the CRH response to Incubation with pro-
caine. Hypothetlcally, we postulate that carbamazepine may exert antl-klndllng
and mood-stabllizlng effects via Inhibition of the effect of stimuli which
activate the CRH neuron. These findings suggest that local anesthetics
are capable of eliciting many of the behavioral and neuroendocrine alterations
seen in depression, and that some of these effects may be mediated by activa-
tion of limbic-hypothalamic pathways, resulting in release of CRH. Moreover,
the therapeutic effects of carbamazepine may confer protection against exces-
sive CRH release by pathogenic stimuli.
C. The Potential Role of CRH in the Pathophysiology of Panic Disorder;
In Vivo and In Vitro Findings
When administered via the intraventricular route to experimental animals
in low to moderate doses, CRH initiates a coordinated series of physiological
and behavioral responses similar to those seen during adaptation to stressful
situations; these include not only pituitary-adrenal activation, but also
activation of the sympathetic nervous system, decreased feeding and sexual
behavior, arousal, enhanced learning and memory, and Increased aggression.
Like many other neuropeptides which produce arousal, the central effects of CRH
seem to follow an Inverted U-shaped dose response curve, so that at higher
doses maladaptive, "anxlogenic" effects are produced. These Include decreased
exploration in an open field, enhanced responsiveness to acoustic startle, dis-
ruption in learning and memory, and immobility. These behavioral effects in
experimental animals stimulated us to explore the potential role of CRH in the
panic disorder syndrome.
In a series of clinical and pre-cllnical studies, we advanced the follow-
ing lines of evidence suggesting a role for CRH in panic disorder: 1) Patients
with panic disorder, who often manifest basal hypercortisolism, show an attenua-
ted ACTH response to ovine CRH (oCRH) analogous to that described in our
patients with depression and anorexia nervosa. 2) Exercise, which can precipi-
tate panic attacks in patients with panic disorder, produces dose-dependent
increases in plasma ACTH and Cortisol secretion in volunteers (when applied in
graduated levels of 50%, 70%, and 90% of the maximal oxygen utilization rate).
As a corollary, this exercise-induced dose-dependent ACTH secretion correlated
positively with levels of plasma lactate, another stimulus of panic attacks.
3) Studies of the in vitro regulation of the rat hypothalamus show that lactate
produces dose-dependent increases in CRH secretion. 4) Alprazolam, one of the
most potent anti-panic agents, inhibits serotonin-induced CRH secretion in
doses as low as 10~° M and was substantially more potent than was diazepam, a
less effective therapeutic agent. 5) Intravenous alprazolam given to non-
restrained primates with indwelling vena cava lines inhibited ACTH and Cortisol
secretion in a dose-dependent fashion.
In summary, we have advanced several lines of circumstantial evidence sug-
gesting the potential role of CRH in the pathophysiology of panic disorder.
The potential involvement of CRH in the hypercortisolism in disorders such as
depression, anorexia nervosa, and panic disorder is of Interest In light of evl-
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dence that the overall group of patients with these illnesses shows a greater
than normal family history for primary affective disorder. This suggests that
these disorders may lie along a spectrum of depressive disorders with CRH dys-
regulation as a common denominator.
D. Studies of the Differential Diagnosis and Pathophysiology of Depres-
sion and Gushing 's Disease: Focus on CRH and Related Neuropeptide
Systems
Depression is often the first sign of Gushing 's disease, preceding the
physical stigmata by months or years, while the depressed phase of primary
affective disorder is often associated with profound hypercortisolism (often
termed a pseudo-Gushing 's state). Hence, the differential diagnosis
between early or mild Gushing 's disease and depression can be impossible to
ascertain, and their clinical and biochemical similarities have suggested that
they share a similar pathophysiological basis.
To explore patterns relating to the differential diagnosis and pathophys-
iology in depression and Gushing 's disease, we utilized either the administra-
tion of GRH or measurement of CRH and related peptides in the cerebrospinal
fluid (CSF). By these means, we hoped to develop a clinically useful means to
distinguish these two entities and to determine if the two illnesses represent
similar or distinct pathophysiological mechanisms.
Our first line of investigation was to test the functional integrity of
the pituitary corticotroph cell in our patients following the administration
of oCRH. Patients with Gushing 's disease showed markedly exaggerated ACTH
response to oCRH despite their profound basal hypercortisolism, indicating
a gross defect in glucocorticoid negative feedback upon the pituitary cortico-
troph cell. This contrasts markedly with the attenuated ACTH responses to
oGRH that our group has shown in depression, which Indicate a pituitary corti-
cotroph cell appropriately restrained by hypercortisolsim. On the basis of
these divergent responses to oGRH, which reflect different pathophysiological
mechanisms, the oCRH test has proven helpful in the differential diagnosis
between depression and Gushing 's disease. This is so even when basal plasma
Cortisol values overlap, and is in marked contrast to all other noninvasive
diagnostic procedures.
The differential pituitary corticotroph cell function in Gushing 's disease
and depression help explain the fundamental differences in the clinical manifes-
tations of hypercortisolism in these disorders. For instance, the fact that
ACTH secretion can proceed relatively unchecked by Cortisol negative feedback in
Gushing 's disease allows for the kind of sustained hypercortisolism required to
produce Cushingoid stigmata. On the other hand, although depressed patients can
have Cortisol levels in the Cushingoid range, their intact Cortisol negative
feedback upon the pituitary means that any centrally mediated rise in plasma
ACTH and Cortisol secretion would lead to a secondary glucocorticoid restraint
of the pituitary, with a subsequent fall in the plasma Cortisol. Hence, it is
highly unlikely that the kind of sustained hypercortisolism required for Cush-
ingoid features could supervene.
We also wished to determine whether the hypothalamic GRH neuron in Gush-
ing's disease was hyperactive as in depression, or was restrained by long-
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standing hypercortisolism. Three lines of evidence support the latter conclu-
sion: 1) Twenty-two of 26 patients with Cushing's disease, whom we studied
one week after transsphenoidal adenomectomy (at a time when basal ACTH levels
were uniformly undetectable), nevertheless showed ACTH responses to endogenous
CRH; from this we surmise that the post-operative adrenal insufficiency in
the patients reflects suppression of CRH neurons by long-standing hypercorti-
solism; 2) Pituitary corticotroph cell function could be restored post-
operatively by repeated priming doses of human CRH for several days (analagous
to the situation in hypothalamic LH-RH deficiency in which spontaneous LH and
FSH secretion could only be elicited in the context of priming doses of LH-RH);
and 3) CSF CRH levels in Cushing's disease were significantly lower than in
depressed patients or controls.
Because CSF somatostatin levels are significantly reduced in depression,
we measured this peptide in the CSF of patients with Cushing's disease. Soma-
tostatin levels in Cushing's disease were also significantly reduced, suggesting
that hypercortisolism may be a factor in the decreased somatostatin levels in
both Cushing's disease and depression. Though the functional consequences of
this reduction in CSF somatostatin are unknown, it may not be coincidental that
Cushing's disease joins depression and Alzheimer's disease as disorders charac-
terized by diminished cognitive performance and decreases in central somatosta-
tin levels.
E. Studies of the Circadian Organization of Pitutiary-adrenal Function in
Patients with Affective Disorder
In an extensive series of depressed patients and control subjects, we have
assessed plasma ACTH and Cortisol levels every 30 minutes for 40 hours (encom-
passing two nights and one day). We noted that although the mean 40-hour Cor-
tisol secretion was significantly greater in depressed patients than in con-
trols, the mean ACTH concentration was similar. However, depressed patients
consistently showed a greater number of ACTH and Cortisol secretory episodes/
24 hours than did controls, thus giving greater total ACTH as well as Cortisol
secretion than did controls. These data support our formulation that the adre-
nal has become hyperresponsive to ACTH in patients with depression and that the
hypersecretion of CRH takes the form of increased pulsatile secretion as at
least one component in the pathophysiology in HPA function in depressed
patients. A potentially important methodologic point was the finding that in
controls the pattern of ACTH and Cortisol secretion was essentially identical
on each of two successive nights. While there was no significant difference in
these parameters on two successive nights of sampling in depressed patients,
there was more variability in this group than in the control subjects.
F. Studies of ACTH Secretion into the CSF in Patients with Depression,
Anorexia Nervosa, Cushing's Disease, and Alzheimer's Disease
The previous sections have reviewed our work with CRH and have included
extensive data concerning the secretion of ACTH into plasma. We have also
explored the secretion of ACTH into the CSF. Our data in stalk-sectioned pri-
mates, indicating substantial levels of ACTH in CSF despite barely detectable
plasma ACTH levels, indicates that the pituitary may not be the source of CSF
ACTH. Rather, the ACTH found in the CSF most likely is derived from the
arcuate nucleus, which contains POMC and such post-translation products as
ACTH, beta-lipotropin, and beta-endorphin.
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In several separate populations of normal volunteers, we have consist-
ently noted a significant positive correlation between CSF CRH and CSF ACTH.
This finding is compatible with anatomic data that CRH cell bodies in the
paraventricular nucleus send nerve terminals to the arcuate nucleus, and indi-
cates that CRH effects upon central POMC may be analogous to its effects upon
the POMC residing within the pituitary corticotroph cell.
Studies in patient populations show that CSF ACTH is markedly reduced in
both patients with Cushing's disease and the ectopic ACTH syndrome. These
data suggest that the secretion of ACTH into the CSF is glucocorticoid-
suppressible. CSF ACTH levels also tend to be low in depressed patients, but
the levels are not nearly as suppressed as in Cushing's disease. In fact,
there is little overlap between the two groups, so that this measure may prove
helpful in the differential diagnosis of depression and Cushing's disease.
Of potentially even greater diagnostic significance is the plasmarCSF ratio of
ACTH in depression and Cushing's disease. In normals and depressed patients,
this ratio is almost always greater than 1.0. Conversely, in patients with
Cushing's disease, the confluence of high basal plasma ACTH and low CSF ACTH
values produces a plasma :CSF ACTH ratio which is almost always less than 1.0.
A marked reduction in ACTH and other POMC fragments was also found in
underweight patients with anorexia nervosa (i.e., ACTH, beta-lipotropin, beta-
endorphin, and N terminal fragment). The cause of these abnormalities is
unknown, but may reflect the restraining influence of hypercortisolism or an
effect of CRH neuron hyperactivity to deplete the pool of POMC fragments which
is ordinarily secreted into the CSF. CSF ACTH levels return to normal upon the
short-term correction of weight loss in anorexia nervosa, and are also normal
in patients studied months to years after restoration of normal weight.
CSF ACTH was also significantly reduced in patients with Alzheimer's dis-
ease. On the other hand, their levels were normal in subjects with schizophre-
nia and in alcoholic patients studied at one and three weeks after abstinence
from drinking.
The functional consequences of the apparent reduction of CSF ACTH or other
POMC fragments in Alzheimer's disease or anorexia nervosa is not known. Data
from studies in experimental animals suggest a possible role for ACTH in media-
ting sexual behavior. Additional studies in experimental animals also suggest
that ACTH may enhance certain components of information processing.
Pre-clinical Studies:
A. Regulation of CRH Secretion
We have recently worked to develop a sensitive in vitro hypothalamic
rat organ culture system to explore the regulation of the hypothalamic neuron.
We have examined the effects of several substances, including serotonin, ace-
tylcholine, epidermal growth factor, and norepinephrine.
Serotonin (5HT) stimulated immunoreactive (IR) corticotropin releasing
hormone (CRH) secretion by rat hypothalamic explants after an overnight pre-
incubation but not immediately after explantation. This response was mediated
primarily by 5HT2 receptors, as suggested by the complete inhibition of sero-
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tonin-lnduced rat CRH (rCRH) secretion by ritanserin. Neither atropine and
hexamethonium nor phentolamine inhibited 5HT-induced IR-rCRH secretion, sugges-
ting that it is not mediated by cholinergic or alpha-adrenergic interneurons.
In contrast to 5HT, KCl-induced depolarization increased IR-rCRH secretion
both after overnight incubation and immediately after explantation. On both
occasions the IR-rCRH content of hypothalami and their histological examina-
tion were similar. The bulk of IR-rCRH secreted from the hypothalamic
explants under basal conditions and after KCl stimulation, coeluted with syn-
thetic rCRH on Sephadex G-75 gel filtration chromatography.
Acetylcholine (ACH) also stimulated hypothalamic IR-rCRH secretion in a
dose-dependent fashion, at concentrations ranging from 10~° M to 10"^ M.
This effect was antagonized by the simultaneous presence of atropine and
hexamethonium, a muscarinic and a nicotinic receptor antagonist, respectively
(p < 0.05). Further evidence for the cholinergic regulation of the CRH neuron
was provided by the findings that both carbachol, a muscarinic receptor agon-
ist, and nicotine, a nicotinic receptor agonist, stimulated IR-rCRH secretion
in a dose-dependent fashion. These effects were antagonized by atropine and
hexamethonium, respectively, suggesting that both muscarinic and nicotinic
receptors are involved in the process. ACH also stimulated hypothalamic IR-
rCRH secretion in the presence of phentolamine and kitanserin. Hence, we
conclude that ACH stimulates hypothalamic CRH secretion via both mus-
carinic and nicotinic receptor mechanisms. This effect is not mediated by a
serotonergic or alpha-adrenergic interneuron. These data suggest that acetyl-
choline may be implicated in the regulation and the stress activation of the
HPA axis in vivo.
Epidermal growth factor (EGF), a polypeptide mitogen that participates
in wound healing, has ACTH-like activity in ewes. We examined its effects on
the primate hypothalami c-pituitary-adrenal (HPA) axis by administering EGF
intravenously (0-100 ug/kg) to rhesus monkeys. EGF caused dose-dependent
elevations of plasma ACTH and Cortisol in these animals. To define whether
the locus of stimulation was the hypothalamus and/or the pituitary gland, we
examined the capacity of EGF to directly stimulate hypothalamic corticotropin
releasing hormone (CRH) or pituitary ACTH secretion in a rat hypothalamic
organ culture system and a rat pituitary cell system. EGF-stimulated hypo-
thalamic CRH release in a dose-dependent manner, but failed to cause pituitary
ACTH release. Hence, EGF, in addition to its previously described ACTH-like
activity, also has CRH-releasing activity in the primate. In this regard, EGF
may participate in the physiologic activation of the HPA axis at times during
which EGF concentrations are raised. These include such states as trauma, sur-
gery, and possibly emotional stress.
We explored the effect of norepinephrine on CRH secretion because of a
growing body of data which indicate that the noradrenergic and CRH systems
may interact to play a principal role in modulating the responses to physical
and emotional stress. Moreover, an extensive series of previous studies have
provided conflicting results regarding the effects of catecholamines on the
central component regulating pituitary-adrenal function.
Norepinephrine (NE) stimulated IR-rCRH secretion in a dose-dependent fash-
ion with peak effects in the nM range. The effect of NE was antagonized by the
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alpha antagonist phentolamlne, by the alpha ^ antagonist prazosin and by the
alpha 2 antagonist yohimbine, but not by the beta blocker L-propranolol. Compa-
tible with these data were the findings that the alpha^ agonist phenylephrine
and the alpha2 agonist clonidine both stimulated IR-rCRH secretion in a dose-
dependent fashion. On the other hand, while the beta agonist isoproterenol
caused a weak, non-dose dependent increase in IR-rCRH secretion, this effect
could not be antagonized by L-propranolol. Despite pre-treatment with antagon-
ists to serotonin and acetylcholine receptors, the effect of NE upon IR-rCRH
secretion was undiminished. On the other hand, pre-treatment with gamma-
amino-butyric acid (GABA) significantly attenuated NE-induced IR-rCRH secre-
tion.
Epinephrine-stimulated IR-rCRH secretion, but only at higher concentra-
tions. This stimulatory effect was antagonized by phentolamlne, but not by
L-propranolol. Dopamine (DA) had a weak stimulatory effect that could be anta-
gonized by the DAj^ receptor antagonist SCH 23390, but not by phentolamlne.
We conclude that NE stimulates hypothalamic IR-rCRH secretion via hypo-
thalamic alpha 1 and alpha2 receptors. The effect of NE upon IR-rCRH secre-
tion does not appear to be mediated by serotonergic or cholinergic interneur-
ons, but is modulated by the inhibitory neurotransmitter GABA. The present
data support the idea that NE is excitatory rather than inhibitory upon CRH
secretion when it acts directly at a hypothalamic locus. These findings may
be of clinical relevance to illnesses such as depression, which are charac-
terized by activation of both central NE and CRH systems. Hence, activation
of the central NE system may contribute to the hypercortisolism characteristic
of this disorder.
B. Studies Utilizing an in vitro Perifuslon System for Studying the
Hormonal Functions of the Human Placenta:
Pregnancy is the only known state in which CRH circulates in plasma at
levels expected to cause activation of the pituitary-adrenal axis (100 pg/ml
up to 4,000 pg/ml). These levels gradually increase even further during labor.
Plasma CRH concentrations return to undetectable (< 15 pg/ml) levels following
delivery. Interestingly, it has been known for years that pregnancy is charac-
terized by hypercortisolism to a degree similar to what has been observed in
severe depression and anorexia nervosa.
To study the potential role of the placenta as a source of plasma CRH dur-
ing pregnancy, we have developed an in vitro perifuslon system in which full
thickness placenta fragments are kept in culture. These fragments contain a
1300-nucleotide-long mRNA which hybridizes with a CRH specific cDNA probe.
Glucocorticoids, prostaglandins, catecholamines, oxytocin and vasopressin have
an inhibitory or stimulatory effect on placental secretion of CRH, which is
chromatographically identical to hypothalamic CRH. Our perifused placenta
fragments also secrete immunoreactive beta-endorphin. Both CRH and oxytocin
at 10~8 M concentrations stimulate the secretion of beta-endorphin by the
placenta. These findings have raised the following questions: 1) Does the
high intra-pregnancy CRH suppress hypothalamic secretion of CRH (via Cortisol
negative feedback) and therefore lead to a brief post-partum adrenal insuf-
ficiency state, followed by a later rebound of endogenous hypothalamic CRH
secretion 6-8 weeks later? Interestingly, the very common "post partum blues"
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occur In the first week, post partum whereas post partum depression occurs 1-3
months later. 2) What regulates the secretion of placental CRH? Mechanical
contraction or ischemia of the placenta may be responsible for the elevations
of plasma CRH seen during labor. 3) What is the function of pregnancy plasma
CRH other than stimulating the maternal pituitary-adrenal axis? Do the vasodi-
latory properties of CRH play role in labor? For instance, CRH-induced super-
ior mesenteric vessel dilation may protect the maternal intestinal tract from
ischemia. Moreover, circulating CRH might regulate uterine blood vessel flow.
C. Studies with an in vivo Primate Model;
We have recently adapted a technique for surgical implantation of a vena
cava cannula which allows continuous access to the intravenous space of non-
restrained non-human primates. The purpose of our primate laboratory is to
allow extensive study of the physiology of hypothalamic-pituitary-adrenal func-
tion and to explore our hypotheses regarding the pathophysiology of illnesses
such as depression and anorexia nervosa. Findings to date include: 1) demon-
stration of dose-dependent effects of alprazolam in suppressing pituitary-
adrenal function, compatible with our in vitro data showing that this agent is
a potent suppressor of the CRH neuron; 2) demonstration of dose-dependent
effects of procaine to stimulate the pituitary-adrenal axis, compatible with
our ^ vitro data that procaine is a potent stimulus to the CRH neuron;
3) demonstration of a potent effect of epidermal growth factor (EGF) to stimu-
late pituitary-adrenal function in doses comparable to CRH itself, compatible
with our in vitro data that this growth factor stimulates the CRH neuron and
the data of others that EGF may antagonize glucocorticoid effects by phosphory-
lating (and hence inactivating) lipocortin, the lipoprotein thought to mediate
many glucocorticoid effects; 4) establishment of dose-response relationships
for vasopressin-induced ACTH secretion; 5) elucidation of the effects of adre-
nalectomy and high-dose RU 486 on pituitary adrenal function (e.g., time course
of ACTH elevation to peak levels, effects on pulsatility, doses of glucocorti-
coids required to restrain the hypothalamic-pituitary components of the axis).
Significance to Biomedical Research and the Program of the Institute
The Section on Clinical Neuroendocrinology has attempted to extend current
concepts regarding the physiology of neuroendocrine regulation in animal models
and healthy volunteers, and to apply this understanding in an effort to unravel
the pathophysiology of neuroendocrine disturbances in a variety of patient
populations. Such clinical populations span multiple disciplines, including
patients with major psychiatric and major neuroendocrine illnesses.
Our work with corticotropin releasing hormone serves to illustrate the
implementation of our basic strategy for research. For instance, with respect
to normal physiology, we advanced the first data that CRH was of physiologic
relevance to the regulation of basal circadian pituitary-adrenal function in
man. Our data in controls, however, also support the idea that stress-induced
ACTH secretion requires factors other than CRH alone. These findings, as well
as our finding that the pituitary corticotroph, like the gonadotroph, requires
the priming effects of its hypothalamic releasing hormone, have added to the
understanding of hypothalamic-pituitary-adrenal physiology in human subjects.
In further studies with volunteers , our group was the first to compare the
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pharmacokinetic properties of ovine and human CRH. Moreover, on the basis of
this data, we concluded correctly that oCRH was best suited for diagnostic
tests while hCRH was most suited for studying pulsatile ACTH secretion. Dose-
response studies, which revealed the lowest maximal stimulatory dose for ACTH
and Cortisol secretion, subsequently facilitated the clinical application of a
CRH stimulation test. Our demonstration that hormonal responses to oCRH were
greater in the evening than in the morning further allowed us to refine this
CRH stimulation paradigm.
Our CRH stimulation paradigm has found widespread clinical application.
Indeed, the results of our studies have clarified some of the most elusive and
difficult clinical problems in endocrinology. For instance, the differential
pituitary-adrenal responses to CRH in depression and Cushing's disease con-
tributed to the conclusion that hypercortisolism in these illnesses reflects
abnormalities at different loci in the adrenal axis. Moreover, the CRH stimu-
lation test represents a substantial advance over any previous diagnostic test
as an aid in determining the differential diagnosis between depression and
early or mild Cushing's disease. The CRH stimulation test has also proved
helpful in distinguishing Cushing's disease from ectopic ACTH secretion and in
distinguishing subtypes of secondary adrenal insufficiency (i.e., those second-
ary to either selective corticotroph cell deficiency vs. corticotroph sparing
or hypothalamic CRH deficiency).
Our conclusion that hypercortisolism in depression reflects hypersecre-
tion of endogenous CRH is of interset, not only because it clarifies the
pathophysiology of HPA dysfunction in depression, but also because CRH admin-
istration to experimental animals reproduces many of the components of the
symptom complex of affective illness, such as hypothalamic hypogonadism,
decreased libido, anorexia, irritability, and changes in motor activity. More-
over, CRH given ICV produces limbic seizures which cross-sensitize with elec-
trically kindled seizures. Our hypothesis that CRH may constitute an important
link between stressful early life experience and the syndrome of recurrent
depression has become one that is being vigorously pursued in many major
research centers.
Our pre-clinical data have helped us to further extend our hypothetical
mode of depression to include potentially synergistic interactions between the
CRH system and the locus ceruleus-norepinephrine (LC-NE) system. Hence, our in
vitro hypothalamic organ culture studies have shown that norepinephrine is a
potent stimulus to CRH secretion. This finding, in association with the find-
ing that CRH markedly increases the LC firing rte, suggests that the CRH and
LC-NE system (i.e., the two major effectors of the stress response) may parti-
cipate in a mutually reinforcing reverberatory loop which may be responsible
for many of the clinical and biochemical manifestations of depression. This
hypothesis is fully elaborated in much greater detail in an invited paper
solicited by The New England Journal of Medicine, entitled "Clinical and Bio-
chemical Manifestations of Depression: Relationship to the Neurobiology of
Stress."
Our studies have also significantly added to an understanding of patho-
physiological mechanisms in anorexia nervosa. For instance, our data that
hypercortisolism in anorexia nervosa reflects pathophysiological alterations
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similar to those seen in depression have considerably strengthened the idea
that depression and anorexia nervosa share important features in common and
may reside together within a broad depressive spectrum disorder. This is
especially so because, as noted, CRH administration to experimental animals
produces many behavioral and physiological features common to both depression
and anorexia nervosa.
Several lines of evidence advanced by our group have led to a testable
hypothesis regarding the role of CRH in the pathophysiology of panic disorder.
These data, reviewed in detail in this summary, suggest that CRH may be
Involved not only in the hypercortisolism of this disorder, but also in exer-
cise and lactate-induced panic episodes. Moreover, we have shown in a series
of in vivo and in vitro studies that alprazolam is a much more potent inhibi-
tor of the pituitary-adrenal axis and the CRH neuron than diazepam, compatible
with its clinical superiority in the treatment of panic disorder.
Our studies of vasopressin regulation in anorexia nervosa have also clari-
fied the pathophysiology of disturbed water metabolism in these subjects. Spe-
cifically, we described a novel defect in plasma vasopressin secretion, in
which this parameter had become entirely uncoupled from osmoreceptor control.
Moreover, we noted that this defect in plasma vasopressin regulation was almost
always associated with either hypersecretion of vasopressin into the CSF space
or a reversal of the normal plasma CSF vasopressin gradient. The finding of
increased central vasopressin secretion in anorexia nervosa is of potential
interest in light of data that vasopressin delays extinction of learned behav-
ior acquired during aversive conditioning, and the observation that a fundamen-
tal abnormality in anorexia nervosa is a perseverative and exaggerated sense
of the adverse consequences of eating. Parenthetically, the findings of en-
hanced central secretion of CRH and vasopressin in anorexia nervosa may con-
tribute to the particularly pernicious, treatment-resistant quality of this
disorder, because these two peptides exert synergistic effects on the pituitary
corticotroph cell, and may do so within the CNS.
Our work has also helped to clarify the pathophysiology of growth hormone
hypersecretion in anorexia nervosa. Our findings, based on the first applica-
tion of GH-RH to the study of anorexia nervosa, suggest that the hypersecretion
of GH in this disorder may, in part, reflect a functional deficiency of somato-
medin C, which exerts a tonic restraining effect on the pituitary somatotroph
cell.
Our pre-clinical studies have helped to extend knowledge regarding the
regulation of the CRH neuron. Hence, we determined the major stimulatory
(norepinephrine, serotonin, acetylcholine) and inhibitory (GABA/benzodiazepine
system) neurotransmitters influencing CRH secretion and demonstrated the pres-
ence of both short- and ultra-short negative feedback loops via ACTH and CRH,
respectively. We have also shown that several substances secreted during
physical stress, such as Interleukin I, interleukin II, epidermal growth fac-
tor, and lactate may be CRH secretogogues of physiological relevance.
Future Directions
A. Comparative Studies of Depression and Anorexia Nervosa
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One of our fundamental hypotheses is that depression and anorexia nervosa
lie on a broad continuum of depressive spectrum disorders with CRH dysregula-
tion as a potential link of relevance to many overlapping components of these
illnesses (e.g., hypercortisolism, hypothalamic hypogonadism, decreased libido,
anorexia, and increased motor activity). Our work in exploring the dysregula-
tion in CRH function common to both illnesses will attempt to further document
both the presence and the pattern of this defect and to explore its possible
underlying causes.
One of the consequences of CRH hypersecretion in depression and anorexia
nervosa is hypercortisolism. Although we have shown that the mechanism of
hypercortisolism in these disorders is distinct from that in Gushing 's disease,
the magnitude of the hypercortisolism can be as severe. In fact, because our
data strongly suggest that patients with anorexia nervosa do not manifest glu-
cocorticoid resistance, it is our sense that the consistently profound hyper-
cortisolism in underweight anorexics would ordinarily produce the physical
stigmata of Cushing's syndrome if only there were sufficient underlying biolo-
gic substrate. Hence, anorexia nervosa may constitute the only known form of
centrally mediated Cushing's syndrome. In light of the fact that glucocorti-
coid receptors are located in many strategic locations in brain to influence
a variety of behavioral and physiologic processes, it seems essential that we
explore the functional consequences of hypercortisolism, especially in anorexia
nervosa, but as well in depression. This is especially so because recent data
strongly suggest that a principal biologic role of the glucocorticoids during
physical and emotional stress is to terminate the stress responses (e.g., inhi-
bit the CRH and LC-NE systems). In this regard, one can conceptualize depres-
sion as an illness in which there is inadequate counter-regulation by hyper-
cortisolism of both CRH and LC-NE activation, whereas in anorexia nervosa the
LC-NE system seems effectively counter-regulated while the CRH neuron is even
more hyperactive than in depression. Whether there is focal glucocorticoid
resistance in specific brain regions in depression or anorexia remains a ques-
tion for future investigation. Parenthetically, it is our sense that the long-
standing suppression of LC-NE function in anorexics even afer correction of
weight loss reflects the prolonged suppression of this system by the prior his-
tory of profound hypercortisolism. We have previously demonstrated that the
adrenal insufficiency which persists for many months after successful trans-
sphenoidal adenectomy in Cushing's disease reflects prolonged suppression of
the CRH neuron by long-standing hypercortisolism.
In addition to common defects in CRH regulation and the asociated hyper-
cortisolism, these and other neuroendocrine abnormalities suggest hyperfunc-
tion of central 5HT function in anorexia nervosa and depression. Hence, we
have recently shown that 5HT is a potent stimulus to in vitro CRH secretion,
while this neurotransmitter could also contribute to the hypothalamic hypogo-
nadism and hypersecretion of growth hormone common to the two disorders.
Despite pronounced similarities between depression and anorexia nervosa,
these lllneses also manifest clear differences. For example, the kind of mono-
symptomatic obsession which consumes the anorexic is less ommmon in depression,
while depressed mood associated with intense dysphoric hyperarousal is less
present in anorexia nervosa. Although many factors could account for these
differences, our initial studies will focus on norepinephrine and vasopressin.
86
ZOl MH 00452-12 BP
This choice reflects, in part, the fact that abnormalities in each are in
opposite directions in depression and anorexia nervosa, while each has the
potential to act synergistically with the common defect in CRH regulation.
Moreover, the confluence of enhanced CRH and central vasopressinergic function
in anorexia could theoretically contribute to the profound obsessionalism of
this disorder, while concomitant activation of the CRH and LC-NE systems in
depression could contribute to the greater dysphoric hyperarousal associated
with this illness.
Plans for Specific Clinical Studies in the Eating Disorders and
Depression;
We are about to commence a study of the effects of prolonged administra-
tion of the potent glucocorticoid antagonist, RU 486, in patients with anorexia
nervosa and depression. Such a study will have relevance to many of the theore-
tical issues raised above. For instance, we predict that patients with anor-
exia nervosa and depression will show a greater 'overshoot' in pituitary-
adrenal function and in CSF CRH levels than controls, further validating hyper-
activity of the CRH system in these disorders. Such a study will also allow us
to determine whether pathophpysiological abnormalities potentially attributable
to hypercortisolism resolve during RU 486 administration (e.g., decreased sen-
sitivity of the osmoreceptor in depression, blunted prolactin responses to
m-CPP in anorexia nervosa, hypothalamic hypogonadism in each disorder). We
shall further assess the impact of this intervention on indices of noradrenergic
and serotonergic functional activity (e.g., CSF NE, MHPG, 5HIAA) and on a vari-
ety of behavioral and cognitive parameters which may also be influenced by sus-
tained hypercortisolism. Finally, we shall assess lymphocyte glucocorticoid
receptor mRNA before and during the trial as a more definitive means of docu-
menting that peripheral glucocorticoid resistance is not associated with either
illness.
Further efforts to evaluate CRH function in patients with anorexic nervosa
and depression will include assessment of the architecture of the secretion of
CRH and related peptides and neurotransmitters into the CSF via continuous
drainage of lumbar CSF through an indwelling lumbar drain and evaluate the
relationship between these parameters and basal circadian pulsatile pituitary-
adrenal function. We also hope to evaluate the potential role of CRH in the
overall symptom complexes of depression and anorexia by administering a lipo-
philic CRH antagonist. As a corollary, we also plan to evaluate the relative
role of CRH and vasopressin by acutely administering CRH and vasopressin recep-
tor antagonists active at the pituitary corticotroph cell.
To evaluate serotonergic function in depression and anorexia nervosa, we
propose to administer IV m-CPP in the lowest maximal stimulatory dose for
pituitary-adrenal activation in the evening, when the HPA is normally quies-
cent. In light of the potent stimulatory effect we have observed for 5HT upon
in vitro CRH release, m-CPP may be both an important means of evaluating the
potential role of serotonin on CRH-lnduced hypercortisolism and also the best
available peripherally administered stimulus for evaluation of the hypothalamic
CRH neuron in clinical populations. In light of a stimulatory 5HT effect on
vasopressin secretion, m-CPP could also function as one of several non-osmotic
stimuli to be applied at various times to further evaluate the functional
integrity of vasopressin secretion in our clinical populations. An additional
87
ZOl MH 00452-12 BP
This choice reflects, in part, the fact that abnormalities in each are in
opposite directions in depression and anorexia nervosa, while each has the
potential to act synergistically with the common defect in CRH regulation.
Moreover, the confluence of enhanced CRH and central vasopressinergic function
in anorexia could theoretically contribute to the profound obsessionalism of
this disorder, while concomitant activation of the CRH and LC-NE systems in
depression could contribute to the greater dysphoric hyperarousal associated
with this illness.
Plans for Specific Clinical Studies in the Eating Disorders and
Depression;
We are about to commence a study of the effects of prolonged administra-
tion of the potent glucocorticoid antagonist, RU 486, in patients with anorexia
nervosa and depression. Such a study will have relevance to many of the theore-
tical issues raised above. For instance, we predict that patients with anor-
exia nervosa and depression will show a greater 'overshoot' in pituitary-
adrenal function and in CSF CRH levels than controls, further validating hyper-
activity of the CRH system in these disorders. Such a study will also allow us
to determine whether pathophpysiological abnormalities potentially attributable
to hypercortisolism resolve during RU 486 administration (e.g., decreased sen-
sitivity of the osmoreceptor in depression, blunted prolactin responses to
m-CPP in anorexia nervosa, hypothalamic hypogonadism in each disorder). We
shall further assess the impact of this intervention on indices of noradrenergic
and serotonergic functional activity (e.g., CSF NE, MHPG, 5HIAA) and on a vari-
ety of behavioral and cognitive parameters which may also be influenced by sus-
tained hypercortisolism. Finally, we shall assess lymphocyte glucocorticoid
receptor mRNA before and during the trial as a more definitive means of docu-
menting that peripheral glucocorticoid resistance is not associated with either
illness.
Further efforts to evaluate CRH function in patients with anorexic nervosa
and depression will include assessment of the architecture of the secretion of
CRH and related peptides and neurotransmitters into the CSF via continuous
drainage of lumbar CSF through an indwelling lumbar drain and evaluate the
relationship between these parameters and basal circadian pulsatile pituitary-
adrenal function. We also hope to evaluate the potential role of CRH in the
overall symptom complexes of depression and anorexia by administering a lipo-
philic CRH antagonist. As a corollary, we also plan to evaluate the relative
role of CRH and vasopressin by acutely administering CRH and vasopressin recep-
tor antagonists active at the pituitary corticotroph cell.
To evaluate serotonergic function in depression and anorexia nervosa, we
propose to administer IV m-CPP in the lowest maximal stimulatory dose for
pituitary-adrenal activation in the evening, when the HPA is normally quies-
cent. In light of the potent stimulatory effect we have observed for 5HT upon
in vitro CRH release, m-CPP may be both an important means of evaluating the
potential role of serotonin on CRH-induced hypercortisolism and also the best
available peripherally administered stimulus for evaluation of the hypothalamic
CRH neuron in clinical populations. In light of a stimulatory 5HT effect on
vasopressin secretion, m-CPP could also function as one of several non-osmotic
stimuli to be applied at various times to further evaluate the functional
integrity of vasopressin secretion in our clinical populations. An additional
88
ZOl MH 00452-12 BP
strategy to be seriously considered Is a therapeutic trial of the 5HT antagon-
ist, metergollne, a short course to assess Its effects on parameters such as
various Indices of HP function or on glucocortlcold-medlated negative feedback.
Our clinical studies In depression and anorexia nervosa will be complemen-
ted with a full program of pre-cllnlcal investigation utilizing our capacity to
apply the techniques of molecular biology, autoradiography, Immunocytochem-
Istry, and behavioral pharmacology. For Instance, we plan a series of studies
to explore the source of neurohormones secreted Into the CSF and to determine
whether the CSF plays a physiologically relevant role for parasynaptlc Informa-
tion processing In brain. Such studies will Include a variety of leslonlng
experiments, the ICV administration of labelled peptides, and the electrical
stimulation of specific peptlde-contalnlng regions, with subsequent evaluation
of anatomic distribution and receptor function. We also plan to employ in
vivo autoradiographic techniques to study the effects of maternal deprivation
or inescapable shock on the regulation of parasynaptlc CRH and opiate recep-
tors. In our behavioral pharmacology laboratory, we plan to evaluate the
potential synergy between CRH and AVP on CNS functions such as locomotion,
feeding, and extinction of averslvely conditioned learning. Moreover, we shall
also attempt to develop animal models of anorexia nervosa utilizing either
taste aversion on mild-moderate averslve conditioning of food intake, with sub-
sequent evaluation of centrally administered AVP or CRH antagonists, or study of
underlying biologic substrates (e.g., via in situ hybridization).
Comparative Studies of Depression and Gushing 's Disease;
In further studies, we shall pursue the development of additional clini-
cal studies to facilitate the differential diagnosis of Cushlng's disease and
depression. These will Include the application of the alpha-2 blocker yohim-
bine which stimulates pituitary-adrenal function via the CNS and, hence, should
fail to provoke a response in patients with Cushlng's disease. We shall also
apply other challenges such as alprazolam, which we have shown suppresses the
hypothalamic-pitultary-adrenal axis via a central rather than a peripheral
mechanism. Similarly, we have commenced a comparative study applying the glu-
cocorticoid antagonist to both depressed and Cushlng's disease patients. Pre-
vious work has shown that RU 486 preferentially augments pituitary-adrenal
function during times when the CRH neuron Is presumably most active. An addi-
tional focus of our comparison of depression and Cushlng's disease has been
our studies of CSF neurohormones and neurotransmitters. Previous studies have
shown significant differences in the levels of CRH and other behavlorally
active peptides, with Implications for both the differential diagnosis and
pathophysiology of these two disorders.
Future Directions of Tissue Culture Studies:
We have previously conducted an extensive series of studies exploring the
regulation of hypothalamic CRH secretion utilizing an In vitro rat hypothalamic
organ culture system. We shall continue with these studies, which will be
extended to Include assessment of effects of psychotropic agents and the meas-
urement of other hypothalamic hormones such as vasopressin. A major new ini-
tiative in the area of tissue culture studies will be led by Dr. Mark Smith,
who has begun to develop tissue culture systems for brain regions other than
the hypothalamus and to assess the response of various tissues not only by stu-
dies of secretion but also by methodologies such as Northern blot analysis and
89
ZOl MH 00452-12 BP
quantitative in situ hybridization.
Future Directions of Primate Laboratory:
We have undertaken a major initiative in collaboration with
Dr. Miles Herkenham and his group to explore the source and regulation of
neurohormonal secretion into primate CSF. To accomplish this goal, we have
developed a procedure for placing an indwelling intraventricular line in
tethered primates who may be studied without chair restraint. An additional
theoretical focus will be on studies to explore whether coherent physiology
governs neurohormonal secretion into the CSF and, if so, whether such secretion
can be construed to have a physiologically relevant function.
90
i
ZOl MH 00452-12 BP
PUBLICATIONS;
Gold, P.W. , Kling, M.A. , Calabrese, J.R. , Post, R.M. , Roy, A., Nieman, L.K. ,
Cutler, G.B., Jr., Lorlaux, D.L. , and Chrousos, G.P.: Corticotropin releasing
hormone in the hypercortisolism of depression and Gushing 's disease. N. Engl.
J. Med. 316: 218-219, 1987.
Gold, P.W. , Gwirtsman, H. , Avgerinos, P.C. , Nieman, L.K. , Gallucci, W.T. , Kaye ,
W.H. , Jimerson, D. , Ebert , M. , Rittmaster, R. , Loriaux, D.L. , and Chrousos, G.P. ;
Abnormal hypothalamic pituitary-adrenal function in anorexia nervosa. N. Engl.
J. Med. 316: 219-220, 1987.
Gold, P.W., Goodwin, F.K. , and Chrousos, G.P.: Clinical and biochemical mani-
festations of depression: Relationship to the neurobiology of stress. N^.
Engl. J. Med. , invited, in press.
Luger, A., Deuster, P., Kyle, S.B., Gallucci, W.T. , Montgomery, L.C. , Gold,
P.W., Loriaux, D.L. , and Chrousos, G.P.: Acute hypothalamic-pituitary adrenal
responses to the stress of treadmill exercise: Physiologic adaptations to
physical training. N. Engl. J. Med. 316: 1309-1315, 1987.
Calabrese, J.R. , Gulledge, A.D. , Hahn, K. , Skwerer, R. G. , Schumacher, O.P. ,
Gupta, M.K. , and Gold, P.W. : Autoimmune thyroiditis in lithium-treated manic
depression. Am. J. Psychiatry 142: 714-718, 1986.
Kaye, W.H. , Gwirtsman, H.E. , George, D.T., Ebert, M. , Jimerson, D. , Tomai , T.P.
Chrousos, G.P. , and Gold, P.W. : Elevated cerebrospinal fluid levels of immuno-
reactive corticotropin releasing hormone in anorexia nervosa: Relation to
state of nutrition, adrenal function and intensity of depression. J. Clin.
Endocrinol. Metab. 64: 203-208, 1987.
May, C, Rapoport, S.I., Cutler, N.R. , Chrousos, G. P. , Tomai, T.P., and Gold,
P.W. : CSF concentrations of corticotropin releasing hormone and corticotro-
pin are reduced in patients with Alzheimer's disease. Neurology 37: 535-538,
1987.
Roy, A., Pickar, D. , Paul, S. , Doran A., Chrousos, G.P. , and Gold, P.W. : CSF
corticotropin releasing hormone in depressed patients and normal control sub-
jects. Am. J. Psychiatry 114: 614-645, 1987.
Roy, A., Pickar, D. , Linnoila, M. , Chrousos, G.P. , and Gold, P.W.: Cerebro-
spinal fluid corticotropin releasing hormone in depression: Relationship to
noradrenergic function. Psychiatry Research 20: 229-237, 1987.
Roy, A., Pickar, D. , Doran, A., Wolkowitz, O.M. , Gallucci, W.T., Chrousos, G.P.,
and Gold, P.W.: Normal ACTH and Cortisol responses to corticotropin releasing
hormone in schizophrenia. Am. J. Psychiatry 143: 1393-1397, 1986.
Avgerinos, P.C, Cutler, G.B., Sr. , Tsokos , G.C., Gold, P.W. , Feuillan, P.,
Gallucci, W.T. , Pillemer, S.R. , Loriaux, D.L. , and Chrousos, G.P. : Dissocia-
tion between Cortisol and adrenal androgen secretion in patient receiving
alternate day prednisone therapy. J. Clin. Endocrinol. Metab. 65: 24-29, 1987.
91
ZOl MH 00452-12 BP
I
Udelsman, R. , Bacher, J., Gallucci , W.T. , Gold, P.W. , Renquist, D. , Loriaux,
D.L., and Chrousos, G.P.: Hemodynamic effects of human corticotropin-releasing
factor (hCRF) in a nonhuman primate. Peptides 7: 465-471, 1986.
Oldfield, E.H., Schulte, H.M., Chrousos, G.P., Gold, P.W. , Benker, G. ,
Peterson, R.E. , Cutler, G.B., Sr. , and Loriaux, D.L.: Corticotropin releasing
factor stimulates ACTH secretion in Nelson's syndrome. J. Clin. Endocrinol.
Metab. 62: 1020-1026, 1986.
Roy, A., Gold, P.W. , Pickar, D. , Wolkowitz, CM., Chrousos, G.P. , and Paul,
S.M.: Pre- and post-dexamethasone plasma ACTH levels in depressed patients
and normal controls. J. Affective Pis. 10: 95-99, 1986.
Gold, P.W. , Kling, A. A., Khan, J., Calabrese, J.R. , Kalogeras, K. , Post, R.M. ,
Avgerinos, P.C., Loriaux, D.L., and Chrousos, G.P. : Corticotropin releasing
hormone: Relevance to normal physiology and to the pathophysiology and dif-
ferential diagnosis of hypercortisolism and adrenal insufficiency. In:
D. Nerozzi, F.K., Goodwin, and E. Costa (Eds.): Hypothalamic Dysfunction in
Neuropsychiatric Disorders. Advances in Biochemical Psychopharmacology, Vol.
42- New York, Raven Press, 1987, pp. 1329-1335.
Gold, P.W. , Kling, M.A. , Calabrese, J.R. , Kellner, C.H., Roy, A., Loriaux,
D.L. , and Chrousos, G.P.: Corticotropin releasing factor in the pathophysiol-
ogy of hypercortisolism in depres,sion. In U. Halbreich, (Ed.): Hormones and
Depression. Raven Press, New York, 1987, pp. 77-89.
Avgerinos, P.C. , Schuermeyer, T.H. , Gold, P.W. , Loriaux, D.L., Cutler, G.B.,
Sr. , and Chrousos, G.P.: Pulsatile administration of human corticotropin
releasing hormone in patients with secondary adrenal insufficiency: Restora-
tion of the normal Cortisol secretory pattern. J. Clin. Endocrinol. Metab. 62:
816-821, 1986.
Rittmaster, R.S., Cutler, G.B. , Sr. , Brandon, D. , Gold, P.W. , Loriaux, D.L. ,
and Chrousos, G.P.: The effects of endogenous vasopressin on ACTH and Cortisol
secretion in man. J. Clin. Endocrinol. Metab. 64: 371-376, 1987.
Calabrese, J.R. , Kling, M.A. , and Gold, P.W.: Special Article: Alterations
in immunocompetence during stress, bereavement and depression: Focus on the
interplay between the immunologic apparatus and neuroendocrine regulation. Am.
J. Psychiatry, in press.
Roy, A., Gallucci, W.T. , Linnoila, M. , Chrousos, G.P. , and Gold, P.W. : Neuro-
endocrine responses to CRH in bereavement: Relationship to previous history of
depression. Br. J. Psychiatry, in press.
Amsterdam, J., Bernstein, W. , Winocur, A., Kling, M.A. , and Gold, P.W. : Abnor-
mal ACTH responses to the morning administration of corticotropin releasing
hormone in patients with depression. Arch. Gen. Psychiatry, in press.
Calabrese, J.R. , Gulledge, A.D. , Hahn, K. , Skwerer, R.G. , Kotz , M. , and Gold,
P.W.: Antinuclear antibodies in psychiatric patients. In J. Clark, (Ed.):
Viruses, Immunity, and Mental Diseases. New York, Plenum Press, in press.
92
ZOl MH 00452-12 BP
Kling, M.A. , Kellner, C.H. , Post, R.M. , Cowdry, R.W. , Gardner, D.L. , Coppola,
R. , Putnam, F.W. , and Gold, P.W.: Neuroendocrine effects of limbic activation
by electrical, spontaneous, and pharmacological modes: Relevance to the
pathophysiology of affective dysregulation in psychiatric disorders. Prog.
Neuropsychopharmacol. Biol. Psychiatry, in press.
Gold, P.W. , Kling, M.A. , Calabrese, J.R. , Gallucci, W.T. , Kellner, C.H. , Post,
R.M. , Cutler, G.B., Sr., Avgerinos, P.C, Loriaux, D.L. , and Chrousos , G.P.:
Clinical applications of corticotropin releasing factor. In C.B. Nemeroff
(Ed.): Handbook of Clinical Neuroendocrinology. New York. Raven Press, in
press.
Gold, P.W. , Kling, M.A. , Khan, I., Kalogeros , K. , Calabrese, J.R. , Whitfield,
H.J., Loriaux, D.L. , and Chrousos, G.P.: Hypercortisolism and depression. In
D. Pfaff and D. Ganten (Eds.): Neuroendocrinology of Mood. Current Topics in
Neuroendocrinology, Vol. 8. Heidelberg, Springer Verlag, in press.
Chrousos, G.P. , Luger, A., Avgerinos, P.C, Kling, M.A. , Oldfield, E.H. ,
Nieman, L.K. , Schuermeyer, T.H. , Loriaux, D.L. , and Gold, P.W. : Corticotro-
pin releasing hormone: Relevance to physiology and pathophysiology. In
R. D'Agata and G.P. Chrousos (Eds.): Recent Advances in Adrenal Regulation and
Function. New York, Raven Press, 1986,' pp. 121-136.
Gold, P.W. , Kling, M.A. , Gwirtsman, H. , Brandt, H.A. , Kalogeras, K. , Loriaux,
D.L., and Chrousos, G.P.: Basic and clinical studies with CRH in depression
and anorexia nervosa. Evidence for a common pathology. In A. Schatsberg and
C.W. Nemeroff (Eds.): Hypothalamic-Pituitary-Adrenal Physiology and Pathophys-
iology. New York, Raven Press, in press.
Gold, P.W. and Rubinow, D.R. : Neuropeptide function in primary affective dis-
order: Corticotropin releasing hormone and somatostatin as model systems. In
D. Meltzer and W.E. Bunney, Jr. (Eds.): Psychopharmacology: A Generation of
Progress. New York, Raven Press, in press.
Gold, P.W. and Chrousos, G.P: Biological effects of CRH: a neurohormonal
link between stress and the pathophysiology of recurrent depressive illness. In
D.S. Palermo and J. Kagan (Eds.): Coping with Uncertainty: Biological, Behavi-
oral and Developmental Perspectives (Center for the Study of Child and Adoles-
cent Development Series). University Park, Pennsylvania State University
Press, in press.
Chrousos, G.P. , Udelsman, R. , Gold, P.W. , Kling, M.A. , Avgerinos, P.C,
Gallucci, W.T., Oldfield, E.H. , Schuermeyer, T.H. , Schulte, H.M. , Doppmann, J.,
and Loriaux, D.L.: Corticotropin releasing factor: physiological and clini-
cal implications. In E.E. Muller and R.M. Macleod (Eds.): Neuroendocrine Per-
spectives, Vol. 2. New York, Raven Press, in press.
Gold, P.W. and Kling, M.A. : Psychoneuroendocrinology. In K.L. Becker (Ed.):
Principles and Practices of Endocrinology and Metabolism. New York, Lippin-
cott Company, in press.
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Avgerinos, P.C., Gold, P.W. , Schuermeyer, T.H., Kllng, M.A. , Udelsman, R. ,
Nieman, L.K. , Loriaux, D.L., and Chrousos, G.P.: Human corticotropin releas-
ing hormone as a tool for studying the pulsatile function of the hypothalamic-
pituitary-adrenal axis. In W. Crowley (Ed.): Episodic Hormone Secretion;
Methods of Analysis and Normative Data. New York, Academic Press, in press.
Calogero, A., Gold, P.W. , Gallucci, W.T. , Loriaux, D.L. , and Chrousos, G.P.:
Effects of norepinephrine, serotonin, acetylcholine, and GABA on in vitro CRH
secretion. In G.P. Chrousos, D.L. Loriaux, and P.W. Gold, (Eds.): Mechanisms
of Physical and Emotional Stress. New York, Raven Press, in press.
Luger, A., Deuster, P., Kyle, S.B., Gallucci, W.T. , Montgomery, L.C., Loriaux,
D.L., Gold, P.W. , and Chrousos, G.P.: Acute hypothalamic-pituitary adrenal
responses to the stress of treadmill exercise: Physiological adaptations to
physical training. In G.P. Chrousos, D.L. Loriaux, and P.W. Gold, (Eds.):
Mechanisms of Physical and Emotional Stress. New York, Raven Press, in press.
Margioris, A., Grino, M. , Gold, P.W. , Chrousos, G.P.: Fetal regulation of
placental endocrine function. J. Clin. Endocrinol. Metab. , in press.
Gold, P.W. , Kling, M.A. , Whitfield, H. , Kalogeras, K. , Loriaux, D.L. , Chrousos,
G.P. : Hypothalamic-pituitary-adrenal factors in depression and anorexia ner-
vosa. In G.P. Chrousos, D.L. Loriaux, and P.W. Gold (Eds.): Mechanisms of
Physical and Emotional Stress. New York, Raven Press, in press.
Laue, L. , Kawai, S. , Udelsman, R. , Nieman, L.K., Brandon, D.D. , Gallucci,
W.T. , Loriaux, D.L. , Gold, P.W. and Chrousos, G.P.: The glucocorticoid anta-
gonist RU 38486 (RU 486): Basic studies and clinical implications with empha-
sis on the immune system. In L.M. Lichtenstein, H. Claman, A. Oronsky and R.P.
Schleimer (Eds.): Anti-inflammatory Steroid Action: Basic and Clinical
Aspects. New York, Academic Press, in press.
Chrousos, G.P. and Gold, P.W. : The hypothalamic-pituitary-adrenal axis in
emotional stress: Is there a vicious cycle? In D.S. Palermo and J. Kagan
(Eds.): Coping with Uncertainty: Biological, Behavioral and Developmental
Perspectives (Center for the Study of Child and Adolescent Development Series).
University Park, Pennsylvania State University Press, in press.
Bernardini R. , Luger, A., Gold, P.W. , Chrousos, G.P.: Cytokines stimulate
pituitary-adrenal function via activation of the CRH neuron. In F.
Petraglia (Ed.): Brain and Female Reproductive Function. London, Parthenon
Press, in press.
94
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01090-11 BP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 chartctars or less. Vth must tit on one Una batwean tha borders.)
Studies of Central Nervous ..System Functional Anatomy
PRINCIPAL INVESTIGATOR (Ust other professional personnel behw ttn Principal Investigator) (Name, title, laboratory, and instttuta attiliation)
Miles Herkenham, Ph.D., Chief, Unit on Functional Neuroanatomy, SCN, BPB, NIMH
Stafford McLean, Senior Staff Fellow, BPB, NIMH
Linda S. Brady, Staff Fellow, BPB, NIMH
John B. Glowa, Research Psychologist, BPB, NIMH
Richard B. Rothman, Guest Worker, LP-DSMHR, NIMH
Kenner C. Rice, Chemist, LN, NIDDK
COOPERATING UNITS (It any)
Neuroscience Branch, Laboratory of Pre-Clinical Pharmacology, SEH, NIMH
LAB/BRANCH
Biological Psychiatry Branch
Clinical Neuroendocrinology, Unit on Functional Neuroanatomy
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
5.2
PROFESSIONAL-
5.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues H (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed ttte space provided.)
A sensitive method for light microscopic localization of brain receptors by
in vitro autoradiography was developed previously in this laboratory. By this
method we have mapped the locations of drug and neurotransmitter receptors in
the brains of rats and other vertebrates, including primates. Immunohistochem-
istry is used to compare the locations of transmitter-specific pathways with
the locations of the relevant receptors. The non-correspondence, or "mismatch"
between the locations of receptors and transmitters in the opiate and tachy-
kinin systems, as well as others, allows hypotheses about parasynaptic circui-
try in the central nervous system. Physiological activation of vasopressin
dynorphin pathways in the posterior pituitary altered kappa opiate receptor
binding in the neurohypophysis, a site of parasynaptic communication. Current
and planned work uses similar strategies to better understand the forms of
functional intercellular communication.
95
PHS 6040 (B^. V64) '■ e^oti4...«'
^^ . ^ . ZOl MH 01090-1 I BP
Objectives :
Over the last decade a major thrust of neuroscience research is the
identification of receptors for drugs, neurotransmitters, and other
"informational substances" . in the brain. An understanding of
receptor function requires knowledge of the biochejnistry and
pharmacology as well as the neuroanatomical localization of
receptors. Receptors are identified by pharmacological criteria in
collaborative studies with Dr. R, B. Rothman, Laboratory of
Preclinical Pharmacology, NIMH. We use immunohistochemistry to
identify the neuronal circuitry that is "plugged into" these
receptors. Our finding that the organization of chemically-defined
pathways is different than the organization of receptor
distributions led to a major proposal for the existence of endocrine
actions of neurotransmitters, apart from their roles in synaptic
physiology. A major objective is to use anatomical, biochemical, and
molecular techniques to show the dynamic relationships between
informational substances and their receptors, focusing on the
corticotropin-releasing hojrmone (CRH) and opiate systems. We have
chosen these neuropeptides because of their well-characterized roles
as central regulators which elicit a coordinated and coherent series
of adaptive responses to stressful or painful stimuli.
Methods Employed:
We have successfully developed an in vitro autoradiographic
technique for visualizing dru^ and neurotransmitter receptors in
slide-mounted tissue sections. The details of this technique were
described previously (Project number ZOl MH 01090-09 LNP) .
Radiolabeled tracer substances can also be mapped
autoradiographically after systemic or intracerebroventricular
injections in order to visualize distribtition channels or target
sites for biologically active drugs. Chronic drug delivery is made
possible by surgical implantation of osmotic minipumps and (if
necessary for delivering neuropeptides which do not cross the blood
-brain barrier) ventricular cannulae. In vivo receptor
autoradiography allows competition for binding between injected
ligand and endogenous ligand, to enable us to make inferences about
the status of endogenous systems during physiological manipulations.
Dynamic activities in specified neurotransmitter systems will be
probed by the technique of in situ hybridization. Quantitative
immunohistochemistry will be used to compare genetic expression of
transmitter witht levels measured in fiber terminals. Thus we are
equipped to simultaneously monitor numerous aspects of transmitter
function during specified physiological conditions. Facilities for
precise behavioral control and measurement have been -set up to
permit study of animal models of stress and mental disorders.
Major Findings :
Several anatomical tools have been used to reach our stated
objectives. Receptor autoradiography informs us about the
relationship of neurotransmitters and their receptors only if it is
done properly, that is to say, with preservation of anatomical
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ZOI MH 01090-1 I BP
detail, with well-defined and validated binding conditions, and
with proper selection of structures and species for demonstration
of the relationships between transmitters and receptors. Likewise,
transmitter distributions are accurately localized only with proper
immunohistochemical techniques. We emphazise the quality of our
work because that is a major reason why our work has helped to
create a shift of awareness within the neuroscience community
towards an appreciation of the parasynaptic mode of intercellular
communication. Our work in the opiate and tachykinin systems and
our use of comparative anatomy has provided the bulk of the
supportive evidence for parasynaptic comunication as a plausible
explanation for the observation of mismatches between the locations
of transmitters and receptors. in brain. In addition, however, we
have argued for the generality of this phenomenon by thorough
analysis of a large variety of data taken from the literature,
resulting in major position papers published as a book chapter and,
more recently, as a 38-page Commentary in Neuroscience.
We have begun an aggressive program of functional approaches to an
understanding of parasynaptic function in brain by combining
behavioral pharmacological techniques with neuroanatomy. After
using subtype-selective binding conditions to show that opiate
receptors in the pituitary are exclusively kappa, we manipulated
levels of endogenous opioid peptides by chronically dehydrating or
salt-loading rats, resulting in massive co-release of vasopressin
and dynorphin in the neural lobe. This manipulation resulted in a
down-regulation of neural lobe opiate receptors, as measured by
binding kinetics performed on slide-mounted tissue sections. There
are no synapses in the neural lobe, and these receptors appear to
be located on nerve terminals of neurosecretory axons as well as on
pituicytes (modified astroglia) , thus qualifying as parasynaptic
receptors .
In a different approach, we are focusing on CRH and its role in the
production of a coordinated series of centrally mediated events
collectively termed the stress response. Projects are underway to
examine this response in rats, marmosets, squirrel monkeys and
rhesus monkeys. Each species offers potentially important insights
into the stress response and concommitant depression in humans.
In coordination with our objectives to obtain a greater
understanding of the neuroanatomical mechanisms of stress response,
our recently-crefeted behavioral pharmacology laboratory has
developed methods to precisely and rapidly establish dose-effect
functions comparing the effect,s. of stress-related peptides on a
variety of different types of behaviors. To date 'the efects of
CRH, arginine vasopressin, oxytocin, and angiotensin II have been
established with the goal of assessing potential interactions
between these peptides. In addition, dose-response functions for
ether-induced stress have been established in oprder to facilitate
correlations between potential endogenous mechanisms of stress and
behavioral effect. Lastly, mathematical modelling procedures have
been refined to allow the determination of the effects of very low
doses of agents.
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ZOI MH 01090-1 I BP
Significance to Biomedical Research and to the Program of the
The visualization by autoradiographic techniques of opiate receptor
locations throughout the CNS has greatly advanced our appreciation
of the richness of opiate functions in normal physiology and has led
to new insights into receptor-mediated brain processes. Receptors
that are not located at sites of synaptically released transmitter
may be mediating transmitter action of a more hormonal nature. This
parasynaptic or endocrine organization of the brain has many
implications for biological psychiatry. For example, centrally
acting drugs exert pervasive effects after diffusion through
extracellular spaces and, thus, may mimic the mode of action of
endogenous neurochemicals far more than was previously thought.
Further work examining parasynaptic mechanisms should help to
elucidate pathophysiological mechanisms in psychiatric illness and
provide a foundation for better understanding of information
transfer and consolidation in the brain. When these phenomena are
understood in better detai^, significant improvements can be
expected in the approach to treatment of neuropsychiatric disease.
Proposed Course of the Project:
With our growing appreciation that many receptors are
nonsynaptically located and, therefore, may mediate parasynaptic
intercellular communication in an endocrine fashion, we will explore
several lines of pertinent inquiry. We propose to trace the
movement of inert as well as biologically active peptides through
the cerebrospinal and extracellular fluids. Other neuroanatomical
work will use comparative and developmental approaches in several
neurotransmitter/receptor systems (we will focus on the opiate and
CRH systems) to gain insights into the rnles of organization of the
respective distributions. Correlative studies of the distributions
of relevant molecules, such as synthesizing and degradative enzymes,
and neural pathways identified by histochemical and physiological
means will serve to generate hypotheses about functional operations
within the systems. Quantitative autoradiographic techniques will
be used to show dynamic relationships between transmitters and
parasynaptic receptors. Molecular probes and in situ hybridization
will be used to show the locations and physiological regulation of
cells expressing the relevant markers. Similar studies carried out
in human tissues will allow inferences to be made about the
pathophysiology of psychiatric illnesses.
We are also investigating the ^functional roles of selected
neuropeptides as components of central neuroendocr-in®. systems . We
plan studies in which transmitter synthesis, transport and release
and receptor regulation are concurrently studied under controlled
behavioral or physiological manipulations. As a result of such
studies, it may be possible to develop animal models of psychiatric
disorders, such as depression, with known endocrine abnormalities
detected by aberrant fluctuations of peptide hormones in the
cerebrospinal fluid or individual brain loci. Our choice of
primates for many of these studies is based on the availability of
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ZOI MH 01090-! I BP
large volumes of CSF for sampling studies, on the ability to take
advantage of complex social behaviors (such as pair bonding in
marmosets and distress vocalizations in squirrel monkeys) , and the
applicability of the results to similar human conditions.
Publications:
Herkenham, M. New perspectives on the organization and evolution
of nonspecific thalamocortical projections. In Jones, E.G. and
Peters, A. A. (Eds.): Cerebral riortex. Vol. 5, Motoy Areas and
Aspects of Cortical Connectivity. New York, Plenum Press, 1986, pp.
403-445.
McLean, S., Rothman, R. B. and Herkenham, M. Autoradiographic
localization of |I and 5 opiate receptors in the forebrain of the
rat. Rrain Research 278: 49-73, 1986.
Herkenham, M., Rice, K. C . ', Jacobson, A. E. and Rothman, R. B.
Opiate receptors in rat pituitary are confined to the neural lobe
and are exclusively kappa. Brain Research 382: 36-371, 1986.
Danks, J. A, Rothman, R. B., Cascieri, M. A., Chicchi, G. G.,
Liang, T. and Herkenham, M. A comparative autoradiographic study
of the distributions of substance P and eledoisin binding sites in
rat brain. Brain Research 385: 273-281, 1986.
McLean, S., Rothman, R. B., Jacobson, A. E., Rice, K. C. and
Herkenham, M. Distribution of opiate receptor subtypes and
enkephalin and dynorphin immunoreactivity in the hippocampus of
squirrel, guinea pig, rat, and hamster. J. Comp . Neurol . 255: 4 97-
510, 1987.
Glowa, J. R. Comparisons of some behavioral effects of d-
amphetamine and toluene. Neu rot oxico logy 8: 237-248, 1987.
Barrett, J. E., Glowa, J. R., and Nader, M. A. Behavioral and
pharmacological history as determinants of drug actions. In
Emmett-Oglesby, M.E. and Goudie, A.J. (Eds.): Tolerance and
Sensitization to Psychoactive Drugs New Jersey, Humana Press,
Clifton, in press.
Glowa J. R., Bergman, J., Insel, T., and Newman, J. D. Drug
effects on primate alarm vocalizations In Newman, J.D. (Ed.) :
Physiological Control of Mammalian Vocalization. New_York, Plenum
Press, in press.
Herkenham, M. Receptor Autoradiography: Optimizing Anatomical
Resolution. In Leslie, F. (Ed.): Receptor Localization. Part A:
Ljqand Autoradiography. Receptor Biochemistry and Methodology
Series . New York, Alan R. Liss, in press.
Gerfen, C. R., Herkenham, M. and Thibault J. The neostriatal
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ZOI MH 01090-1 I BP
mosaic: II. Patch and matrix directed mesostriatal dopaminergic and
non-dopaminergic systems. J. Neuroscience, in press.
Brady, L. S. and Herkenham^ M. Dehydration reduces kappa opiate
receptor binding in the neurohypophysis of the rat^. Rrain Research,
in press.
Herkenham, M. Mismatches between neurotransmitter and receptor
localizations in brain: observations and implications.
Neuroscience, in press.
McLean, S. Axonal transport of opiate receptor subtypes. Peptides,
in press.
Rothman, R. B. and McLean, S. An examination of the opiate
receptor subtype labeled by [3H] cyclof oxy : an opiate antagonist
suitable for positron emission tomography. Biol. Psychiatry, in
press .
100
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 00180-05 BP
TITLE OF PROJECT (80 characters or less. We must tit on one line t>etween ttie borders.)
Psychobiology and Treatment of Menstrually-Related Mood Disorders
PRINCIPAL INVESTIGATOR (List ottter professional personnel t>elow the Principal Investigator.) (Name, title, latxirator/, and institute affiliation)
David R. Rubinow, M.D., Chief, Unit on Peptide Studies, BPB, NIMH
P. Schmidt, BPB, NIMH; M.C. Hoban, BPB, NIMH; K. Denicoff, BPB, NIMH; G.
Merriam, ERRB, NICHD; R. Elin, CPD, CC, NIH; H, Weingartner, BPB, NIMH; B.
Both-Ortmann, BPB, NIMH; F. Putnam, St. Elizabeth's Hospital; D. Raben, ERRB,
NICHD; N. Hall, George Washington University; E. Bou , CC, NIH; N. Rosenthal,
CPB, NIMH; R. Anderson,
COOPERATING UNITS (if any)
BPB, CPB, NIMH; ERRB, NICHD; HEB , NHLBI; CPD, CC, NIH; St. Elizabeth's Hospital;
George Washington University
UVB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Peptide Studies
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
D (a1) Minors
S (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The occurrence of dramatic changes in mood, behavior, cognition and somatic func-
tioning in some women in relation to the menstrual cycle has recently been the
focus of a great deal of public scrutiny. This project is designed to study the
psychobiology and treatment response of women with well-defined
menstrually-related mood disorders. The longitudinal screening methods developed
by this group are capable of distinguishing women with menstrually-related mood
syndromes from those who only believe that they have such a syndrome. With these
methods, we have identified the following: 1) menstrual cycle phase dependent
changes in perception and cognitive performance in patients with menstrually-
related mood disorders but not controls; 2) an increased tendency to dissociate
in patients versus controls; 3) an increased prevalence of abnormal basal and
stimulated thyroid measures in patients; 4) a higher than expected frequency
of hypoglycemic episodes following glucose tolerance testing in women with
premenstrual syndrome irrespective of menstrual cycle phase; and 5) preliminary
evidence of the efficacy of alprazolam in the treatment of premenstrual
syndrome relative to placebo or other treatments employed. The goals of this
project are to detect and accurately describe menstrually-related mood disor-
ders, explore their pathophysiology and response to pharmacological and
environmental manipulation, and to document the relationship between reproduc-
tive endocrine change and disorders of mood as a way of further investigating
the neurobiology of psychiatric illness.
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PHS 6040 (Rev. 1/84)
ZOl MH 00180-05 BP
I. Project Description
A. Objectives
This project has as its main intent the selection of subjects with care-
fully documented menstrually-related mood changes who can then undergo
psychological and biological evaluation as well as participate in double-blind,
placebo-controlled trials of several widely prescribed treatment modalities.
B. Methods Employed
1. Subjects
a. Subjects are self- and physician-referred women between the
ages of 18 and 55 who meet study criteria as described in detail in Project
#Z01 MH 00180-03 BP.
b. Normal controls for this study include women with no
complaints nor evidence of menstrually-related mood disorder and who are
without primary psychiatric illness, and women who have complaints of, but no
visual analogue scale evidence of, menstrually-related mood changes.
2 . Procedures
Phase 1 . An extensive screening phase that has been described in
detail in Project ZOl MH 00180-02 BP.
Phase 2. This is an intensive psychobiological evaluation phase
for patients meeting entry criteria for the study.
a. Patients are given a thorough physical and laboratory
examination in order to rule out the presence of unknown medical illness.
b. Ongoing studies of longitudinally obtained basal and
stimulated hormonal levels have been previously described in Project #Z01 MH
00180-03 BP. In addition, we are performing the following studies:
1) Psychometrics : Previously described cognitive and mood
selfrating batteries are currently being supplemented by evaluation of the
frequency of and response to life events over the course of the menstrual cycle
(M.C. Hoban, Dr. P. Schmidt) , comparison of performance on the Raven
Progressive Matrices Tests during the symptomatic and symptom-free phases of
the menstrual cycle, determination of menstrual cycle phase-dependent
associations, and longitudinal self-evaluation of mood state satisfaction (Drs.
H. Weingartner and F. Putnam) .
2) Diet: In collaboration with Dr. N. Rosenthal and E.
Bou, we are evaluating dietary patterns over the course of the menstrual cycle
in order to test hypotheses about altered carbohydrate or salt intake in men-
strually-related mood disorders.
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ZOl MH 00180-05 BP
3) Glucose tolerance test: Because of reports of altered
carbohydrate tolerance in premenstrual syndrome, we are performing glucose
tolerance tests in patients with menstrually-related mood disorders in both
cycle phases (Dr. K. Denicoff) .
4) Corticotropin releasing hormone (CRH) stimulation tests:
With Dr. D. Raben, we are performing CRH stimulation tests during the
follicular and luteal phase in patients and controls to see if there is a
biological marker for the reported altered stress sensitivity in women with
premenstrual syndrome.
5) Clonidine stimulation tests: Because of the
hypothesized role of endogenous opiate withdrawal in the precipitation of
menstrually-related mood symptoms and a preliminary report of the efficacy of
clonidine in the treatment of premenstrual syndrome, we have performed
clonidine infusions during the symptomatic and asymptomatic states in order to
assess menstrual state-related symptomatic and endocrine response.
6) Immune system: In light of reports of both altered
T-cell function as a function of estrogen levels and abnormal allergic
responses in women with premenstrual syndrome, we are investigating T-cell
function in relation to menstrual cycle phase in women with premenstrual
syndrome and normal controls, in collaboration with Dr. N. Hall.
Phase 3. This is a multi-modality treatment phase for patients
who have completed Phase 2. Double-blind, placebo-controlled crossover
evaluation of progesterone, medroxyprogesterone acetate, pyridoxine,
carbamazepine, alprazolam, and nalmefene are currently being conducted.
Rationales for the selection of these particular compounds have been previously
described. Additionally, protocols have been submitted for the evaluation of
the anti-progesterone agent RU 486 and "square wave" progesterone withdrawal in
the treatment of premenstrual syndrome. It is believed that these protocols
will provide critical information regarding the role of the reproductive
endocrine profile during the luteal phase in the production of mood changes
that occur during the latter phase of the menstrual cycle.
C. Findings
A variety of menstrual cycle phase-dependent psychological and
cognitive changes have been identified in women with premenstrual syndrome
compared with controls. Thus, women with premenstrual syndrome tend to report
a greater number of negative life events and a smaller number of positive life
events during the luteal phase, and further, experience the same events as more
negative during the luteal phase. Baseline measures of the tendency to
dissociate are higher in women with premenstrual syndrome than controls, and
are higher in the luteal than in the follicular phase. Cognitive performance,
as measured by the Raven Progressive Matrices Text, is more impaired in
premenstrual syndrome women during the luteal phase while control women tend to
show more impaired performance during the first test administration,
irrespective of menstrual cycle phase. State complacency scale measures of
affective change reveal that change along dimensions of mood state stability,
sense of control, and satisfaction with mood state can occur independently in
103
ZOl MH 00180-05 BP
women undergoing mood state switches. These data, along with data from our
previously described endocrine studies, are consistent with the hypothesis that
premenstrual syndrome represents a biologically facilitated state change, with
the altered cognitive and perceptual characteristics a product of the altered
state, rather than the normal reproductive endocrine changes.
Efforts to characterize the biology of premenstrual syndrome have included
the performance of TRH and CRH infusions, as well as performance of glucose
tolerance tests and measurement of progesterone metabolites. We have extended
our earlier demonstration of a high prevalence of abnormal TSH responses to TRH
in patients with premenstrual syndrome, but have also demonstrated that,
despite reports to the contrary, premenstrual syndrome is not simply
hypothyroidism and the occurrence of thyroid autoantibodies in women with
premenstrual syndrome is a rare rather than a common event. Glucose tolerance
testing revealed a high frequency of hypoglycemic episodes in women with
premenstrual syndrome irrespective of menstrual cycle phase. However, the
symptoms experienced were typical of hypoglycemic attacks and were atypical of
the symptoms ordinarily experienced during the luteal phase. CRH tests have
been performed in both menstrual cycle phases in eight patients and eight
controls to date. The results of this testing are not currently available. In
collaboration with Dr. R. Anderson, several progesterone metabolites have been
identified in the plasma of women with premenstrual syndrome, but not in
controls. We are currently pursuing this finding with larger groups of
patients and controls.
Studies of alterations in immune function and osmoregulatory hormones over
the course of the menstrual cycle in patients and controls have been completed,
although the final results have not been obtained at present. Initial
experience with the 11 patients participating in a double-blind,
placebo-controlled trial of alprazolam are encouraging and superior to those
observed in similar studies of vitamin B6, progesterone, and the opiate
antagonist nalmefene. Finally, we observed that luteal phase-related increases
in appetite are observed in both patients and controls, although the increase
in appetite in patients is threefold greater than that observed in controls.
Additionally, the changes in appetite observed in patients are highly
correlated with changes in mood, while the same is not observed in the
controls .
D. Proposed Course of Project
With a group of well-defined patients, we hope to explore the natural
course of menstrually-related mood disorders as well as their phenomenology and
biological correlates in relation to treatment response. Early endocrine
findings will be pursued and specific hypotheses regarding the etiology of
premenstrual syndrome (e.g., endorphin addition/withdrawal, carbohydrate
intolerance, electrolyte dysregulation) will be tested with endocrine challenge
studies. Treatment protocols will be completed, and evaluation of putative
therapeutic agents will be undertaken. Cognitive testing will be continued
with the addition of distractors during testing. We will explore the state-
and stress-related characteristics of premenstrual syndrome through
implementation of state-dependent learning paradigms and continued performance
of CRH stimulation testings. Protocols for the administration of RU 486 and
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ZOl MH 00180-05 BP
"square wave" progesterone withdrawal will help define, better than any
currently available evidence, the nature of the relationship between luteal
phase endocrine events and mood changes. These protocols have great
explanatory potential because they effectively blind the patient to her
position in the menstrual cycle, a methodological problem that was previously
uncorrectable. We wish to expand our investigation of the effects of menstrual
phase on mood to include patients hospitalized at the Clinical Center with
major affective disorder, panic anxiety disorder, and anorexia-bulimia, as well
as patients with hereditary angioedema. Our early experience with a number of
women with these disorders suggests that symptoms may be exacerbated or may
cluster during the premenstruum; these clinical impressions require prospective
confirmation. Finally, studies of women with post-partum and menopausal
depression are being designed.
E. Significance to Biomedical Research and the Program of the Institute
Despite the current lack of clear understanding of the nature of the
relationship between mood disorders and the menstrual cycle, numerous studies
of this phenomenon suggest its importance to the psychiatrist on many levels:
practically (as a problem about which the psychiatrist may be called to consult
or as a factor which may influence the course of the treatment of patients) ;
heuristically (as a model for learning about state changes, a process of clear
relevance to studies of other mood state disorders such as manic-depressive
illness or panic anxiety disorder) ; and conceptually (as a potential means for
providing biological-phenomenological isomorphs and further understanding the
role of entrainment in episodic or cyclic psychiatric disorders) .
Menstrually-related mood disorders in their own right are important to better
understand, if only for the fact that there are large numbers of women who feel
that they suffer from such syndromes and seek treatments that are unproved and
potentially dangerous. In addition, it would appear that menstrual cycle phase
is a variable that has been all too frequently ignored in studies of
traditional psychiatric and medical illnesses. It is our belief, therefore,
that this project will provide information that will be of immediate clinical
relevance and that will further our understanding of the complex relationship
between endocrine system activity and mood.
PUBLICATIONS
Roy-Byrne, P.P., Rubinow, D.R., and Linnoila, M. : Relation between plasma
prolactin and plasma homovanillic in normal subjects. Neuropsychobiology 16:
85-87, 1986.
Roy-Byrne, P.P., Rubinow, D.R., Gwirtsman, H. , Hoban, M.C., and Grover, G.N.:
Cortisol response to dexamethasone in women with premenstrual syndrome.
Neuropsychobiology 16: 61-63, 1986.
Roy-Byrne, P.P., Hoban, M.C., and Rubinow, D.R.: The relationship of
menstrually related mood disorders to psychiatric disorders. Clin. Obstet
Gynecol. 30: 386-395, 1987.
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ZOl MH 00180-05 BP
Rubinow, D.R., Hoban, M.C., and Grover, G.N. : Menstrually-related mood
disorders. In Nerozzi, D., Goodwin, F.K., and Fragese, G. (Eds.):
Hypothalamic Dysfunction in Neuropsychiatric Disorders. New York, Raven Press,
1987, pp. 335-346.
Roy-Byrne, P.P., Rubinow, D.R., Hoban, M.C., Grover, G.N., and Blank, D. : TSH
and prolactin responses to TRH in patients with premenstrual syndrome. Am. J.
Psychiatry 144: 480-484, 1987.
Rubinow, D.R. and Schmidt, P.J.: Mood disorders and the menstrual cycle. J.
Reprod. Med. 32: 389-394, 1987.
Frankel, B.L. and Rubinow, D.R. : The premenstrual syndromes. In Howells, J.G.
(Ed.): Modern Perspectives in Psychiatry. New York, Brunner/Mazel , in press.
Rubinow, D.R., Hoban, M.C., and Grover, G.N.: Premenstrual syndromes - medical
and psychiatric perspectives. In Keye, W.R. (Ed.): The Premenstrual
Syndromes. New York, Grune and Stratton, in press.
Rubinow, D.R. : PMS : Practical and ethical aspects of pharmacotherapeutic
evaluation. In Ginsberg, B. and Frank-Carter, B. (Eds.): Legal and Ethical
Implications of the Biobehavioral Sciences. New York, Plenum Press, in press.
Rubinow, D.R., Hoban, M.C. , Grover, G. , Roy-Byrne, P.P., and DeJong, J.: The
relationship between menstrually-related mood changes and major depressive
disorder. In Shagass, C, Josiassen, R.C., Bridger, W.H., Weiss, K.J., Stoff,
D., and Simpson, G.M. (Eds.): Biological Psychiatry, 1985 (Developments in
Psychiatry, Vol. 7). Amsterdam, Elsevier, in press.
106
DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00181-04 BP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must' fit or\ or)e line between the borders.)
Hormonal Studies of Affective Disorder
PRINCIPAL INVESTIGATOR (List other protessional personr^el betow the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
David R, Rubinow, M.D., Chief, Unit on Peptide Studies, BPB, NIMH
P. Sunderland, M.D., LCS, NIMH; M. Linnoila, M.D., LCS, NIAAA; M. ; S. Bracha, M.D
M, SMRC; S.R.B. Weiss, Ph.D., BPB, NIMH; W. Kaye , M.D., Univ. of Pittsburgh; J.
Crawley, Ph.D., NSB, NIMH; K. Denicoff, M.D., BPB, NIMH; P. Hauser, M.D., BPB,
NIMH; J. Hill, Ph.D., NSB, NIMH
COOPERATING UNITS (if any)
BPB, LCS, NSB, NIMH; LCS, NIAAA; NCI; M, SMRC; SUNY, New York; University of
Pittsburgh
UB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Peptide Studies
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
d (a) Human subjects
D (a1) Minors
1] (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies of somatostatin and Cortisol in relation to affective and other
neuropsychiatric disorders have continued. Additionally, further studies of the
mechanisms of inter leukin- 2 -induced neurotoxicity have been performed in rodents.
A) Somatostatin - CSF somatostatin has been measured in an expanded group of neuro
psychiatric patients including patients with multiple sclerosis and neurological
controls, patients with alcoholism, pathological gambling, and Huntington's demen
tia. Significant correlations were observed between somatostatin and performance
on the Wechsler Memory Scale, supporting our earlier observations (and perhaps th^
clinical relevance) of decreased somatostatin levels in patients with neuropsychi
ric disorders characterized by cognitive impairment. Studies of the regional con
centrations of brain somatostatin in post-mortem schizophrenics, suicide victims,
and accident victims have been completed with the sample code not yet broken.
B) Cortisol - A salivary Cortisol method, developed and validated in our
laboratory, has been used to longitudinally follow patients with affective and
anxiety disorders. This method shows great promise for studies of ambulatory
patients with affective disorder.
B) Interleukin-2 - No changes in blood-brain-barrier permeability were observed i|i
rats treated with interleukin-2, irrespective of the size of the tracer employed
These findings contradict previous reports and leave unresolved the mechanism of
lymphokine-induced neurotoxicity .
107
PHS 6040 (Rev, 1/84)
GPO BI4-»lt
ZOl MH 00181-04 BP
I. Project Description
A. Objectives
The goal of this project is to study neuroendocrine and immune soluble
products in patients with neuropsychiatric disorders in order to expand our
understanding of the mechanisms and significance of reported abnormalities in
somatostatin, Cortisol, and immune system activity in affective illness.
B. Methods Employed
1. Subjects
a. Subjects include inpatients on NIMH clinical units meeting
criteria for major depressive disorder, Alzheimer's disease, Huntington's
dementia, pathological gambling, anorexia nervosa, and bulimia as well as
patients with substance abuse disorder (alcoholism). Gushing 's syndrome, and
multiple sclerosis.
b. Normal controls are volunteers selected from the normal
volunteer program at the NIH. Neurological controls were employed for studies
of patients with multiple sclerosis.
2. Procedures
Lumbar punctures are performed to obtain CSF samples for somato-
statin, CRF, and other related CNS peptides/neurotransmitters. Regional
analysis of brain somatostatin is performed in post-mortem human brain samples.
Brain slices in animals are punched and assayed for somatostatin content.
Whole brains are dissected and radioactivity measured in animal lymphokine
studies.
II. Findings
A. Somatostatin
Preliminary evidence obtained in collaboration with Dr. P. Hauser
suggests both a decrease in CSF somatostatin in patients with multiple
sclerosis relative to neurologic controls, as well as an increase in CSF
somatostatin in patients with multiple sclerosis during clinical remission. In
collaboration with Dr. M. Linnoila, we observed no syndromal alterations in CSF
somatostatin in patients with alcoholism, pathological gambling, or
Huntington's dementia. However, we did observe a highly significant
correlation between CSF somatostatin and performance on the Weschler Memory
Scale in patients with Huntington's dementia, a finding previously observed by
us in Alzheimer's patients, and one that further suggests the relevance of
diminished somatostatin levels in patients with neuropsychiatric disorders
characterized by cognitive impairment. In collaboration with Dr. W. Kaye, we
have observed normal CSF somatostatin concentrations in patients with anorexia
nervosa (at low weight and after weight recovery) and in bulimic women.
However, when normal weight bulimics stop binging, they display a modest but
significant increase in CSF somatostatin. CSF somatostatin was not related to
plasma growth hormone levels, but did show relationships to the
108
ZOl MH 00181-04 BP
hypothalamic-pituitary-adrenal (HPA) axis. Thus, as we previously
demonstrated, a significant positive relationship was observed in healthy
controls between CSF somatostatin and CSF CRH. Additionally, in underweight
anorectics, CSF somatostatin was negatively related to both 24-hour urinary
free Cortisol and plasma Cortisol levels after dexamethasone. The differences
in the relationship between somatostatin and the HPA axis in anorectics and
bulimics may constitute or reflect pathophysiological distinctions between
these disorders.
B. Cortisol
We have demonstrated the practical utility of the salivary Cortisol
measure in the evaluation of the function of the hypothalamic-pituitary-adrenal
axis. Appropriate increases and decreases in salivary Cortisol were observed
during CRH stimulation tests and dexamethasone suppression tests, respectively,
and circadian alterations were also observed in both normal volunteers and
patients with affective illness. Longitudinal studies of several patients with
affective illness prior to and during treatment with carbamazepine revealed no
evidence of carbamazepine-induced Cortisol hypersecretion as indicated by
elevated salivary Cortisol (and hence free Cortisol) levels.
C. Interleukin-2
In collaboration with Drs. K. Denicoff and J. Crawley, we observed no
alterations in blood-brain-barrier permeability in animals treated with
interleukin-2 (IL-2) demonstrating behavioral toxicity. Both high and low
molecular weight radio tracers (albumin and alpha-amino isobutyric acid) were
employed, but no increased brain accumulation was seen with either tracer at
any time-point during the week of IL-2 administration. These findings
contradict those previously reported in mice. Finally, in collaboration with
J. Hill, we were unable to demonstrate the induction of brain IL-2 receptors
following IL-2 treatment in rodents. Thus, the mechanism of the IL-2-induced
neuropsychiatric toxicity that we observed in humans remains unresolved.
III. Proposed Course of Project
We hope to: 1) examine the effects of electrical kindling and learned
helplessness on brain somatostatin and m-RNA in rodents; 2) develop assay
techniques to permit evaluation of the contribution of somatostatin fragments
to the total syndromal alterations in CSF somatostatin observed; 3) employ
salivary Cortisol measures to characterize the mood state switches that occur
in patients with affective illness in ambulatory settings; 4) explore
alternative mechanisms for enabling somatostatin to pass the
blood-brain-barrier so as to be able to test its efficacy as a treatment for
cognitive disturbances in patients with Alzheimer's disease and affective
illness; and 5) continue investigations of the neuroimmunoendocrine concomi-
tants and inducers of impaired cognitive performance.
IV, Significance to Biomedical Research and the Program of the Institute
Depression-related dysregulation of somatostatin and Cortisol may provide
a window into the central neurochemical lesions responsible for depression.
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ZOl MH 00181-04 BP
Further, specific behavioral or physiological disturbances (e.g., cognitive
impairment or Cortisol dysregulation) may be products of abnormal
neuroendocrine activity. It may prove to be the case that depression-related
reductions in somatostatin are mechanistically relevant to depression-related
disturbances in hypothalamic-pituitary-adrenal activity, the most commonly
reported biological abnormality in depression. Determination of the mechanisms
of the profound behavioral and cognition altering effects of interleukin-2
would fill a major gap in our knowledge of the ways in which the immune system
can regulate central nervous system activity. Further study may not only en-
hance our knowledge of the neurobiology of depression, but may, as well, more
generally inform us about the relationship between hormones and human behavior.
PUBLICATIONS
Rubinow, D.R., Post, R.M. , Davis, C.L. and Doran, A.R.: Somatostatin and GHRH:
Mood and behavioral regulation. In Nerozzi, D., Goodwin, F.K. and Fragese, G.
(Eds.): Hypothalamic Dysfunction in Neuropsychiatric Disorders. New York,
Raven Press, 1987, pp. 137-152.
Rubinow, D.R,, Post, R.M. , Davis, C.L. and Doran, A.R.: Somatostatin and de-
pression. In Reichlin, S. (Ed.): Somatostatin. New York, Plenum Press, 1987,
pp. 183-192.
Rubinow, D.R.: Somatostatin in depressive disorders. Actualites
Psychiatriques , in press.
Kahn, J. P., Rubinow, D.R., Davis, C.L., Kling, M, , and Post, R.M. : Salivary
Cortisol: A practical method for evaluation of adrenal function. Biol.
Psychiatry, in press.
110
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00182-04 BE
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line Ixtween the borders.)
Behavioral Medicine
PRINCIPAL INVESTIGATOR (Ljst other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
David R. Rubinow, M.D., Chief, Unit on Peptide Studies, BPB, NIMH
Dr. Kirk Denicoff, Biological Psychiatry Branch, NIMH
Dr. David Pickar, Clinical Neuroscience Branch, NIMH
Dr. Steven Rosenberg, Surgery Branch, NCI
Dr. Pirn Brouwers, Georgetown University, Washington, D.C.
Dr. Clifford Lane, Laboratory of Immunoregulation, NIAID
COOPERATING UNITS (If any)
BPB, NSB, LPP, CPB, NIMH; SB, PB, FCRF, NCI; LIR, LCI, NIAID; CCM, CC; OD , CE,
A&R, NIADDKD; MD, CB, NHLBI; St. Michael's Hosp. , Toronto; Georgetown Univ.
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Peptide Studies
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
2
PROFESSIONAL:
1
CHECK APPROPRIATE BOX(ES)
[E (a) Human subjects
D (a1) Minors
[J (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Eight protocols are currently -active and conducted out of the Consultation-Liaison
Service-based behavioral medicine research program. These protocols examine the
phenomenology and biological correlates of illness or treatment-induced mood, behav-
ioral, and cognitive changes. The protocols address such areas as: a) the effects
of previous psychiatric history on the psychiatric morbidity associated with certain
diseases and their treatment; b) the psychiatric phenomenology of certain diseases
and their treatment; c) the treatment response characteristics of psychiatric disor-
ders associated with diseases or their treatment; d) biochemical factors that may
serve as predictive diagnostic markers for illness or for treatment-associated
mood/behavioral or cognitive syndromes; e) the effects of mood state alterations or
immunologic function. Significant findings to date include demonstration of the
following: 1) significant neuropsychological cognitive impairment in patients with
AIDS compared with seropositive patients, chronically medically ill patients, or
controls; 2) significant cognitive and affective deterioration following removal of
thyroid hormone replacement, with no differential effects of T and T on mood and
cognition; 3) absence of evidence of acute immune changes following hypnotically-in
duced affective states; 4) a significant effect of dose on interleukin-2-induced
neuropsychiatric toxicity; 5) preliminary confirmation of alpha-delta intrusion in
patients with fibromyalgia; 6) preliminary evidence of exaggerated response to
novelty in patients with chest pain and normal coronary arteries.
Ill
PHS 6040 (Rev. 1/84)
SPO BI4-SI«
ZOl MH 00182-04 BP
Other Professional Personnel (continued)
Dr. Jacob Robins, Clinical Endocrinology Branch, NIADDKD
Dr. Stanley Pillemer, Arthritis & Rheumatism Branch, NIADDKD
Dr. Richard Cannon, Cardiology Branch, NHLBI
Dr. Russell Joffe, St. Michael's Hospital, Toronto, Canada
Dr. Nicholas Hall, Georgetown University, Washington, D.C.
Dr. Peter Schmidt, Biological Psychiatry Branch, NIMH
Dr. Daniel Longo, Frederick Cancer Research Facility, NCI
112
ZOl MH 00182-04 BP
I. Project Description
A. Objectives
This project has as its main intent the identification of mood and cog-
nitive symptoms that appear in the context of specific medical illnesses and their
treatment, determination of the relationship between these symptoms and both the
primary medical disorder and prior psychiatric history, and utilization of the
occurrence of these symptoms in a medical context as models for the occurrence of
similar symptoms in a primarily psychiatric context.
Protocols
Active:
1) Neuropsychiatric dysfunction in patients with Acquired Immune
Deficiency Syndrome (AIDS) .
2) The effect of thyroid replacement and withdrawal on cognition and mood in
patients with carcinoma of the thyroid.
3) Assessment of neuropsychiatric concomitants of metoclopramide
administration .
4) The effect of hypnotically-induced affect on immune function in normal
subjects.
5) Neuropsychiatric effects of alternate day steroid administration in
patients with systemic lupus erythematosis.
6) The cognitive and behavioral effects of interleukin-2/lymphokine activated
killer cell therapy.
7) Mood and cognitive toxicity of gamma-Interferon administration in
seropositive patients.
8) Fibromyalgia.
Completed:
1) A prospective study of the behavioral, cognitive and neurochemical effects
of chronic, systemically-administered corticosteroids.
2) The correlation of hypnotizability, dissociation, and absorption in normal
subjects.
Planned:
1) Conditioned immunosuppression and immunoenhancement in cancer patients.
2) Development of endocrine correlates of the "intensive care unit syndrome".
B. Methods Employed
1. Subjects
a. Subjects are NIH patients who are referred for participation in
these protocols by collaborators from the Institute responsible for the primary
care and treatment of these patients.
113
ZOl MH 00182-04 BP
b. Controls for the individual studies are selected in a way that
allows for stratification of populations with respect to the relevant variables
under study.
2 . Procedures
a. Psychiatric Diagnostic Evaluation
The primary methodology employed is that of evaluating the psychiat-
ric history of all subjects and their families utilizing a semistructured psychiat-
ric interview, the Schedule for Affective Disorders and Schizophrenia (SADS-L) ,
which provides information from which an RDC diagnosis can be made.
b. Longitudinal Evaluation
Most studies utilize a "self as own control" design employing longi-
tudinal assessment of mood ratings, physical symptoms, and cognitive performance.
Detailed description of methodologies employed can be found in Project #Z01 ^4H
00182-02 BP.
c . Laboratory Assessment
Urine and/or blood samples are collected in order to permit evalua-
tion of those biological substances believed to be related to the development of
affective or cognitive disturbances.
3. Findings
1) We have replicated earlier findings of cognitive impairment in AIDS
patients without evidence of central nervous system opportunistic infection.
Similar impairment was not observed in seropositive patients, controls, or patients
with chronic active hepatitis (a chronic medically ill control group)
(collaborator: Dr. C. Lane and P. Brouwers) .
2) In addition to providing the first description of neuropsychiatric
toxicity of interleukin-2 (IL-2) (during systemic administration) , we have observed
the following: increased incidence of toxic effects with high dose IL-2; absence of
a relationship between prior personal or family psychiatric history and the likeli-
hood of developing IL-2-induced neuropsychiatric changes; a clear latency between
initiation of treatment and development of neuropsychiatric side effects, suggest-
ing that a secondary rather than a direct effect of IL-2 is being observed;
significant decreases in ACTH but not Cortisol in association with IL-2
administration (collaborators: Drs. K. Denicoff and S. Rosenberg).
3) Preliminary analysis of a comprehensive immune profile performed on
blood samples drawn prior to, during, and subsequent to a hypnotically-induced
affective state change in highly hypnotizable patients under four different
conditions has revealed no obvious changes in immune variables, including natural
killer cell activity, response to mitogen stimulation, and IL-2 concentrations.
These data will help to interpret immune alterations reported to occur during major
depressive episodes (collaborators: Drs. N. Hall and P. Schmidt) .
114
ZOl MH 00182-04 BP
4) No differential effects of T and T were observed on mood or
cognition in thyroidectomized patients post carcinoma of the thyroid. However,
significant differences were observed in mood and cognition between either
treatment and the absence of thyroid hormone replacement; specifically, marked
increases in anxiety and depression, along with decreased cognitive functioning,
were observed soon after removal of thyroid hormone. These data provide the first
prospective, longitudinal demonstration of the effects of thyroid hormone on mood
and cognition in nonpsychiatric patients. Additionally, a high prevalence of past
history of affective disorder was observed in the group who were most severely
symptomatic during thyroid hormone withdrawal (collaborators: Drs. J. Robins and K.
Denicoff ) .
5) Lactate infusions performed in a small group of normal volunteers to
date suggest that the panic attacks that we observed during lactate infusion in
patients with chest pain and normal coronary arteries may represent an unusual
response to novelty rather than a special vulnerability to lactate. We are
continuing these studies in order to further define the phenomenological and
biochemical similarity between patients with chest pain and normal coronary
arteries and patients with major anxiety disorders (collaborators: Dr. R. Joffe, P.
Schmidt, and R. Cannon) .
6) Early work with patients with fibromyalgia has confirmed the presence
of the alpha wave intrusion during sleep. Attempts to demonstrate these findings
in a larger group of patients with fibromyalgia and compare them with arthritic
controls and normal volunteers will be undertaken in order to determine whether the
symptoms and EEG abnormality in fibromyalgia are specific to that syndrome or
merely secondary to disturbed sleep (collaborators: Dr. S. Pillemer and K.
Denicoff) .
D. Proposed Course of Project
The studies noted above will be continued until adequate numbers of sub-
jects are obtained. Attempts to identify neuroendocrine correlates of or biologic
mechanisms for the lymphokine- induced neuropsychiatric disturbances have been
initiated. Studies investigating the immunoregulatory potential of
hypnotically-induced mood states and of the phenomenologic, somnographic , and
treatment response characteristics of patients with fibromyalgia have similarly
been initiated. Completion of these descriptive studies should permit design of
focused investigations of the neurobiology of specific mood, behavioral, and
cognitive disorders. Finally, studies are currently being undertaken to
investigate the psychobiology of pathological grief in spouses of cancer patients,
describe the prevalence of major anxiety disorders in patients with Graves '
disease, describe the development and endocrine concomitance of the "intensive care
unit syndrome", and condition immunosuppression and immunoenhancement in cancer
patients.
E. Significance to Biomedical Research and the Program of the Institute
The studies in this project are hypothesis-generating as well as hypo-
thesis-testing. Thus, they should not only help to expand the behavioral phenom-
enology of many medical disorders, but should, as well, suggest optimal studies for
the application of modern neuroscientif ic techniques to disorders of regulation of
115
ZOl MH 00182-04 BP
mood and cognition. Detection of conditioned immunosuppression in patients should
have profound effects on both our understanding and treatment of many medical
disorders. The utilization of medical disorders as models for the development of
mood and cognitive disturbances in the context of biological dysregulation should
clarify the meaning of these biological alterations already observed in psychiatric
disorders.
PUBLICATIONS
Pincus, H. and Rubinow, D.R.: Research at the interface of medicine and
psychiatry. In Pincus, H. (Ed.): The Integration of Neuroscience and Psychiatry.
Washington, D.C., American Psychiatric Press, Inc., 1985, pp. 77-94.
Rubinow, D.R. and Joffe, R.T. : Psychiatric and psychosocial aspects of AIDS. In
Broder, S. (Ed.): AIDS: Modern Concepts and Therapeutic Challenges. New York,
Marcell Dekker, Inc., 1986, pp. 123-134.
Joffe, R.T., Rubinow, D.R., Denicoff, K.D., Maher, M. , and Sindelar, W.F.:
Depression and carcinoma of the pancreas. Gen. Hosp. Psychiatry 8: 241-245, 1986.
Joffe, R.T., Rubinow, D.R., Squillace, K. , Lane, C.H., Duncan, C. , and Fauci, A.:
Neuropsychiatric manifestations of the acquired immune deficiency syndrome (AIDS) .
Psychopharmacol . Bull. 22: 684-688, 1986.
Rubinow, D.R., Peck, G.L., Squillace, K.M., and Gant, G.T.: Reduced anxiety and
depression in cystic acne patients after successful treatment with oral
isotretinion. J. Am. Acad. Dermatol., in press.
Denicoff, K.D., Rubinow, D.R., Papa, M.Z., Simpson, C. , Seipp, C.A., Lotze, M.T.,
Chang, A.E., Rosenstein, D., and Rosenberg, S.A.: The neuropsychiatric effects of
interleukin-2/lympokine-activated killer cell treatment. Ann. Intern. Med. , in
press.
Joffe, R.T. and Rubinow, D.R.: Are there neuropsychiatric features of AIDS?
Harvard Mental Health Letter, in press.
Rubinow, D.R., Joffe, R.T., Brouwers, P., Squillace, K. , Lane, C, and Mirsky, A.:
Neuropsychiatric impairment in patients with AIDS. Adv. Biochem. Psychopharmacol.,
in press.
Rubinow, D.R. , Berrettini, C.H., Brouwers, P., and Lane, H.C.: Neuropsychiatric
consequences of AIDS. Ann. Neurol. , in press.
Loewenstein, R.J. and Rubinow, D.R. : Psychiatric aspects of AIDS: The organic
mental syndromes. In Pincus, H. (Ed.): The Integration of Neuroscience and
Psychiatry. Washington, D.C., American Psychiatric Press, in press.
116
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00147-12 BP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the ttorders.) „
oehavioral, and.
Physiological Effects of Brain Peptides and Other Psycnoac"tive Compounds
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute aflillation)
Agu Pert, Ph.D., Chief, Unit on Behavioral Pharmacology, BPB , NIMH;
Lorna Estal, visiting Fellow, BPB, NIMH; R.J. Weber, Sr. Staff Fellow, SNB,
NINCDS; K.C. Rice, LC, NIADDK; B. DeCosta, Visiting Fellow, LC, NIDDKD;
A. A. Hagen, American U.; T. Seeger, Pfizer Central Research, Groton, Ct.; H.D.
Everist, Guest Worker, BPB, NIMH; C.C. Chiueh, Staff Fellow, LCM, NIMH;
S.R.B. Weiss, Staff Fellow, BPB, NIMH; C.B. Pert, CNB, NIMH; P. Glue, LCS , NIAAA;
D. Nutt, LCS, NIAAA
COOPERATING UNITS (it any)
BPB, CNB, LCM, NIMH; LC , NIADDK; LC , NIDDKD; LCS, NIAAA; SNB, NINCDS; Pfizer Cen-
tral Research, Groton, Ct. , American University, Washington, D.C.
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Behavioral Pharmacology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.5
PROFESSIONAL:
1.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues @ (c) Neither
n (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Phencyclidine produces behavioral excitation through a dopamine independent mech-
anism in the nucleus accumbens, probably by blocking the excitatory amino acid
NMDA receptor. Motor asymmetries produced by phencyclidine are caused by activa-
tion of pathways projecting caudally from the substantia nigra. Opiate-induced
suppression of immune function appears to be mediated, at least in part, through
the periaqueductal gray matter. The periaqueductal gray matter in general seems
to be an important region of the brain for regulating the immune function.
Nicotine produces behavioral excitation by activating nigrostriatal and mesolimbic
dopamine pathways. Locomotor activation produced by amphetamine and cocaine in
the nucleus accumbens is mediated through activation of a variety of efferent
pathways projecting to the striatal fundus, ventral pallidum, zona reticulata,
and the medial and lateral olivary complex. Kappa opiate receptors modulate loco-
motor behaviors as well as a variety of homeostatic mechanisms. The depressant
actions of kappa opiate agonists are not produced through the opiate receptor but
appear to be nonspecific.
117
PHS 6040 (Rev. 1/84) CPOSi4-«it
ZOl MH 000147-12 BP
I. Project Description
A. Objectives
1 . Nicotinic Modulation of Nigrostriatal and Mesolimbic Dopamine
Functions
We have previously reported the presence of stereospecific, saturable,
and reversible binding of [^H] nicotine to rat brain sections, which is of high
affinity and is selectively displaced by nicotinic agonists including acetylcholine.
Autoradiographic analyses revealed heavy nicotine labeling in a variety of thalamic
nuclei as well as the cortex and substantia nigra. A substantial concentration of
nicotine receptors was also found in the nucleus accumbens and striatum. Subse-
quent lesion studies revealed that nicotine receptors are localized on both the
perikarya as well as terminals of the mesolimbic and nigrostriatal dopamine neurons.
The purpose of these studies was to determine if any of the behavioral effects of
nicotine could be mediated through either of these dopamine pathways.
The rotational model was utilized in order to assess the effects of nicotine
on nigrostriatal dopaminergic function. Rats were lesioned unilaterally in the
medial forebrain bundle with 6-hydroxydopamine . Animals prepared in this manner
rotate ipsilaterally to the lesion following indirectly acting sympathomimetics
like amphetamine and contralaterally to directly acting sympathomimetics like apo-
morphine. Following recovery, the animals were injected subcutaneously with 0.5,
0.25 or 0.125 1-nicotine tartrate and placed in automated ratometers. In subsequent
studies, interactive effects between nicotine and amphetamine and the ability of
mecamylamine to antagonize the effects of nicotine were also studied. Since nico-
tine has initial behavioral depressant actions to which animals become tolerant,
it was of interest to examine the effects of chronically administered nicotine on
rotational output in lesioned rats.
Since the mesolimbic dopamine system appears to be involved in modulating loco-
motor activity, it was conceivable that the locomotor excitatory effects of nicotine
were mediated through this system. Rats were implanted with cannulae guides aimed
for the nucleus accumbens and the ventral tegmental area which are the brain regions
containing the terminals and perikarya of the mesolimbic dopamine pathway, respec-
tively. Following recovery, the animals were injected in these brain regions with
cytisine, a potent nicotinic agonist, and observed for alterations in locomotor
activity. Various pharmacological controls were utilized to ascertain whether the
effects of these manipulations were specific to the dopaminergic and nicotinic
systems .
We have previously found that the dopaminergic neurons of the substantia nigra
zona compacta are excited by nicotine and acetylcholine. The dopaminergic perikarya
in the zona compacta also possess high-affinity nicotine binding sites and intense
acetylcholinesterase activity which are consistent with a cholinoceptive role. In
previous studies we utilized retrograde tracing with histochemical visualization
of cholinergic neurons to demonstrate that pedunculopontine efferents project to
the substantia nigra zona compacta. In the present studies, kainic acid was micro-
infused into the tegmental region in order to stimulate local cholinergic perikarya.
A functional activation of cholinergic input into the substantia nigra was assessed
with extracellular recording of dopaminergic neurons in the zona compacta.
118
ZOl MH 000147-12 BP
2. Functional Outflow of the Nucleus Accumbens
It is certain that the nucleus accumbens represents a critical focus for
the excitatory effects of sympathomimetic compounds. Injections of indirectly- as
well as directly-acting dopaminergic agonists into this structure increase locomotor
output, while 6-OHDA lesions attenuate the excitatory effects of amphetamine and
cocaine. Recently, Swerdlow et al (Brain Res., 1986; 306:141-143) have demonstrated
that the efferent pathway from the nucleus accumbens to the subpallidal region ap-
pears to serve as one important output of mesolimbic activity for the expression
of locomotor behavior. Whether the pallidal output is solely involved in translat-
ing the sympathomimetic activation of the nucleus accumbens into locomotor excita-
tion remains to be determined. The purpose of this investigation was to assess the
functional outputs of the nucleus accumbens following direct activation with sym-
pathomimetics using 2-deoxyglucose autoradiographic procedures. Rats were implanted
with unilateral cannulae guides aimed for the nucleus accumbens. Following re-
covery, the animals were injected in the nucleus accumbens with either 15 nmoles
of d-amphetamine sulfate or 50 nmoles of cocaine HCl. These doses were chosen on
the basis of previous studies which revealed that they were effective in increasing
locomotor behavior. Three minutes .following nucleus accumbens injections the rats
were administered 100 yCi/kg of [ C] -2-deoxyglucose (2DG) through indwelling
jugular catheters. Forty-five minutes following 2DG injections, the animals were
prepared for autoradiographic analyses using standard procedures.
3. Modulation of Immune Function by the CNS
Numerous observations indicate that opiates can affect immune function.
The presence of opiate receptors on leukocytes and endorphin production by these
cells suggest a role for the opiate system in internal regulation of immune func-
tion. Direct ^ vitro effects of opiates on antibody production, lymphocyte prolif-
eration induced by mitogens, and cytotoxic responses support this notion. Effects
of opiates on the immune system in vivo, however, suggest that opiates may also act
indirectly to modulate immune function. We have previously shown that chronic ad-
ministration of morphine in mice alters T-cell functions involved in antibody produc-
tion. Others have demonstrated morphine suppression of natural killer (NK) cell
activity in rats and, moreover, have shown that intracerebroventricular (ICV) admin-
istration of morphine at a much lower dose produced a similar suppression of NK cell
function, suggesting that this effect was mediated through the CNS.
In our present studies, we have attempted to localize the actions of opiates
in suppressing immune function. Rats were implanted with cannulae guides aimed for
the lateral ventricles or for various brain sites. One week following surgery, the
animals received bilateral injections of morphine (5 pg in 1 ul saline) into the
brain sites or unilateral injections (20-100 yg) into the ventricles. Three hours
following injection, the rats were sacrificed, spleens removed, and both NK cell
activity and mitogen-stimulated lymphocyte proliferation were measured.
One region of the brain that appeared to be uniquely involved in modulating
immune function following the direct application of morphine was the periaqueductal
gray matter (PAG) . We have recently attempted to further define the participation
of the PAG in the regulation of immune function with other procedures as well.
Rats were implanted with bipolar electrodes in various mesencephalic sites includ-
119
ZOl MH 000147-12 BP
ing the PAG and reticular formation. Following recovery, the animals were stimu-
lated through the electrodes for 10-15 minutes with intermittent stimulation.
Following stimulation the rats were sacrificed, spleens removed, and both NK cell
activity and mitogen-stimulated lymphocyte proliferation were measured.
4. The Effects of Phencyclidine on Nigrostriatal and Mesolimbic Functions
Phencyclidine (PCP) is a powerful psychotomimetic substance that produces
psychopathological effects that mimic the primary symptoms of schizophrenia. Many
of the effects of PCP have been thought to involve dopaminergic mechanisms. We
have utilized a number of neurobiological techniques to ascertain the precise
interactive effects of PCP with mesolimbic as well as nigrostriatal dopaminergic
neurotransmission. In the first series of studies, rats were implanted with
cannulae guides aimed for the terminal and cell body areas of the mesolimbic and
nigrostriatal dopamine pathways. Rotational behavior was used to assess the
interactive effects of PCP with the nigrostriatal dopamine system while locomotor
activity was used to assess the effects of PCP on the mesolimbic dopamine system.
In order to further define the functional outputs of the nigrostriatal system
that are activated by PCP, rats were implanted with unilateral cannulae guides aimed
for the substantia nigra as well as with i.v. jugular catheters. Following re-
covery, the animals were injected in the substantia nigra with 25 nmoles of PCP.
Three minutes later, the same rats were also injected i.v. with 100 uCI/kg of
2DG. Forty-five minutes following administration of 2DG, the animals were
sacrificed. The brains were prepared for autoradiographic analyses using standard
procedures.
5. Kappa Opiate Receptor-Mediated Effects
A great deal of effort has been devoted to the development of non-addic-
tive opiate analgesics. Originally, it had been hoped that certain benzomorphan
analogs that interact selectively with kappa opiate receptors might have the de-
sired characteristics to suit this need. Recently, a very selective kappa receptor
agonist (U-50,488) became available which was subsequently resolved into (1) and
(d) enantiomers. Preliminary findings indicate that the 1-enantiomer is 4,000 times
more potent than the d-enantiomer in kappa receptor binding assays, making the
d-enantiomer an ideal substance to control for nonspecific U-50,488. In our
initial series of studies, we evaluated and compared the effects of (1) and (d)
U-50,488 on locomotor behavior, feeding, and drinking following intraventricular
injections.
B. Major Findings
Acute injections of nicotine to rats lesioned unilaterally in the substantia
nigra with 6-hydroxydopamine had little effect on rotational output during the first
30 minutes following injection. During the second 30 minute period following injec-
tion of 0.5 mg/kg of nicotine, however, a modest but significant increase in rota-
tional behavior ipsilateral to the lesion was noted. Lower doses were ineffective
in modifying this behavior. Injections of 0.5 mg/kg of nicotine in animals pre-
treated with amphetamine were found to potentiate rotational behavior ipsilateral
to the lesion induced by amphetamine. Mecamylamine pretreatment prevented nico-
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ZOl MH 000147-12 BP
tine-induced rotational behavior. Chronic injections of nicotine induced rotation-
al behavior that increased in intensity over days. These data suggest that
nicotine induces a functional activation of the nigrostriatal dopamine system.
Cytisine injections into the ventral tegmental area were found to increase
locomotor output. This effect was antagonized by 6-OHDA lesions of the nucleus
accumbens. Injections of cytisine into the VTA were also accompanied by increases
in dopamine as well as DOPAC in the n. accumbens, while injections of cytisine
into the n. accumbens had no effect on dopamine metabolites in this structure.
Unilateral nicroinfusions of kainic acid into the pedunculopontine nucleus
increased the firing rate of dopaminergic neurons in the ipsilateral substantia
nigra. Excitation was dose-related and occurred within seconds, indicating that
the drug action was indirect. The kainate-induced excitation of dopaminergic neu-
rons was prevented by intravenous administration of the centrally-acting nicotinic
cholinergic antagonist mecamylamine. These results support the notion that cholin-
ergic parikarya in the vicinity of the pedunculopontine tegmental nucleus innervate
dopaminergic neurons in the substantia nigra zona compacta via nicotinic receptors.
Furthermore, it appears that nicotine functionally activates both nigrostriatal as
well as mesolimbic dopamine neurons in brain. It is suggested that the addictive
properties of nicotine, like other drugs of abuse, are mediated through an activa-
tion of central dopaminergic neurons.
Injections of amphetamine and cocaine were found to decrease metabolic activity
in the head of the caudate nucleus, olfactory tubercle, cingulate cortex, as well
as the nucleus accumbens ipsilateral to the injection. Significant increases in
metabolic activity, however, were found in the ipsilateral ventral pallidus and the
striatal fundus. The activity in some thalamic nuclei decreased ipsilateral to the
injection. In the hindbrain, increases in activity were found in the zona reticu-
lata and ipsilateral medial and lateral olivary complex. Combined with lesioning
procedures, the 2DG methodology may allow a more comprehensive analysis of the func-
tional outflow from behaviorally relevant neural systems.
Injections of 20-100 yg of morphine into the lateral ventricle produced a dose-
dependent suppression of NK cell activity, suggesting that the effects of morphine
on the immune system are probably mediated, at least in part, through the CNS. In
subsequent studies, we have found that opiates act specifically in the periaqueduc-
tal gray matter (PAG) to produce suppression of two parameters of immunocompetence.
Injections of morphine into the anterior hypothalamus, arcuate nucleus, medial amyg-
dala, medial thalamus, and dorsal hippocampus had no significant effect on the
parameters of immunocompetence examined when compared to uninjected controls. In-
jections of morphine into the PAG, however, produced a significant suppression of
NK cell activity and T-cell proliferation in response to mitogen, when compared to
saline-injected animals. These findings suggest that the central actions of
opiates on immune function are mediated through the PAG. The precise neural
mechanisms involved, however, remain to be elucidated. More recently, we have
found that direct electrical stimulation of the PAG also compromises immune
function in rats. It appears that this primitive core of the brain is a critical
region for regulating the immune system.
Our findings have revealed that although PCP appears to produce behavioral ef-
fects indicative of DA activation, there is no evidence of direct dopamine involve-
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ZOl MH 000147-12 BP
ment. Injections of PCP into the nucleus accumbens (the terminal region of the
mesolimbic dopamine system) produced locomotor excitation characteristic of dopamine
receptor activation or dopamine release. This behavior, however, was not modified
by 6-OHDA lesions of the nucleus accumbens or by haloperidol, indicating that PCP
was producing behavioral effects independent of dopamine function.
PCP appeared to have little direct effect on striatal function although injec-
tions of PCP into the substantia nigra also produced behaviors indicating altera-
tion of the DA nigrostriatal system. Further analyses using 2DG as well as selec-
tive lesioning techniques, however, have revealed that the effects of PCP at the
levels of the substantia nigra are not mediated through the ascending dopamine
nigrostriatal pathways, but involve systems projecting caudally to the pontine
reticular structures.
While PCP does not appear to produce the behavioral effects through dopamine
pathways, there is recent evidence suggesting that some of the effects of PCP may
be mediated through an excitatory amino acid receptor. It has been found that PCP
is a noncompetitive antagonist at the NMDA excitatory amino acid receptor. We have
recently found that APS (a phosphoric acid analog which is a competitive antagonist
at the NMDA receptor) produces behavioral effects in rats which are identical to
those produced by PCP.
In a 75-minute test of motor activity, repeated administration of different
doses of d-U-50,488 (10, 25, 50, 100 nmol) had no effect on rats' horizontal activi-
ty; however, 50 nmol did significantly decrease vertical activity (p < .05) .
Repeated administration of different doses of l-U-50,488 (10, 25, 50, 100 nmol)
significantly increased horizontal activity at the 25 (p < .05) and 100 nmol
(p < .01) doses. The effects of 100 nmol of l-U-50,488 on horizontal activity was
antagonized by 5.0 mg/kg (i.p.) naloxone.
In a separate study, the acute administration of 100 nmoles of d-U-50,488 pro-
duced a significant depression of locomotor activity which was not antagonized by
naloxone. The 1-enantiomer also produced a modest but not statistically signifi-
cant depression of locomotor output which was surprisingly enhanced by naloxone.
The initial depressant effects of the l-U-50,488 were followed by locomotor excita-
tion which was antagonized by naloxone. The d-enantiomer again had no excitatory
effect on locomotor output.
Increases in the intake of a highly palatable food were seen in non-food-de-
prived animals after ICV injections of l-U-50,488 (25 S 100 nmoles). The increase
in food intake induced by l-U-50,488 (100 nmoles) was antagonized by 1.0 mg/kg
(i.p.) naloxone. The d-enantiomer had no effect on food intake.
Depression of water intake was seen in 21-hour water-deprived animals follow-
ing repeated administration of different doses of l-U-50,488 (25 & 100 nmoles).
Repeated administration of different doses of the d-enantiomer had no effect on
deprivation-induced drinking.
These findings clearly demonstrate that some of the centrally mediated behav-
ioral effects of the kappa agonist U-50,488 are stereospecif ic. The 1-enantiomer
enhanced locomotor activity, increased food intake, and decreased water intake.
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ZOl MH 000147-12 BP
The d-enantiomer did not produce similar effects. These compounds will undoubtedly
prove useful in analyzing the specific effects of kappa receptor-mediated behaviors.
Significance to Biomedical Research and the Program of the Institute
Since opiate alkaloids produce some of the most profound behavioral and physi-
ological effects, endogenous opiates (which are mimicked by the alkaloids) must
serve an important role in regulating emotions as well as physiological and sensory
processes. The use of our newly developed autoradiographic procedures, which allow
a measurement of ongoing activity in these systems, may reveal the functional sig-
nificance of opiate pathways in brain.
Since nicotine is one of the most abused substances in society, understanding
its neuronal mechanisms of action will aid in understanding the abuse properties
of this substance.
Phencyclidine is also an increasingly abused substance. Furthermore, phencyc-
lidine produces effects in man very similar to some of the primary symptoms of
schizophrenia. For these reasons, it is valuable to understand the mechanisms of
action of this class of compounds.
It has been suggested that the cholinergic system also plays an important role
in mental disorders. It is therefore necessary to understand the functions of this
system in brain, and to analyze its interactive effects with other neurotransmitter
systems such as dopamine.
Besides endorphins, the brain contains numerous other peptides which undoubt-
edly serve important regulatory functions. It is important to identify the distri-
bution of binding sites for other substances in brain and to analyze their physio-
logical and behavioral effects -with micro-injection mapping techniques. Altera-
tions in the activity of these systems may underlie a number of neurological and
psychiatric disorders.
Proposed Course of Project
1. The in vivo autoradiographic technique will continue to be used to define
endorphinergic circuitry activated by various behavioral and physiological manipu-
lations.
2. Microdialysis procedures will be introduced and utilized to assess the
activity of catecholamine systems in rat brain following the introduction of neuro-
peptides and other psychoactive compounds.
3. Attempts will be made to assess the functional activity of catecholamine
systems during various behavioral states in the unanesthetized, freely-moving rat.
4. Functional metabolic activity of specific brain circuits will be assessed
during various behaviors and following focal injections of various drugs using 2-DG
procedures.
5. The evaluation of behavioral and physiological effects of neuropeptides
will continue.
123
ZOl MH 000147-12 BP
6. The specific brain circuits regulating immune function will be defined
using electrical stimulation and microinjection procedures.
7. Further attempts will be made to localize opiate receptor subtypes on
serotonergic, noradrenergic, and dopaminergic systems.
PUBLICATIONS
Clarke, P.B.S., Hamill, G.S., Nadi, S.N., Jacobowitz, D.M., and Pert, A.:
3H-Nicotine and I-alpha-bungarotoxin labeled nicotinic receptors in the
interpeduncular nucleus of rats. II. Effects of habenular deaf ferentation. J.
Comp. Neurol. 251: 407-413, 1986.
Hamill, G.S., Clarke, P.B.S., Pert, A. and Jacobowitz, D.M. : ^n-nicotine and
I-alpha-bungarotoxin labeled nicotinic receptors in the interpeduncular nucleus
of rats. I. Subnuclear distribution. J. Comp. Neurol. 251: 398-406, 1986
Gaudreau, P., Quirion, R. , St. -Pierre, S., Chiueh, C.C., and Pert, A.: Localiza-
tion of cholecystokinin receptors in relation to the nigrostriatal and mesolimbic
dopaminergic pathways. Neuropeptides 9: 283-293, 1987.
Ostrowski, N.L., Burke, T.R., Jr.,, Rice, K.C., Pert, A., and Pert, C.B.: The pat-
tern of [3H]cyclofoxy retention in rat brain after in vivo injection corresponds
to the in vitro opiate receptor distribution. Brain Res. 402: 275-286, 1987.
Pert, A.: Cholinergic and catecholaminergic modulation of nociceptive reactions:
interactions with morphine. In Akil, H. (Ed.): Pain and Headache, Vol. 9.
Basel, Karger, 1987, pp. 1-63.
Sforza, G.A., Seeger, T.F. , Pert, C.B., Pert, A. and Dotran, CO.: In vivo opioid
receptor occupation in the rat brain following exercise. Med. Sci. Sports and
Exercise 18: 380-384, 1987.
Weber, R. J. , Ikejiri, B., Rice, K.C., Pert, A., and Hagan, A.A. : Opiate
receptor-mediated regulation of the immune response in vivo. In Harris, L.S.
(Ed.): Problems of Drug Dependence. Washington, D.C., NIDA Research Monograph,
Vol. 76, 1987, pp. 341-348.
Clarke, P.B.S., Hammer, D.W., Pert, A., and Skirboll: Innervation of substantia
nigra dopaminergic neurons by cholinergic afferents from pedunculopontine nucleus
in rats: neuroanatomical and electrophysiological evidence. Neuroscience, in
press.
Clarke, P.B.S, and Pert, A.
rat brain. In Martin, W.R.
Tobacco Smoking and Health:
in press.
Autoradiographical evidence of nicotinic receptors in
Van Loon, G.R., Davis, D.L., and Iwamato (Eds.):
A Neurobiological Approach. New York, Plenum Press,
124
ZOl MH 000147-12 BP
Clarke, P.B.S., Skirboll, L. and Pert, A.: Anatomical and electrophysiological
evidence for the nicotinic cholinergic innervation of the dopaminergic neurons of
substantia nigra pars compacta. Neuroscience, in press.
Namura, I., Douillet, P., Sun C.J., Pert, A., Cohen, R.M. , and Chiueh, C.C.: MPP+
(l-methyl-4-phenylpyridine) is a neurotoxin to dopamine, norepinephrine and
serotonin containing neurons. Eur. J. Pharmacol., in press.
Ostrowski, N.L., Hill, J.M. , Pert, C.B. and Pert, A.: Autoradiographic
visualization of sex differences in the pattern and density of opiate receptors in
hamster hypothalamus. Brain Res. , in press.
Ostrowski, N.L., Pert, C.B. and Pert, A.: visualization of opiate receptors in
vivo. In Leslie, F. (Ed.): Receptor Localization: Ligand Autoradiography. New
York, Alan Liss Inc., in press.
Pert, A. and Clarke, P.B.S.: Nicotinic modulation of dopaminergic neurotransmis-
sion: functional implications. In Martin, W.R., Van Loon, G.R., Davis, D.L., and
Iwamato (Eds.): Tobacco Smoking and Health; A Neurobiological Approach. New
York, Plenum Press, in press.
125
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00400-05 BP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Protein Phosphorylation in Brain
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attillation)
Jitendra Patel, visiting Associate, Unit on Neurochemistry, BPB, NIMH
Sheela vyas, BPB, NIMH; Anne-Marie O'Carroll, LCS, NIMH; Douglas Kligman, LCB,
NIMH; Savella Detera, CNB, NIMH; Peter Fishman, DMNB, NINCDS; John Bishop, ET,
NINCDS
COOPERATING UNITS (if any)
CNB, LCS, LCB, NIMH; DMNB, ET, NINCDS
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Neurochemistry
INSTITUTE AND LOCATION
NIMH, NIB, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.4
PROFESSIONAL:
1.2
0.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues (El (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have extended our characterization of the major protein kinase C sub-
strates of 87,000 dalton (Mr 87) . We have purified this phosphoprotein to homo-
geneity. Purified Mr 87 was used to elucidate its amino acid composition, site
of phosphorylation and partial amino acid sequence. Polyclonal antibodies to Mr
87 were raised and were used to perform immuno-localization of this phosphopro-
tein. Cloning of Mr 87 and other studies attempting to elucidate the function of
MR 87 are in progress.
Our previous work had demonstrated that protein kinase C is involved in the
regulation of receptor activity and neurotransmitter release. This work was fur-
ther extended.
Further characterization and isolation of the 94,000 dalton cyclic AMP-de-
pendent protein kinase substrate was performed.
127
PHS 6040 (Rev. 1/84)
SPO •I4-«I*
ZOl MH 00400-05 BP
PROJECT DESCRIPTION:
Objectives: The objective of this laboratory is to elucidate the chain of
biochemical events, in particular protein phosphorylation, that is triggered
ensuing receptor activation.
A number of different approaches are adopted in such investigations and they
include :
1. Identification of particular protein kinase and phosphoproteins involved
in specific receptor-triggered response either at the cellular level (e.g., recep-
tor desensitization, stimulus-secretion coupling) or the behavioral level (e.g.,
kindling) .
Methods Employed: protein purification, tissue culture, enzyme kinetics
Major Findings: A number of attempts have been made to identify the protein
substrates that mediate the effect of protein kinase C. One such substrate that
appears to be phosphorylated in a wide variety of tissues, including the brain, is
a protein with apparent molecular weight of 87,000 daltons. In an attempt to eluci-
date the function of this phosphoprotein, we have purified Mr 87K phosphoprotein
to apparent chemical homogeneity and have performed the following (in collaboration
with Drs. Kligman and Detera) .
1. We have raised highly specific polyclonal antibodies to Mr 87K.
2. Immunocytochemical and immunohistochemical localization of Mr 87K.
3. Using a gt-11 expression library from rat brain, we have identified
a number of cDNA clones that selectively hybridize with the Mr 87L
antibodies. The nucleotide sequence of the cDNA inserts has been
determined.
4. We have determined partial amino acid sequence of a number of puri-
fied trypic fragments of Mr 87K. Based on this sequence oligonucleo-
tides have been synthesized and are now being used to screen gt-11
cDNA library from rat brain. A number of positively hybridizing
clones have been identified and are now being characterized.
The role of protein kinase in stimulus-secretion coupling in endocrine cells
was investigated using CRF-stimulated endorphin release from AtT-20 cells as a model
system. A procedure to deplete AtT-20 cells of protein kinase C was elucidated and
characterized. Using these cells, the role of protein kinase C in the receptor-
activated neurotransmitter secretion and biosynthesis was investigated. This work
was performed by Dr. Vyas in collaboration with Mr. Bishop and will shortly be sub-
mitted for publication in the Journal of Neurochemistry. A similar experimental
approach was also undertaken in collaboration with Dr. Fishman, to elucidate the
role of protein kinase C in the regulation of beta-adrenergic receptor-coupled
adenylate cyclase activity. This work has now been published (Fishman et al.,
Biochem. Biophys. Res. Commun. , 144:620-627, 1987).
128
ZOl MH 00400-05 BP
In an attempt to elucidate the role of protein phosphorylation in the
"kindling" process, the modulation of kinase activities were investigated in the
hippocampus of lidocaine-kindled rats. A significant elevation in cyclic
AMP-dependent protein kinase and protein kinase C activities were observed in
kindled animals. Unlike protein kinase C, the elevation of cyclic AMP-dependent
protein kinase activity did not appear to be associated with the seizures that the
animals experience during the kindling process.
Dr. O' Carroll has continued attempts to define a protocol to purify a 94,000
dalton cyclic AMP-dependent protein kinase substrate. Recently, polyclonal
antibodies to 94,000 dalton phosphoprotein were raised and these are now being
characterized .
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE:
The study of protein phosphorylation is of vital importance in the quest
to better understand how the neurotransmitters mediate their action. Such an
understanding is the necessary prerequisite for the appreciation of the
biological basis of normal and abnormal behavior.
PROPOSED COURSE OF PROJECT:
These studies are expected to continue for the next several years.
PUBLICATIONS:
Patel, J. and Kassis, S.: Concanavin A prevents phorbol-mediated redistribution of
protein kinase C and beta-adrenergic receptors in rat glioma C6 cells. Biochem.
Biophy. Res. Commun. 144: 1265-1272, 1987.
Patel, J. and Kligman, D. : Purification and characterization of an Mr 87,000 (Mr
87) protein kinase C substrate from rat brain. J. Biol. Chem. . 1987, in press.
129
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01831-11 BP
PERIOD COVERED
October 1. 1986
September 30, 1987
TITLE OF PROJECT (80 characterz or less. Title must fit on one line between the borders.)
:3asic and Clinical Studies of Neuronal and Glial Enolases
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute attillation)
;>aul J. Marangos, Ph.D. Chief, Unit on Neurochemistry, BPB, NIMH
J.M. Polak Chairperson, Histochemistry Dept. , Royal Medical School, London
A.G. Pearse Professor Emeritus, Royal Medical School, London
:). Schmechel Assoc. Professor, Neurology, Duke University
il. Ginns Neurologist, NSB, NIMH
3. Martin Biochemist, NSB, NIMH
Van Lubitz Physiologist, Georgetown Univ. Medical School
COOPERATING UNITS (if any)
?oyal Med. School, London; Hoffmann La Roche; Duke U. ; Vanderbilt U.; U. of Texas;
of Virginia; UCLA Med. School; U. of Ulm, Germany; Georgetown Med. School; NSB,
^IMH; Children's Hosp.. Phila.; Johns Hopkins U. ; Finsen Inst., Copenhagen.
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Jnit on Neurochemistry
INSTITUTE AND LOCATION
^IMH. ADAMHA, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
1.4
PROFESSIONAL
0.5
0.9
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
13 (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the spece provided.)
The basic neurobiology and potential clinical utilization of neuron specific
enolase (NSE) has been a long-standing interest in our laboratory. During the
past year, studies have begun to focus on the molecular biology of NSE in an
effort to use the brain enolase isoenzyme system as a model to study neuron
specific gene expression. We now have cDNA probes for both NSE and non-neuronal
enolase (NNE) . The probes have been characterized and in situ hybridization
studies have been performed in human brain. Our cDNA probe for NSE only labels
neurons whereas the probe for NNE preferentially labels glial cells. Studies
performed using northern blots in both rat and guinea pig brain reveal that the
mRNA for NSE is substantially larger than that for NNE. There is approximately
75% homology within the coding region for human NNE and NSE (some 1700 base
pairs). The 3 'non-coding region is much larger for NSE and has very little
homology to that of NNE, a fact we made use of to generate specific probes.
Studies are in progress regarding the NNE to NSE switch that occurs during neural
differentiation .
Studies are also in progress to determine the effect of cerebral ischemia on brain
NSE levels in gerbil brain. In these studies we will attempt to demonstrate that
NSE levels can be used as an index of neural damage post ischemia. The effect of
various protective agents such as adenosine receptor agonists will also be assessed
Clinical studies assessing NSE levels in both human CSF and serum consequent to
stroke are also in progress. The goal here is to determine whether NSE levels are
correlated with the degree of post-ischemic neurologic damage.
131
PHS 6040 (Rev. 1/84)
SPO »\*-9\»
other Professional Personnel
ZOl MH 01831-11 BP
s.
Cohen
M.
Usetegi
P.
Zeltzer
D.
Johnson
A,
Greco
L.
Rubinstein
R.
Seeger
A.
Evans
Neurologist
Director of Diagnostic Research
Assoc. Prof., Pediatric Oncology
Associate Professor, Oncology
Assoc. Professor, Oncology
Chairman, Dept. of Pathology
Pediatric Oncologist
Chief, Pediatric Oncology
V. Balasubramanian Assistant Professor
A. Pedersen
K. Schilling
C. Pilgrim
D.T. Nakajima
Clinical Oncologist
Cell Biologist
Chairman, Dept. of Anatomy
visiting Fellow
Georgetown Med . School
Hoffmann La Roche,
Nutley, NJ
U. of Texas, San Antonio
Vanderbilt Med. School
Vanderbilt Med. School
University of Virginia
UCLA Medical School
Children's Hosp. , Phila.
Div. of Nuclear Med.,
Johns Hopkins University
Finsen Institute,
Copenhagen , Denmark
Univ. of Ulm, W. Germany
Univ. of Ulm, W. Germany
BPB, NIMH
132
ZOl MH 01831-11 BP
Project Description
A. Objectives
The central theme of the Unit on Neurochemistry continues to be the characteri-
zation and potential clinical application of brain proteins. In this regard, our
studies over the past decade have focused on both brain membrane proteins (neuro-
transmitter receptors) and on soluble neuron specific proteins such as the neuron
specific enolase (NSE) and the glial specific protein S-100. The importance of
cell-specific proteins is difficult to overstate, since it is clear that they prob-
ably are intimately involved in the specific differentiated functions of their parent
cell. The characterization of such proteins, therefore, provides a useful approach
to the study of neural physiology. The potential clinical applications of such
proteins is also obvious, since antisera to these can serve as specific probes for
the parent cell type.
Work done in our laboratory during the past decade has established that NSE is,
in fact, specific to neurons and neuroendocrine cells, making it a highly useful
marker for both cell types. We have also shown that NSE or the gamma enolase sub-
unit, only appears in differentiated neurons. This makes it a highly useful marker
for neural differentiation. We have also, at the clinical level, shown that NSE
levels in various biological fluids can be of diagnostic importance in stroke pa-
tients and in two neuroendocrine cancers, small cell lung cancer, and pediatric
neuroblastoma. Our studies concerning both the lung cancer and neuroblastoma pa-
tients have now been confirmed in several other laboratories and serum NSE assays
are now becoming a part of clinical work-ups in both of these neuroendocrine can-
cers. The clinical information obtained from serum NSE levels has been shown to be
highly useful for diagnosis, charting the clinical course of the illness, and deter-
mining the response to chemotherapy of individual patients. Our radioimmunoassay
has become the standard to which other assays are compared, and several cancer
diagnostic facilities are now using it to routinely screen patients.
Our major objectives in the NSE studies are two-fold, the first being to gain
a better understanding of the role of NSE in neural physiology; i.e., why does the
neuron require a unique form of this glycolytic enzyme? The second is to utilize
the NSE methodology in neurologic and psychiatric patient populations. For the
short term, we are now focusing on the utilization of serum and CSF NSE levels in
stroke patients as an index of neurologic damage and prognosis,
B. Methods Employed
Gel electrophoresis, chromatography, northern, southern and western blotting,
radioimmunoassay, immunocytochemistry, in situ hybridization, isoelectric focusing,
and enzyme assays.
C. Major Findings
During the past year, the Unit has expended a major portion of time trying to
incorporate a molecular biological component into the laboratory. This has involved
the recruitment of a Fogarty Fellow, Dr. Takashi Nakajima, and the purchase of some
equipment. Dr. Nakajima spent several months learning techniques related to RNA
isolation, northern blotting, and cDNA probe processing in the laboratory of Dr.
Edward Ginns, one of our major collaborators in these studies. We now have these
procedures ongoing in our own laboratory, although the experiments using radioactive
133
ZOl MH 01831-11 BP
tective effect on post-ischemic brain damage. The protective agents are adenosine
receptor agonists such as cyclohexyladenosine. These studies utilizing NSE as an
index of post-ischemic brain damage, therefore, interdigitate quite well with the
adenosine receptor studies that are currently in progress in our laboratory. We
have succeeded in working out the radioimmunoassay for gerbil NSE and are currently
preparing our first group of animals where we will look at NSE levels in seven dif-
ferent brain areas at times ranging up to seven days post ischemia. We have plans
for following up these experiments with immunocytochemical analysis where neural
staining patterns with our anti-NSE serum will be analyzed.
In collaboration with Dr. Stanley Cohen at Georgetown, clinical studies are
also in the planning stages concerning serum and CSF NSE levels in stroke patients.
Previous work in our laboratory has documented an elevation in CSF NSE levels conse-
quent to stroke. In the present study, we seek to extend these observations and
attempt to determine whether a correlation exists between the severity of the stroke
and the NSE level in CSF. We also will look at serum in the study in the hope that
we will see elevated NSE levels. If this works out, it will make the application
of the NSE methodology much more feasible in stroke patients.
To summarize, the past year has been one in which NSE-related studies have been
refocused at both the basic and clinical levels. We are rekindling our basic
studies, which will now focus on molecular biology and the mechanism of the alpha
to gamma developmental switch process. Our efforts in the basic science of NSE have
been very few in the past five years, with the major focus having been the clinical
applications of NSE to neuroendocrine cancers (lung cancer and neuroblastoma) , as
mentioned in previous annual reports. In the clinical area, we intend to put more
emphasis on neurologic and psychiatric disorders such as stroke and schizophrenia.
We are currently planning not only to assess NSE levels in patient populations, but
we would also like to be able to measure antibodies to NSE in patient sera. In this
regard, we are in the process of developing an ELISA for NSE and hope to have it
operational within several months. This will enable us to determine more of a bio-
logic record on a patient related to neural tissue degeneration rather than having
to rely on catching an elevation in actual NSE levels that is probably closely cor-
related temporarily with tissue destruction.
D. Significance to Biomedical Research and the Program of the Institute
The enolase isoenzyme system in brain represents an ideal system for studying
neural differentiation. With NSE, we have a means of assessing neural function both
chemically (RIA) and histologically (immunocytochemistry) . Utilization of this
unique methodology should greatly facilitate our understanding of neural development
and function. Clinically, the NSE methodology has been shown to be of considerable
utility for neuroendocrine cancers, with the potential also existing for its utili-
zation in neurologic and psychiatric disorders. The major change of the IRP is to
engage in novel neuroscience research that has the potential for increasing our
understanding of brain function in both health and disease. It is clear that our
work over the past decade closely parallels these goals.
E. Proposed Course of the Project
The studies will likely continue for at least the next two years.
134
ZOl MH 01831-11 BP
PUBLICATIONS
Polak, J.M. and Marangos, P.J.: Neuron specific enolase, a marker for neuroendo-
crine cells. In Falkner, S., Hakanson, R. and Sundler, F. (Eds.): Evolution and
Tumor Pathology of the Neuroendocrine System. Amsterdam, Elsevier, 1985, pp.
433-480.
Marangos, P.J. and Schmechel, D.R.: Neuron specific enolase, a clinically useful
marker for neurons and neuroendocrine cells. Ann. Rev. Neurosci. 10: 269-295,
1987.
Giannone, L. , Johnson, D.H. , Grosh, W.W. , Davis, B.W., Marangos, P. J. , and Greco,
F.A.: Serum neuron specific enolase in metastatic Merkel cell tumors. Med.
Pediatr. Oncol., in press.
Schmechel, D.R., Marangos, P.J., Martin, B. and Ginns, E. : Localization of neuron
specific enolase (NSE) mRNA in human brain. Neurosci. Lett. , in press.
Marangos, P.J,: Neuron spcific enolase, a neural and neuroendocrine protein. In
Marangos, P.J., Campbell, I.C. and Cohen, R. (Eds.): Neurobiological Research,
Vol. II. New York, Academic Press, in press.
135
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
:201 MH 01833-07 BP
PERIOD COVERED
October 1, 1986 - September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on or^e lir\e betweer\ the borders.)
Adenosine Receptors in the CNS
PRINCIPAL INVESTIGATOR (List other prolessional persortnel below the Phncipal Investigator.) (Name, title, laboratory, and institute alfillatlon)
Paul J. Marangos, Ph.D. Chief, Unit on Neurochemistry, BPB, NIMH
J. Patel Biochemist, BPB NIMH
R. M. Post Chief BPB NIMH
S. Cohen Neurologist Georgetown Univ.
Dag Von Lubitz Neurologist Georgetown Univ.
N. Sperekalis Chairman, Dept. of Physiology Univ. Cincinnati
K. Jacobsen Staff Fellow LBC, NIADD
COOPERATING UNITS (if any)
BPB, CNB, NIMH; LBC, NIADD; CP , NCI; U. of Cincinnati, Georgetown Univ.
LAB/BRANCH
Biological Psychiatry Branch
SECTION
Unit on Neurochemistry
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAL
0.6
OTHER:
1.1
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects S (b) Human tissues D (c) Neither
D (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The pre-clinical evaluation of both adenosine receptor and adenosine uptake site
continue to occupy a major portion of the Unit's resources. We have described the
complete anatomical distribution of the adenosine uptake site using
[^H] dipyridamole autoradiography and shown by these binding experiments that there
are multiple populations of brain adenosine uptake sites. Studies measuring
adenosine uptake in synaptoneurosomes support the binding and autoradiography.
More evidence has been gathered indicating that carbamazepine is acting as an
adenosine antagonist. Xanthine-induced seizures have been studied using the new
A -specific antagonist, XAC. Evidence was generated showing that XAC-induced
seizures differ from those induced by caffeine in that they are not blocked by
RO-15-1788, a benzodiazepine receptor blocker. We have also shown increased aden-
osine uptake sites in drug resistant human breast cancer cell cultures, suggesting
that the adenosine uptake site may be involved in the phenomenon of multi-drug re-
sistance.
Recent studies have focused on the role of adenosine agonists as protective agents
during cerebral ischemia. The post-ischemic neuropathy is being characterized as
it relates to adenosine, glutamate, and benzodiazepine status at both the binding
and autoradiographic level. These studies will hopefully assess the potential
utility of adenosine agonists in the treatment of stroke patients.
137
PHS 6040 (Rev. 1/S4) CPO <i4-«ii
ZOl MH 01833-07 BP
Other Professional Personnel:
P. F. Morgan
J. Deckert
J-L. Daval
T. Nakajima
S.R.B. Weiss
T. Insel
K. Jacobson
R. Fine
Fogarty Visiting Fellow
Guest Researcher
Guest Researcher
Fogarty visiting Fellow
Staff Fellow
Staff Fellow
Staff Fellow
Oncologist
BPB, NIMH
BPB, NIMH
BPB, NIMH
BPB, NIMH
BPB, NIMH
CNB, NIMH
LBC, NIADD
CP, NCI
138
ZOl MH 01833-07 BP
I. Project Description
A. Objectives
The characterization of specific receptor systems in the CNS is now recog-
nized as a desirable first step in pre-clinical neuropharmacologic research.
The rationale is that the elucidation of such systems will greatly facilitate the
development of specific pharmacologic agents that interact with only the receptor
system in question. Specificity of interaction is of obvious importance for the
development of new drugs that will have a minimum of side effects. The radiorecep-
tor binding methodology has proven to be of great utility in the discovery of new
neurotransmitter and neuromodulator receptor systems in brain and can be highly
useful in pre-clinical studies. We have, for the past ten years, applied this
approach to the study of the benzodiazepine, GABA, and adenosine receptor systems
and have, in the process, increased our understanding of the basic biology of each
as well as the potential for pharmacologic intervention.
A major focus of our laboratory has centered around the mechanisms involved
in the depression of neural activity. In this regard, both the benzodiazepine and
adenosine systems play major roles. Since an endogenous ligand for the benzodiaze-
pine receptor remains illusive, it is difficult to postulate an actual physiologic
role for the benzodiazepine receptor. It is, however, quite clear that this is
not the case as regards the adenosine system, and adenosine is, in fact, now widely
conceptualized as being the brain's own natural depressant or sedative. Since the
importance of the benzodiazepine receptor as an agent of in vivo physiologic signi-
ficance is open to question (due ^o the lack of an endogenous ligand) , we have be-
come increasingly involved with the adenosine system as an object of study as re-
gards the modulation of CNS arousal states. There is now ample evidence that aden-
osine is a major neuromodulator in brain having marked effects on cyclic AMP levels,
neurotransmitter release, calcium fluxes, neuronal firing, and behavior. It is also
clear that adenosine serves as the endogenous ligand, so all the elements of the
system are at least identified. Also, the types of behaviors mediated by this
system (arousal, sedation, convulsions, and post-ischemic protective effects) make
it a good candidate for the development of new drugs of potential relevance to psy-
chiatry and neurology.
B. Methods Employed
Scintillation counting, autoradiography, radioreceptor assays, tissue extrac-
tion, animal surgery, and chromatography.
C. Results
Our laboratory is virtually the only one in the world actively engaged in
studies of both the adenosine receptor and the adenosine uptake site. We were the
first to demonstrate that brain adenosine uptake sites could be labeled using [^H]-
nitrobenzylthioinosine (NBI) and proceeded to characterize this site showing that
it was totally distinct pharmacologically from the adenosine receptor. During the
past year, we have worked further with a new ligand probe for the adenosine uptake
site which we had custom labeled. This probe is [^H] dipyridamole (DPR) , which we
synthesized because we suspected that [^HJNBI was not recognizing all of the adeno-
sine uptake sites present in brain.
Using [3H]DPR, we have shown by three different approaches that this ligand
binds to several-fold more sites in the brain than does [^HJNBI, and have, in the
139
ZOl MH 01833-07 BP
process been able to put forth the postulation that heterogeneity exists in brain
adenosine uptake sites. In binding studies, we consistently show a higher number
of binding sites for [SHJDPR compared to [3H]NBI (2- to 4-fold) and the inhibition
of [3H]DPR binding by unlabeled NBI is distinctly biphasic in nature. These re-
sults clearly suggest that an NBI-sensitive and an NBI-insensitive site is present.
We have shown that this is the case in guinea pig, dog, and human brain.
In autoradiographic studies, Dr. Deckert has shown that [3H]DPR is able to la-
bel more sites in cerebellum, hippocampus, and the various ependymal membranes lin-
ing the ventricles. Also, unlabeled NBI is not as effective in displacing [^HJDPR
as is unlabeled DPR, further supporting the binding studies as regards adenosine
uptake site heterogeneity. Dr. Deckert has also shown a substantially better co-
localization of adenosine uptake sites with the adenosine receptor when he uses
[3H]DPR as the ligand probe. This has been very important, since it is a major
criterion for the identification of adenosinergic neurons; i.e., the co-localiza-
tion of both the receptor and the uptake site.
Dr. Philip Morgan has shown, using actual measurements of [^H] adenosine into
synaptoneurosomes, that DPR has different properties from NBI. Dr. Morgan's stud-
ies show a biphasic inhibition of [^H] adenosine uptake by NBI and a monophasic in-
hibition of DPR. These elegant studies again totally complement the binding and
autoradiographic studies in that they strongly suggest the existence of NBI-sensi-
tive and NBI-insensitive sites and, consequently, adenosine uptake site multiplici-
ty.
All of the above mentioned studies are currently in press in various journals
in a total of five manuscripts.
In an effort to further extend our understanding of the adenosine uptake site,
we undertook a study of the ontogenic development of adenosine uptake sites and
how they relate to the receptor to the cyclase. Here, Dr. Morgan has shown that
in rat brain the uptake site is high from very early in development, followed by
the appearance of the receptor and the coupling mechanism. We are currently extend-
ing these studies to look at the development of adenosine uptake site subtypes in
guinea pig brain. Preliminary results show that the ratio of [^HJDPR to [^HJNBI
sites increases during guinea pig brain development.
In all of the above studies, the goal is at some point to be able to develop
the ability to selectively modulate each sub-population of adenosine uptake sites
in brain. Such selective drugs may prove to have beneficial sedative, anxiolytic,
or anti-ischemic properties and would be highly specific.
We have begun to shift our focus this past year toward more clinical applica-
tions as regards the adenosine system. Basically, this has been in three areas
which are: 1) adenosine as a protective agent against brain ischemia; 2) multi-
drug resistance of cancer cells and the adenosine uptake site; and 3) adenosine as
an anticonvulsant and its relationship to carbamazepine.
We have recently begun a collaboration with Drs. Von Lubitz and Cohen from
Georgetown University dealing with the effects of adenosine agonists on post-ischem-
ic neurologic damage. These investigators have shown dramatic protective effects
of cyclohexyladenosine (CHA) when given up to 30 minutes after ischemia in gerbils.
140
ZOl MH 01833-07 BP
CHA- treated animals (>90%) survive ischemia, whereas >80% of untreated animals die
within 8 hours after the ischemia (30 minutes of carotid artery occlusion) . We are
currently investigating the pharmacology of this effect as well as the neuropharma-
cologic, neurochemical, and neuroanatomic consequences of ischemia, comparing both
the treated and untreated animals. Our hypothesis is that adenosine agonist treat-
ment prevents glutamate release post-ischemia, thereby protecting these neurons
from the excitotoxic syndrome. We hope to show that CHA and adenosine uptake
blocker treatment actually does preserve neural function as judged by adenosine and
glutamate receptor autoradiography. We will also attempt to show blockade of the
effect by adenosine antagonists and to compare the potency of adenosine agonist
effects with those of glutamate antagonists. These studies will constitute a
major research effort in our laboratory and it is expected that they will be of
major clinical relevance.
In collaboration with Dr. Robert Fine from NCI, Dr. Morgan has just completed
a rather interesting study which has shown, at the level of both [3H]NBI and [3H]DPR
binding, as well as by direct [^H] adenosine uptake, that cultured human breast
cancer cells which are resistant to chemotherapeutic drugs manifest a dramatic in-
crease in adenosine transport. This is an intriguing finding which suggests that
the tone of the adenosine transporter may be significant as it relates to the
sensitivity of a cell towards chemotherapeutic drugs. This finding is currently
being written up for publication with future studies planned to dissect out the
potential significance of these observations. It would certainly be intriguing if
we could demonstrate a reversal of drug resistance with adenosine uptake blockers.
Our long-term studies regarding the interaction of the anticonvulsant and anti-
manic agent carbamazepine with adenosine receptors have also continued during the
past year and yielded some new and interesting results. We have shown, in colla-
boration with Drs. Post and Weiss, that the upregulation of adenosine receptors pro-
duced by carbamazepine is quite long-lasting and possibly irreversible, since it
persists for at least eight weeks after carbamazepine withdrawal. We have also
provided convincing biochemical evidence that carbamazepine acts as an adenosine
antagonist in that it is more potent at lower temperatures as an inhibitor of adeno-
sine agonist binding, and that its potency as an inhibitor of adenosine agonist
binding increases in the presence of a guanosine triphosphate analog. Both the
pharmacologic and the biochemical study are in press and both are consistent with
an antagonist interaction of carbamazepine with adenosine receptors. Future studies
will focus on the mechanism of carbamazepine-induced adenosine receptor upregula-
tion and its brain regional aspects in comparison with the effect of chronic
caffeine. In this regard, we have currently embarked on a major effort to estab-
lish a cell culture facility within the Unit. Although we have been severely
hampered by space limitations, we have managed to set up an incubator and a sterile
hood in another laboratory, and have begun to grow some transformed cells such as
rat PC-12 and human LAN-1 neuroblastomas. We are now attempting to grow primary
rat neural cultures. Our goal in these efforts is to study the effects of chronic
and acute carbamazepine and caffeine treatment at the molecular level and ask ques-
tions such as whether the adenosine receptor is being phosphorylated and whether
modulators such as phorbol esters can affect carbamazepine-induced adenosine recep-
tor upregulation. The cell culture approach offers distinct advantages for mechan-
istic studies in that variables can be more effectively controlled.
141
ZOl MH 01833-07 BP
D. Significance to Biomedical Research and the Program of the Institute
There is a great need for new and more effective psychotherapeutic drugs.
It is only through an increased understanding of brain function that we can hope
to determine the potential sites for drug intervention. Characterizing defined
brain systems that mediate important neural mechanisms is therefore an important
preclinical research strategy. In this regard, both the adenosine and benzodiaze-
pine systems are good targets for increasing our conceptions of the mechanisms in-
volved in seizures, sedation, and anxiety, all of which are of clinical importance.
E. Proposed Course of the Project
Studies should continue for several years.
PUBLICATIONS
Bisserbe, J.C., Deckert, J. and Marangos, P.J.: Autoradiographic distribution of
[^H] dipyridamole binding sites in guinea pig brain. Neurosci. Lett. 66: 341-345,
1986.
Deckert, J., Bisserbe, J.-C, and Marangos, P.J.: [^h] dipyridamole and [3H]nitro-
benzylthioinosine binding sites in guinea pig brain: a comparison. Pflugers Arch.
Eur. J. Physiol. 407 (Suppl 5): 29, 1986.
Deckert, J. and Marangos, P.J.: Hormonal interactions with benzodiazepine binding
sites in vitro. Life Sci. 39: 675-683, 1986.
Marangos, P.J.: Biochemical and pharmacologic properties of brain adenosine recep-
tors and uptake sites. Pflugers Arch. Eur. J. Physiol. 407 (Suppl 5), 1986.
Marangos, P.J.: Calcium antagonists and the brain adenosine system. In Shagass,
C, Josiassen, R.C., Bridger, W.H., Weiss, K.J. , Stoff, D. and Simpson, G.M.
(Eds.): Biological Psychiatry, 1985 (Developments in Psychiatry, Vol. 7).
Amsterdam, Elsevier, 1986, pp. 308-311.
Marangos, P.J., Deckert, J., and Bisserbe, J.C.: Central sites of adenosine action
and their interaction with various drugs. In Gerlach, E. and Becker, B.F. (Eds.):
Topics and Perspectives in Adenosine Research, 1986, pp. 1-634.
Tamborska, E. , Insel, T., and Marangos, P.J.: "Peripheral" and "central" type
benzodiazepine receptors in Maudsley rats. Eur. J. Pharmacol. 126: 281-289, 1986.
Marangos, P.J. and Deckert, J.: [^H] dipyridamole binding to guinea pig brain mem-
branes, possible heterogeneity of central adenosine uptake sites. J. Neurochem. 48:
1231-1237, 1987.
Marangos, P.J., Patel, J., Smith, K. , and Post, R.M.: Adenosine antagonist
properties of carbamazepine. Epilepsia 28: 387-394, 1987.
Deckert, J., Bisserbe, J.-C, and Marangos, P. J. : Quantitative [^h] dipyridamole
autoradiography: evidence for adenosine transporter heterogeneity in guinea pig
brain. Naunyn-Schmiedebergs Arch. Pharmacol., in press.
142
ZOl MH 01833-07 BP
Deckert, J., Estal, L.B., Marangos, P.J. and Cooper, S.J.: CGS-8216 treatment
decreases central type benzodiazepine receptors in rat brain. Eur. J. Pharmacol.,
in press.
Klein, E., Marangos, P.J., Montgomery, P., Bacher, J., and Uhde, T.W.: Adenosine
receptor alterations in nervous pointer dogs, a preliminary report. Clin. Neuro-
pharmacol . , in press.
Marangos, P.J., Campbell, I.e. and Cohen, R.M. (Eds.): Neurobiological Research,
Vol. II; Functional and Clinical Aspects of Neuronal and Glial Proteins. New York,
Academic Press, in press.
Marangos, P.J., Insel, T.R., Montgomery, P. and Tamborska, E. : Brain adenosine re-
ceptors in Maudsley reactive and non-reactive rats. Brain Res. , in press.
Marangos, P.J., Montgomery, P., Weiss, S.R.B., Patel, J., and Post, R.M. :
Presistent upregulation of brain adenosine receptors in response to chronic
carbamazepine treatment. Clin. Neuropharmacol. , in press.
Morgan, P.F. and Marangos, P.J.: Ontogenetic appearance of the adenosine receptor
precedes N-protein coupling in rat forebrain. Develop. Brain Res., in press.
Morgan, P.F. and Marangos, P.J.: Comparative aspects of nitrobenzylthioinosine
and dipyridamole inhibition of adenosine accumulation in rat and guinea pig
synaptoneurosomes. Neurochem. International, in press.
Morgan, P.F., Montgomery, P., and Marangos, P.J.: Ontogenetic profile of the
adenosine uptake site in rat forebrain. J. Neurochem., in press.
Morgan, P.F., Patel, J., and Marangos, P. J. : Characterization of [3H]R0 5-4864
binding to calmodulin using a rapid filtration technique. Biochem. Pharmacol., in
press.
143
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
nrtnh^r 1 1 ORfi fn .^ppfpmhpr ^O 1 0«7
PROJECT NUMBER
ZOl MH 00450-13 CP
TITLE OF PROJECT (80 charactert or less. Title must lit on one line between the borders.)
Riolncriral Rhyfhms in Affertivc Tllnpcs
:>gir,ai Khy
pXl investiS/
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Harm, title, laboratory, and institute affiliation)
PI: D. Sack Chief, Inpatient Services CPB/NIMH
Others: W.Mendelson Chief, Unit on Sleep Studies CPB/NIMH
W. Duncan Research Psychologist CPB/NIMH
N. Rosenthal Chief, Outpatient Services CPB/NIMH
R. Skwerer Medical Staff Fellow CPB/NIMH
F. Jacobsen Medical Staff Fellow CPB/NIMH
T. Wfihr Chief, riiniral Psyr.hnhinlogy Branch CPB/NIMH
COOPERATING UNITS (If any)
LAB/BRANCH
riiniral Psyr.hrthinlogy Branr.h
INSTITUTE AND LOCATION
NIMH. NTH. Be.thesda. Maryland 7089?.
TOTAL MAN- YEARS:
1
PI
ROFESSIONAL:
0.5
0.5
CHECK APPROPRIATE BOX(ES)
(^ (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In our present study we have been comparing the circadian system of depressed patients and
normal controls under routine ward conditions and then repeating these measurements in
conditions where the internal and external sources of masking have been controlled by holding
diet, activity, posture, lighting and wakefulness constant.
Our prehminary data indicated that the temperature rhythm in patients with affective disorders is
increased in both depression and mania compared to controls and the timing, or phase of the
temperature is shifted to an abnormally early time in both phases of the iUness. We have studied an
additional 5 patients and 5 normal controls and the results or these experiments are being analyzed.
We have confirmed oiu* previous observation that nocturnal TSH secretion is decreased and the
response to sleep deprivation is dimished in depression, in a new study of 34 predominantly
unipolar patients. "Rius, diminished nocturnal TSH secretion does not appear to be restricted to a
particular diagnostic subgroup of depression but is a characteristic feature of this disorder.
In order to better understand the basis for the circadian and sleep dependent changes in TSH
secretion, we have investigated the response of TSH to TRH administered intravenously in the
morning and at night with subjects awake and asleep. A TSH was significantly higher when
subjects were awa^e at night compared with asleep whereas circulating free and total thyroid
hormones did not differ on the two conditions. These data suggest that lower TSH levels during
sleep are due to changes in pituitary sensitivity to TRH which are central in origin, perhaps due to
the inhibitory effect of one or more neuropeptides that affect TSH secretion.
145
PHS 6040 (Rev. 1/84)
SPO ai4-Bia
ZOIMH 00450-13 CP
Project Description:
The details of this project have been extensively described in annual report ZOl MH 00450-11
CP and will only be reviewed briefly here. Abnormalities in the circadian rhythms of
hormones, neurotransmitters, and body temperature have been previously described in
depressed patients compared with controls. Many of these observations have not been
consistently replicated and this may have been due to artifacts related to diet, activity, sleep and
posture. In addition apparent differences in circadian rhythms in patients and normals may
occur secondary to changes in behavior rather than to a change in the properties of the intrinsic
biological clock or clocks regulating these systems. In order to determine the basis for
apparent differences in the circadian rhythms in patients with affective disorders we have
measured the circadian rhythms in a number of physiological variables under our usual ward
conditions and have repeated these measurements under conditions where activity, sleep,
temperature, diet and wakefulness were held constant. An additional purpose has been to
determine the relationship between disturbances in circadian rhythms and the antidepressant
effects of sleep deprivation.
Methods:
The methods have been described in detail in ZOl MH 00450-1 1 CP.
Findings to Date:
As part of a collaborative study with the University of Heidelberg, we have studied 34 patients
with major depression and 12 normal controls using a new method derived from our previous
studies. TSH and prolactin levels were measured at 2 am at baseline and during a night of total
sleep deprivation. TSH levels and the increase in TSH secretion with sleep deprivation were
significantly lower in depressed patients compared with normal controls. Sleep deprivation
increased TSH levels in all controls subjects but levels actually decreased in 1/3 of the
depressed subjects. Although sleep deprivation significantly reduced the severity of depression
in the patients, there was no correlation between clmical improvement and hormone levels at
baseline or their change during sleep deprivation.
In order to further understand the basis for abnormal nocturnal TSH secretion in depressed
patients, we studied the nocturnal responses of TSH to TRH in 9 healthy controls. Subjects
underwent IV TRH challenge tests (500 meg) on three conditions: awake in the morning,
asleep at night, and awake at night. TRH responses were significantly greater at night when
subjects were awake than on either of the other two conditions but free and total thyroid
hormone concentrations did not differ. These data suggest that lower TSH levels during sleep
may be due to inhibition of TSH responsiveness which is central in origin.
Significance to Biomedical Research:
1. The disturbances in temperature regulation and in the hypothalamic-pituitary-thyroid axis
suggest that patients with depression may suffer from an underlying disturbance of metabolism
and thermogenesis and that die symptoms of depression and mania may reflect a physiological
adaptation to this metabolic disturbance. Additional metabolic studies are required to elaborate
on this hypothesis.
2. Contrary to our hypothesis, there was no correlation between the cUnical response to sleep
deprivation and changes in the hypothalamic-pituitary-thyroid axis. However, we have shown
that low nocturnal TSH and a diminished response to sleep deprivation is a characteristic
146
ZOIMH 00450-13 CP
hormonal abnormality in depression and have developed a simple and reliable method for
assessing nocturnal TSH levels in a clinical population.
3. In our most recent experiment we have found that pituitary sensitivity to TRH varies as a
function of time of day and state of consciousness (asleep versus awake). Sleep inhibits TSH
responses to TRH via a central mechanism. Several peptides and neurotransmitters have been
shown to inhibit TSH and night, and excessive inhibition by one or more of these may be
responsible for the abnormal HPT responses in depressed patients.
Proposed Course:
We intend to study additional subjects in order to elaborate on our preliminary findings and in
particular to clarify the relationship between these circadian disturbances and improvement with
sleep deprivation and other antidepressant therapies.
Publications
Sack, D.A., James, S.P., Scherer, M.A., Linnoila, M., Wehr, T.A.: The diurnal variation in
MHPG is aboUshed but a variation in HVA persists under constant conditions. Arch Gen
Psychiatry in press.
Sack, D.A., James, S.P., Rosenthal, N.E., Wehr, T.A.: Deficient nocturnal surge of TSH
during sleep and sleep deprivation in rapid-cycling bipolar patients. Psychiatry Research in
press.
147
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02193-05 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. We must tit on arte line between the tx>rders.)
Clinical Studies of Insomnia
PRINCIPAL INVESTIGATOR (List other professional personnel behw the Principal Investigator.) (Name, title, laboratory, and institute etfillation)
PI: W. B. Mendelson Chief, Section on Sleep Studies CPB/NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Psychobiology Branch
SECTION
Section on Sleep Studies
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
0.3
OTHER:
1.2
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects D (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Intervievi/s
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our work has shown insomniacs have some disturbance of perception. We have reported
insomniacs have cognitive difficulties during the day. Their sleep is not different when compared
to normals, but insomniacs report only half as much sleep as their normal controls. This suggests
insomniacs have some misperception of their state of consciousness.
In an earlier study, we used meaningful (subject's name) and meaningless(electronic tone) stimuli
to arouse the subject from different stages of sleepCjust after lights out, stage II, stage IV, REM,
after a movement time). Subjects were most difficult to arouse from REM and stage IV. The
subjects were insomniacs and their matched controls.
In our current study, we are interested in the effects of hypnotics on this paradigm. Although the
effects of hypnotic on subjective and objective measures of sleep is well documented for
continuous sleep, little is known about their effects on sleep with forced awakenings. We found
the hypnotic, flurazepam, raised the arousal threshold for all stages of sleep, but did not alter
subjective measures of sleep when compared to placebo.
This project is no longer being pursued by the Branch and was terminated in March, 1987.
149
PHS 6040 (Rev. 1/84) OPO •<4.sii
ZOIMH 02193-05 CP
Project Description:
Insomniacs were screened for chronic insomnia, then given a Polysomnogram to rule out sleep
apnea and other sleep disorders. The study consists of four nights (2 stimulus conditions each
with placebo and drug).
Methods:
Ten insomniacs were given a PSG and then adapted for one night. The study consisted of four
nights. Subjects were awakened from 5 different stages of sleep. The arousal stimuli were the
subject's name repeated in a monotone voice for 2 minutes (maximum stimulus duration) or a
continuous electronic tone. Subjects were given drug and placebo in both stimulus conditions.
When the subjects were awakened, they were asked a series of questions about their sleep and
how they felt.
Findings to Date:
The arousal threshold differs across the stages of sleep. This is consistant with previous
studies on arousal threshold. The arousal threshold was greatest for stage IV and REM. They
were greater for meaningless than for meaningful stimuli. Flurazepam increased the arousal
thresholds in both stimulus conditions and decreased sleep latency, but it did not effect the
subjects perceptions of estimated time asleep.
Significance to Biomedical Research:
These data suggest while hypnotics raise arousal thresholds and decrease sleep latency, they do
not alter an insomniac's preception of his sleep.
150
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl m 02197-01 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. TiOe must tit on one //ne betwe€r\ the borders.)
Study of the Effects of Light and Triazolam on Delayed Sleep Phase Syndrome
PRINCIPAL INVESTIGATOR (List ottier professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: N.Rosenthal, Chief, C)uQ)atient Services CPB/NIMH
Others: K.Kelly Medical Staff FeUow CPB/NIMH
J.Vanderpool Medical Staff Fellow CPB/NIMH
P. Schulz Social Worker CPB/NIMH
T. Wehr Chief, Clinical Psychobiology Branch CPB/NIMH
E. Souetre Visiting Fellow CPB/NIMH
COOPERATING UNITS (if any)
Richard Allen, Ph.D., Johns Hopkins Sleep Center, Upjohn Pharmaceutical Company
UAB/BHANCH
CHnical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.5
PROFESSIONAL:
0.5
2.0
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Delayed sleep phase syndrome (DSPS) is a condition characterized by a delay in the timing of sleep onset and
waking. Generally present from an early age, DSPS usually produces considerable difficulties for those who are
required to function on a 9-to-5 schedule. Most subjects have tried unsuccessfully to correct their sleep-wake cycle
many times. One type of currently available treatment, chronotherapy, involves delaying the timing of sleep onset
further each day untU it reaches the desired clock time. Although this is quite effective in some cases, it is
inconvenient and the benefits are often transient.
The object of the present study is to recruit subjects who complain of DSPS, to determine the clinical and
demographic characteristics of these individuals, and to try to correct their circadian rhythm abnormalities by
judicious use of exposure to bright light and darkness, and by using the short-acting benzodiazepine, triazolam. Both
of these strategies are derived from studies of circadian rhythms in animals, the timing of which is ordinarily
influenced by the light-dark cycle. In recent years we have found that light of intensity greater than ordinary indoor
lighting has a variety of effects on brain function. For example, it is capable of suppressing melatonin, reversing
depression in seasonal affective disorder, altering norepinephrine and serotonin metabolism, and affecting resting
metabolic rate and immune function. There is also evidence that bright light can influence the timing of circadian
rhythms in humans.
We identified 93 subjects with DSPS and noted their repcMted patterns of sleeping and waking, as weU as the
difficulties encountered as a result of their sleep problem. We have studied sleep architecture in 11 cases and have
not found patients to be suffering from another type of sleep disturbance. Three patients have been treated with both
active (bright light in the morning plus dark goggles in the evening) and control (dimmer light in the morning and
transparent goggles in the evening) light treatment conditions, and it appears that the active but not the control
condition has a beneficial effect on the timing of sleeping and waking. However, the results are preliminary and
I more subjects will have to be run before a definitive statement can be made as to the value of bright light in the
I treatment of DSPS. We have yet to explore the value of triazolam in this condition.
151
PHS 6040 (Rev. 1/B4)
spo si4-aii
ZOIMH 02197-01 CP
Project Description and Methods:
A group of patients, who have difficulty going to sleep and waking up at conventional times,
has previously been described. This condition, termed delayed sleep phase syndrome (DSPS),
has been shown to cause significant unhappiness in some individuals, who are unable to get to
work on time and suffer the social consequences of being on a different sleep-wake schedule
from most other people. Typically these individuals have made many unsuccessful attempts to
correct their abnormal sleefHwake cycle; indeed, it often appears to be rigidly set in place. A
non-pharmacological treatment, called chronotherapy, in which the patient is advised to go to
sleep at progressively later times each day until the desired sleep onset is reached, has proven
successful in some individuals. However, it is an inconvenient treatment, involving several
days of disrupted schedules and, furthermore, the benefits appear to be short-lived in some
cases, who soon find their sleep onset time drifting progressively later again.
As our understanding of the non-visual, biological effects of light (particularly bright light) has
expanded, we have become aware that light may well play a significant role in setting the
timing of sleeping and waking in humans. If this is so, then perhaps bright light could be used
therapeutically to help patients with DSPS entrain to a more normal sleep schedule. Recent
studies in rats have shown that the short-acting benzodiazepine, triazolam, is capable of
shifting the timing of free-running biological rhythms. It is conceivable that this effect of the
drug could be put to use in helping patients with DSPS entrain to a desired sleep-wake
schedule. In fact, an optimal solution might be to use triazolam to help patients to comply
initially with an unusual sleep schedule and, later, to use bright light alone to maintain the new
sleep schedule.
In the present study, we plan to recruit a population of patients with DSPS, who are motivated
to have their sleep schedules altered, and who are willing to participate in a research study in
order to do so. The initial phase of the study will involve describing the clinical population by
means of specially prepared screening instruments, clinical interviews and standardized
structured psychiatric interviews. Patients will need to meet certain predetermined inclusion
criteria in order to be included in the study. They will then be given daily rating forms for
recording their sleep pattern and be asked to wear a wristwatch-like activity monitor for two
weeks, in order to corroborate prospectively their history of delayed sleep phase. Patients will
be given a physical examination and a polysomnogram in order to rule out other sleep disorders
that could interfere with our making an accurate diagnosis, or with our ablility to make effective
interventions.
After the initial phase of the study, subjects will be treated in a crossover design in which
conditions of light and dark will be manipulated. Subjects will be assigned to two two-week
treatments in random order: 1) two hours of bright (2500 lux) full-spectrum light in the
morning, in conjunction with dark goggles, worn for several hours in the evening; and 2) two
hours of dim (300 lux) light in the morning, in conjunction with transparent goggles, worn for
several hours in the evening. We predict that the former condition will be active and will have
the effect of bringing dawn and dusk earlier; the latter condition should be inactive and thus
serve as a control. If the timing of light and dark are important for the entrainment of circadian
rhythms in DSPS patients, then one would expect that the active treatment would help entrain
subjects to earlier sleep onset and wake times.
152
ZOl MH 02197-01 CP
Findings to Date:
Ninety-three subjects who responded to our initial publicity met criteria for DSPS. Of these 69
(74%) were women and 24 (26%) were men. Mean age of responders (± S.D.) was 34.4 ±
10.4 years; Mean age of onset of noticeable, abnormally delayed sleep onset was 10.1 ± 9.0
years; and patients reported that their sleep schedules became a problem at 15.0 ± 10.8 years.
Sixty -eight subjects (72%) reported that their DSPS interfered with work functioning to a
moderate or marked degree; a similar degree of impairment in interpersonal relationships was
reported by 52 subjects (56%). On average subjects reported going to bed at 1.25 a.m. on
weekdays and 2.26 a.m. on weekends but noted that sleep onset occurred somewhat later
(2.15 and 3.05 a.m. respectively). Corresponding average wake-up times for week and week-
end days were 9.20 and 10.50 a.m. Most patients (86%) described the quality of their sleep as
sound.
Polysomnograms were run on 1 1 subjects and no sleep abnormality, other than the shift in
timing, was noted in 9 of these cases. Three subjects have been run through the two treatment
conditions thus far. They have observed that the bright light in the morning, combined with
the use of dark goggles in the evening, has induced a significant improvement in daily
rhythms. It has been easier for them to fall asleep and wake up earlier, and they have felt more
alert in the morning. Preliminary analysis of sleep latency testing, performed at 9.00 a.m.
shows that patients have longer sleep latencies after the bright light than after the dim light
treatment. This latter, control condition was not experienced as helpful in any way.
Significance to Biomedical Research:
If bright light should prove to be helpful in re-entraining the timing of daily rhythms in DSPS,
this will add a new clinical application to its increasing number of potential uses, as well as
provide us with further insights into the effects of light on circadian rhythms in humans.
Besides helping individuals with chronic abnormalities in circadian rhythms, this information
may be helpful in the treatment of the more common temporary shifts in circadian rhythms
found in shift-workers and in jet-lag, both of which produce dysphoria and functional
impairment
Proposed Course:
We plan to continue to run DSPS patients through the crossover study to determine whether
bright light can indeed induce a statistically and clinically significant effect on the entrainment
of their daily rhythms. In those people who are only partially helped by bright light we plan to
use the short-acting benzodiazepine, triazolam, to improve compliance and enhance the phase-
shifting effects of bright light.
153
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02201-05 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT fflO characters or lass. Title must lit on one line between the borders.)
'. ^arly Versus Late Partial Sleep Deprivation in the Treatment of Depression
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
'I: D. Sack Chief, Inpatient Services CPB/NIMH
Others: T.Wehr Chief, Clinical Psychobiology Branch CPB/NIMH
N. Rosenthal Chief, Outpatient Services CPB/NIMH
W. Mendelson Chief, Unit on Sleep Studies CPB/NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
PROFESSIONAL;
CHECK APPROPRIATE BOX(ES)
[^ (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Total sleep deprivation, partial sleep deprivation, and shifting the time of the sleep period several
hours earlier than usual can improve depression and can induce mania. Also, recovery sleep after
these interventions can induce depression. Thus, changes in the timing and duration of sleep could
trigger episodes of depression and mania during the natural course of affective illness, and
manipulations of sleep could be used to treat and to prevent affective episodes. This project was
designed to investigate the importance of the timing of sleep in patients' clinical responses to sleep
manipulations. In particular, we hypothesized, on the basis of previous findings, that being awake
or asleep in the second half of the night was critical for the responses. Identification of a critical
circadian phase for these effects of sleep would provide an important clue to their mechanisms and
would facilitate the design of effective and practical sleep deprivation treatments for depression.
The antidepressant response to partial sleep deprivation early in the night (PSD-E) was compared
with the response to partial sleep deprivation late in the night (PSD-L) in 16 drug-free depressed
inpatients using a balanced order crossover design. PSD-L had a significantly greater
antidepressant effect than PSD-E. The response to PSD-L was sustained and enhanced by a second
night of treatment. Patients had significantly shorter sleep durations and reduced REM sleep on
PSD-L that did not occur on the PSD-E condition. There was a significant negative correlation
between response to PSD and sleep duration, and in particular, REM sleep duration, on the late
sleep deprivation condition. Thus, the amount and timing of sleep appeaar to be factors in the
response to PSD, and the effect of this procedure on REm sleep may be important
155
PHS 6040 (Rev. 1/84)
CPO 9l4-at(
ZOl MH 02201-05 CP
Project Description:
This project has been extensively described in ZOl MH 02201-04 CP. Briefly the objectives of
this study were:
1) To determine the importance of the timing of the sleep period in the antidepressant response to
PSD.
2) To describe the diagnostic, biochemical and neuroendocrine predictors of sleep deprivation.
3) To assess the therapeutic efficacy of a sustained course of treatment (3 weeks) with PSD.
Methods:
The methods have been extensively described in ZOl MH 02201-04-CP.
Significance to Biomedical Research:
1) The spontaneous changes in sleep that occur in depression and mania are closely asociated with
switches from one mood state to the other and affective disorders may alternatively be characterized
as disorders of the sleep-wake regulatory system. Treatment with TSD and PSD exogenously
impose a 'manic' like sleep schedule and have acute antidepressant effects in both bipolar and
unipolar patients. These studies will contribute to our understanding how changes in the sleep-
wake cycle result in alterations in mood perhaps through changes in neurochemistry and
neuroendocrinology .
2) Current pharmacotherapy of depression requires approximately three weeks to work whereas
the response to PSD begins immediately. If the clinical effects of PSD were sustainable, it would
considerably shorten the period of morbidity and potentially decrease the risk of suicide in
depressed patients.
Proposed Course:
The study has been completed, and the project is terminated.
Pubhcations:
Sack DA, Duncan W, Rosenthal NE, Mendelson WB, Wehr, TA: The timing and duration of
sleep in partial sleep deprivation therapy of depression. Acta Psychiatric Scandinavica, in press.
156
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02205-02 CP
PERIOD COVERED
October 1. 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit or> orte line tietween the borders.)
Antidepressant Effects of Light in Seasonal Affective Disorder and Normal Controls
PRINCIPAL INVESTIGATOR (List other prolessional personrtel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: N.Rosenthal Chief, Unit on Outpatient Studies CPB/NIMH
Others:
R. Skwerer,
K. Kelly,
S. Kasper
P. Schulz
S. Rogers
A. Yancey
Medical Staff Fellow
Medical Staff Fellow
Guest Researcher
Social Worker
Registered Nurse
Guest Worker
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
CC/NURS
CPB/NIMH
COOPERATING UNITS (If any)
Morris Waxier, Ph.D., Food and Drug Administration
George Brainard. Ph.D.. Jefferson Medical College
LAB/BHANCH
CUnical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
2JL
PROFESSIONAL
JL5_
±
CHECK APPROPRIATE BOX(ES)
13 (a) Human subjects
D (a1) Minors
D (a2) lnterviev>?s
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have previously shown that exposure of the eyes to bright light, but not ordinary room light,
can reverse the winter depressive symptoms in patients witih seasonal affective disorder (SAD).
This light treatment may be effective during the morning, the evening or even during ordinary
daylight hours. Normal subjects with no history of winter difficulties do not seem to benefit from
this light treatment. This past year we have gone on to study whether light in a narrow band of the
visual spectrum (blue or red) is as effective as the full-spectrum light used previously. We have
also asked whether normal subjects who complain of mild winter difficulties might benefit from
conventional bright light treatment
In a crossover study on 21 subjects we found that although full-spectrum light tended to be be
more effective than either blue or red light, this did not reach statistical significance, perhaps
because the narrow-band light was clinically active to some degree. If tiiis is so, it would suggest
that the antidepressant effects of light in SAD are mediated by wavelengths that span the full visual
spectrum. A separate study of full-spectrum light in 40 normal subjects, 20 with and 20 without a
history of mild winter difficuties, such as low energy level or decreased productivity, showed that
the former group benefited from light treatment whereas the latter group did not The degree of
improvement was related to the duration of treatment, 5 hours per day proving superior to 2 hours
per day. Since there seems to be a high prevalence of normal subjects with mild winter difficulties,
this finding may have widespread public health implications.
157
PHS 6040 (Rev. 1/84)
SPO «I4-»II
ZOl MH 02205-02 CP
Project Description and Methods:
1. The colored light studv:
We have previously found that patients with SAD show antidepressant responses to bright full-
spectrum light exposure mediated via the eye. In this study we attempted to evaluate which
part of the spectrum was important in mediating this antidepressant response. Twenty-one
patients with SAD were followed in our outpatient clinic until they became depressed, at which
time they entered a balanced-incomplete-block treatment study, in which each patient was given
two out of three possible treatments for one week each with at least one week of withdrawal
between treatments. The three treatments consisted of full-spectrum, red and blue light. All
sources used were fluorescent and the intensity as measured by number of photons per second
per unit of surface area was kept constant across the three conditions by modifying the number
of fluorescent tubes used and the distance away from the light source at which the subject was
asked to sit. Light treatments were administered for two hours each morning and two hours
each evening. Subjects were asked not to alter the times of sleeping and wa^ng during the
treatment week. Mood was monitored by Hamilton Depression Rating Scale (HDRS) ratings,
administered by blind raters. In order to evaluate patients' expectations of treatment, we
administered appropriate questionnaires before each treatment condition was started.
2. The effects of light on mood in normal subjects
This study was planned as a follow-up on the study of the previous year, in which neither
bright nor dim light, administered for two hours in the early morning, was shown to have any
mood-altering effects in 22 normal subjects. In that study subjects with any history of affective
vulnerability or difficulties in relation to the changing seasons were specifically excluded.
However, it had come to our attention that many individuals generally regardai as normal, may
nonetheless experience problems in relation to the changing seasons. We also wondered
whether the amount of light used in the earlier study, namely two hours per day, was
insufficient to produce mood changes in normal subjects.
In this study we recruited forty subjects, twenty of whom were regarded as completely normal,
having no personal or family history (first-degree relatives) of any psychiatric disturbance, nor
any complaint of regular difficulties in mood or behavior during the winter months. The other
twenty, which we designated as subsyndromal SAD (S-SAD) subjects, met the following
inclusion criteria: (1) They had never been treated for depression during the winter months,
had never regarded themselves as suffering from a disorder, nor had anyone else suggested
that they should seek treatment; (2) They had a history of winter difficulties, such as impaired
quality of life or decreased ability to function, for at least four weeks during at least two
consecutive winters. Both types of normal subjects were recruited by appropriate, separate
advertisements in the media. Before receiving Hght treatment all subjects were given a
standardized, self-administered questionnaire, inquiring into their history of seasonal changes
in mood and behavior. This questionnaire, the Seasonal Pattern Assessment Questionnaire
(SPAQ) was developed in the Intramural Research Program of the National Institute of Mental
Health.
Each group of subjects was further subdivided into two groups, one of which received a total
of two hours of light per day (one hour in the morning and one hour in the evening), and the
other of which received five hours of light per day (2.5 hours twice a day). The study was
conducted as a parallel design during the winter months, each group receiving only one
treatment regimen. Subjects were asked not to alter the times of sleeping and waking during
158
ZOIMH 02205-02 CP
as well as by self -ratings (Profile of Mood States and Visual Analog-type ratings). Subjects' a
priority predictions were again assayed.
Findings to date:
1. The colored light study:
Fourteen subjects in each study condition, a total of 21 subjects, were studied. There was a
trend towards a superior response to the full-spectrum condition. However, this did not reach
statistical significance. Although some response was noted following both red and blue light
conditions, it was not possible to say whether this represented some biological effect of these
wavelengths of light or whether the results were simply due to a placebo effect. It is possible
that the lack of statistical significance was a function of the small sample size and that the
negative result was a Type n error. Further studies of this kind would clearly require large
numbers of subjects. In addition, green-yellow light, the color perceived as brightest by the
retina or green Ught, the color to which the rods are most sensitive, would be interesting
controls for the red and blue light in future studies.
There was no positive correlation between the subjects 'a priori expectations, as determined by
standardized questionnaires, and outcome. In fact, there was a negative correlation between
expectations of both red and blue light and outcome following these treatments. This fmding
adds to the mounting evidence that prior expectations do not adequately explain the
antidepressant effects of Ught therapy.
2. The effects of light on mood in normal subjects
We found that neither 2 nor 5 hours per day of light treatment produced a significant change in
the normal group, who had reported a low level of changes in mood and behavior on history.
This corroborated our finding of the year before that Ught does not act as a euphoriant in
asymptomatic normals, Uke cocaine or amphetamine. Rather, it appears to help only those
individuals who complain of decreased energy or other behavioral changes in the winter, even
if these changes faU into the range of functioning generally regarded as normal. Such
functional impairment can be determined retrospectively by means of a simple self-administered
questionnaire, the Seasonal Pattern Assessment Questionnaire (SPAQ), which eUcits
retrospective information about level of functioning in different seasons. In these
subsyndromal SAD individuals it appears that a total duration of five hours of Ught exposure,
divided into two daily dosages, is more effective than two hours of treatment, similarly
administered.
Significance to Biomedical Research:
Of the two studies of Ught therapy performed this past year, the study of light in normal
subjects has more far-reaching implications. The findings of this study suggest that the scope
of individuals who stand to benefit from light treatment is greater than was formerly
appreciated. It appears likely that the subsyndromal form of SAD is more prevalent than the
more extreme form that has been the focus of previous studies. However, the individuals who
suffer from the less extreme form of the condition are less likely to seek medical help for their
problems. In fact, one of our criteria for selecting this population was that the individuals had
never previously specificaUy sought help for their winter difficulties. Thus the problem of
treating these people, by definition, faUs within the realm of public health workers rather than
cUnicians. It is important for these workers to be aware of the existence of numerous
159
ZOl MH 02205-02 CP
individuals whose performance and quality of life might well be improved by enhancing their
environmental light. The development of a simple self-administered retrospective
questionnaire, the SPAQ, is a valuable tool in the definition of this population. It has already
been widely used by fellow researchers, who have found it to be of value. Christopher
Thompson, at Charing Cross Hospital in London, has found that the questionnaire
differentiates between SAD patients, other affective disorder patients and normal individuals.
Dr. Michael Terman, at Columbia University in New York City, has performed an
epidemiological survey in which he has mailed this questionnaire to people randomly chosen
from the Manhattan telephone directory. He has found that at least 25% of those who returned
their questionnaires (57% of the surveyed population responded) complain of seasonal mood
and behavior problems of severity equal to or greater than the S-SAD individuals described
above. Based on our studies of the effects of bright light in normals. Dr. Terman has projected
that approximately 1.9 million people in the New York Metropolitan Area would benefit from
enhancement of their envirormiental Ught. The SPAQ is currently being used in several other
studies in the United States. It has also been translated into French, German and Russian, and
is being used in ongoing studies in Europe and the United States.
While our recent study of normal individuals showed that some normals benefit from enhanced
Ught exposure, it also confirmed our earlier finding that normal individuals without a history of
seasonal changes in mood or behavior do not appear to benefit from enhanced environmental
lighting. This finding would suggest that indiscriminate enhancement of environmental
lighting in the work and living environments of all people is not warranted. Rather, some way
of selecting those individuals who would benefit from enhanced environmental lighting is
desirable, and the SPAQ would seem to be a logical instrument to use in such a selection
process. Enhanced environmental lighting should be recommended for those with high scores
on this questionnaire.
The results of the colored light study were equivocal. Although there was a suggestion that the
full-spectrum light was superior to both red and blue lights, this did not reach statistical
significance perhaps, in part, because the colored lights had some real biological activity of
their own. The partial efficacy of blue and red light might imply that the antidepressant effects
of hght in SAD are induced by a wide array of wavelengths witiiin the visual spectrum.
However, it remains to be determinedwhich wavelengths exert the most powerful effect in this
regard.
Proposed Course:
Further studies of the antidepressant effects of light can be conceptualized along two broad
lines: 1. the ongoing definition of which individuals are most likely to benefit from light; and 2.
continued investigations of the formal properties of light involved in phototherapy. These
broad areas will be addressed in turn.
1. Who stands to benefit from enhanced environmental light?
A. The General Population: As discussed above, we have more closely defined the range of
individuals who may benefit from enhanced environmental lighting. We plan to extend 5ie
approach taken by Terman in his Manhattan project with two modifications: (1) We plan to use
state-of-the art epidemiological techniques for studying the prevalence of seasonal changes in
mood and behavior in the population of Montgomery County. This would involve the use of
random-digit dialling and telephone interviewing, using the SPAQ, of a randomly selected
sample by an outside group of experts in these techniques. Once the sample has been defined,
160
ZOIMH 02205-02 CP
we intend to approach randomly selected individuals within subsamples of the larger
population, based on the severity of their seasonality as determined by SPAQ scores, and offer
them light treatment in the winter months. In this way we hope to project accurately what
proportion of the population, who do not seek out any medical or psychiatric intervention,
would benefit from having their environmental light enhanced.
In addition to the above study, less ambitious surveys involving questionnaires sent by mail
and administered to patients waiting to be seen in doctors' offices, are in progress, in
collaboration with the Psychiatric Institutes of America, to evaluate the prevalence of seasonal
problems at three different locations at different latitudes: Nashua, New Hampshire;
Washington, D.C.; and Sarasota, Florida.
B. The elderly: We are planning to study the effects of bright light in an elderly population,
not selected on the basis of prescreened seasonaUty. It has come to our attention that the
symptoms of SAD frequently become worse over time. It is unclear what factors are involved
in this symptomatic deterioration but declining eyesight and decreased perception of brightness
may be relevant. The migration of the elderly to Florida and other milder climates, especially in
the winter months, is a well-recognized phenomenon. This migration has popularly been
attributed to difficulty with the cold rather than to deprivation of environmental light.
However, it is quite conceivable that the latter factor plays an important role in the discomfort
that prompts migration to the south. We recognize that not all elderly individuals are fortunate
enough to be able to move south. The institutionalized elderly come to mind immediately in
this regard. Unable to get outdoors easily during the winter, they are particularly vulnerable to
the behavioral and mood effects of light deprivation. We plan to study the effects of light on a
selected group of elderly individuals and to explore the degree to which this may improve the
quality of their lives. It would be important in such a study, as in all efficacy studies, to
control for the influence of non-specific placebo factors in the behavioral response to lighting
interventions.
2. Studies of the Formal Properties of Light Required for Effective Phototherapy
Studies previously undertaken in the Intramural Research Program have examined several
aspects of the formal properties of effective phototherapy. For example, we have found that,
in order for light to be effective, it should be of high intensity and that the eyes rather than the
skin should be exposed to the light. We have also shown, in a series of studies, that the timing
of light treatment is not crucial for its effects. Most recently we explored the spectrum of Hght
required for effective phototherapy. At this time we have not decided whether we shall
undertake further studies of the formal properties of light required for effective phototherapy in
the coming year.
An important unresolved issue is whether the small amount of ultraviolet light present in the
full-spectrum light used in most studies performed to date, is necessary for its antidepressant
effects. Preliminary findings both from Dr. Alfred Lewy in Oregon and from Dr. John
Docherty in New Hampshire suggest that this is not the case. Our group has been involved as
collaborators in the latter of the two studies. We are planning to continue the New England
collaboration in the forthcoming year, and Dr. John Docherty has recruited the support of
several mental health centers in comparing light sources with and without ultraviolet light. The
importance of this question grows as it becomes clear that some individuals require enhanced
environmental lighting for several hours a day for the larger part of the year, perhaps for most
of their Uves. We know that in excessive dosage ultraviolet light has potentially harmful effects
on both the eye and the skin. If it can be definitively established that ultraviolet light is
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unnecessary for the mood-altering and behavioral effects of enhanced environmental lighting,
UV light should be excluded from the light sources in future. At present, however, there is
inadequate data on which to base such widespread recommendations.
There are many outstanding questions pertaining to the formal properties of light treatment
What is the optimal duration, intensity, timing, and spectrum of light? What is the safest,
cheapest and most convenient way to administer light therapy? All of these questions are
eminently testable and would almost certainly lead to useful practical information. The major
question in setting our research priorities is whether, given finite resources, we wUl be able to
pursue these research areas as well as investigate the biological effects of light therapy
administered in a standard fashion. This latter approach has yielded exciting preliminary
findings (see Annual Report # ZOl MH 02206-03 CP) and we plan to pursue it further. Now
that the efficacy of phototherapy has been widely confirmed by a number of groups both in the
U.S. and Europe, several groups are in the process of undertaldng the types of formal studies
mentioned above. We have not as yet decided whether we in the Intramural Program of the
NIMH should continue in this direction or whether, at this time, it is better to leave these
cUnical trials to others whUe we pursue the more specialized psychobiology studies we are
equipped to do.
Publications:
James, S. P., Wehr, T. A., Sack, D.A., Parry, B. L., Rosenthal, N.E. Treatment of
seasonal affective disorder with light in the evening. British Journal of Psychiatry, 147:
424 - 428, 1985.
Rosenthal, N.E., Carpenter, C.J., James, S.P., Parry, B.L., Rogers, S.L.B., Wehr, T.A.
Seasonal affective disorder in children and adolescents. American Journal of Psychiatry,
143: 3 :356 - 358, 1986.
Jacobsen, P.M., Rosenthal, N.E. Seasonal affective disorder and the use of light as an
antidepressant. Directions In Psychiatry, 6 (3): 1-7, 1986.
Wehr TA, Skwerer RG, Jacobsen FM, Sack DA, Rosenthal NE. Eye- versus skin-
phototherapy of seasonal affective disorder. American Journal of Psychiatry, 144: 753-757,
1987.
Parry BL, Rosenthal NE, James SP, Wehr TA. Treatment of a patient with seasonal
premenstrual syndrome. American Journal of Psychiatry, 144: 762-766, 1987.
Hellekson CJ, Rosenthal NE. New light on seasonal mood changes. Harvard Medical School
Mental Health Letter, 3(10): 4-6, 1987.
Wehr TA, Sack DA, Rosenthal NE. Antidepressant effects of sleep deprivation and
phototherapy. Acta Psychiatrica Belgica, S5: 593-602, 1985.
James SP, Wehr TA, Sack DA, Rosenthal NE, Mendelson WB. Experimental modalities in
the treatment of seasonal and non-seasonal affective disorder. Biological Psychiatry, Shagass
C, Josiassen RC, Bridger WH, Weiss KJ, Stoff D, Simpson GM (eds.), Elsevier, New York,
1985, pp. 144-146.
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ZOl MH 02205-02 CP
Mendelson WB, James SP, Rosenthal NE, Sack DA, Wehr TA, Gamett D, Weingartner H.
The experience of insomnia. Biological Psychiatry, Shagass C, Josiassen RC, Bridger WH,
Weiss KJ, Stoff D, Simpson GM (eds.), Elsevier, New York, 1985, pp. 1005-1006.
Rosenthal NE, Sack DA, Jacobsen FM, Parry BL, James SP, Tamarkin L, Arendt J,
Wehr TA: Consensus and controversy in seasonal affective disorder and phototherapy.
Biological Psychiatry, Shagass C, Josiassen RC, Bridger WH, Weiss KJ, Stoff D, Simpson
GM (eds.), Elsevier, New York, 1985, pp. 987-989.
Rosenthal NE, Sack DA, Jacobsen FM, Skwerer RG, Wehr TA. Seasonal affective
disorder and light: past, present and future. Clinical Neuropharmacology, Bunney WE, Jr.,
Costa E, Potkin S (eds), 9(4): 193-195, Raven Press, New York, 1986.
Hellekson CJ, KUne JA, Rosenthal NE. Phototherapy for seasonal affective disorder
in Alaska. Am J ournal of Psychiatry, 143(8): 1035-1037, 1986.
Rosenthal NE. Seasonal affective disorders: Seasonal energy syndrome? In Reply.
Archives of General Psychiatry, A3: 188-189, 1986.
Rosenthal NE, James SP. Reply to letter on seasonal affective disorder. Br itish J ournal of
Psychiatry, 148: 478-479, 1986.
Rosenthal NE, Sack DA, Wehr TA. Seasonal effects on mood: The role of Ught, in Adelman
G (ed.). Encyclopedia ofNeuroscience, Vol II, Birkhauser, Boston, pp. 586-588.
Wehr TA, Rosenthal NE, Sack DA. Sleep deprivation, phototherapy and other non-
pharmacological treatments of affective illness, in Extein I (ed). Treatment of Drug-Resistant
Depressed Patients, APA Press, Washington DC in press.
Jacobsen FM, Wehr TA, Sack DA, James SP, Parry BA, Rosenthal NE. Seasonal affective
disorder: A review of the syndrome and its public health implications. American Journal of
Public Health, 77:57-60, 1987.
163
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02206-03 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. We must lit on one line t>etwBen the borders.)
Neurobiology of Seasonal Affective Disorder and Light Therapy
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atfiliatlon)
PI: N.Rosenthal Chief, Unit on Outpatient Studies CPB/NIMH
Others: R. Skwerer Medical Staff FeUow CPB/NIMH
F. Jacobsen Medical Staff Fellow CPB/NIMH
K. Kelly Medical Staff Fellow CPB/NMH
L. Tamarkin Research Biologist CPB/NIMH
T. Wehr Chief, Clinical Psychobiology Branch CPB/NIMH
R. Mghir Guest Researcher CPB/NIMH
COOPERATING UNITS (If any)
W. Berretini, BPB/NIMH, D. Jimerson, M.D., SBP/NIMH
M. Rudorfer, M.D., LCS/NIMH, E. Obarzanek, SBP/NIMH, C. Duncan, Ph.D., LPP/NIMH
L*B/BRANCH
Clinical Psychobiology Branch ^
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2
PROFESSIONAL
1
CHECK APPROPRIATE BOX(ES)
(3 (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Although it has been established that bright light is an effective treatment for SAD, the mechanism
of its action remains unknown. We have shown that our previous theory, that light works by
suppressing melatonin secretion, is an unsatisfactory explanation of this form of treatment
Another theory, proposed by Lewy and colleagues, that light exerts its antidepressant effects by
modifying crrcadian rhythms, is similarly unsatisfactory. We have continued to pursue our
investigations by measuring relevant biological variables in SAD patients and normal subjects and
evaluating the effects of light on these measures.
We have found several differences in patients with SAD and normals: (1) Increased mitogen
stimulation of peripheral lymphocytes in both summer and winter in patients compared to normals;
bright light suppresses this response in patients but enhances it in normals. (2) SAD patients show
increased resting metabolic rate (RMR) in winter compared to normals, and light significantly
suppresses the RMR. (3) Plasma norepinephrine levels in SAD patients are inversely proportional
to the severity of their depressive symptoms, and increased norepinephrine levels are observed in
response to light treatment in direct proportion to its antidepressant efficicacy. (3) A similar direct
relationship between treatment efficacy and a biological effect is observed in the amplitude
enhancement of the P300 component of the event related potential in SAD patients, a change seen
within 48 hours of beginning light therapy. (4) Brain serotonin pathways appear to be abnormally
sensitive in SAD patients as measured by psychological and hormonal responses (prolactin and
Cortisol) to injection of the serotonin agonist M-CPP. (5) Overnight plasma melatonin is low in
SAD, as in other depressed populations. (5) Light treatment appears to delay the nadir of Cortisol
secretion. (6) Recent sleep studies show a shorter total sleep time and greater REM percentage
and movement time in patients compared to controls. Light increases delta sleep time and sleep
efficiency in patients but decreases these parameters in control subjects. These biological findings
are discussed.
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SPO ai4-si(
ZOl MH 02206-03 CP
Project Description and Methods:
1. Study of the Psychobiological Effects of Light
This study was continued from the previous winter. Since light is capable of inducing rapid
and marked antidepressant effects in SAD patients, we evaluated the effects of light on systems
we regard as relevant to the psychophysiology of SAD, by comparing "on-light" and "off-
light" conditions in patients. Similar measurements in normal controls permitted us to evaluate
patient-normal differences.
SAD patients were followed longitudinally from the summer and fall, when they were
euthymic, into the winter. As they became depressed, they were assigned to one of two
conditions on a random basis: off-lights first or on-lights first, to avoid an ordering effect.
This assigimient determined whether psychobiological tests were performed before Ught
treatment in the first instance or on light treatment at first. In order to be included in the study,
patients were required to have been off lights or on lights for at least 10 days before coming
into our inpatient unit. Most of the patients were drug-free but a few patients on medications
were included provided they had been on the medications for an extended period of time and
the dosage could be held constant throughout the study. After the initial set of studies, patients
were crossed over to the alternate condition. Age- and sex-matched controls were recruited and
studied in the same way as the patients.
Studies consisted of 24-hour profiles of a variety of plasma hormones, drawn via an
indwelling intravenous catheter, EEG-recorded sleep studies for 2 nights (excluding an
adapatiation night), mitogen stimulation testing of lymphocytes in vitro, plasma norepinephrine
measurements in both recumbent and standing positions, event related brain potentials and
reaction time, and a lumbar puncture. An additional study performed this year but not last was
the resting metabolic rate (RMR), measured by indirect calorimetry. Not adl subjects agreed to
go through all studies. The lumbal' puncture was performed on the last day in the morning,
after a night of bedrest and with patients in a fasting state. Thirty-two mis of CSF were
removed and these were assayed for the monoamine metabolites, MHPG, 5-HIAA and HVA,
and the peptide neuromodulators, neuropeptide Y, peptide YY and growth hormone releasing
hormone (GHRH). Blood specimens were analyzed for prolactin, growth hormone, Cortisol,
melatonin and thyroid stimulating hormone (TSH). The immunological studies that were
performed as part of this study are reported elsewhere (See Annual Report #Z01 MH 0235-02
CP).
In order to obtain event related brain potentials (ERPs), subjects were tested with auditory and
visual versions of three reaction time tasks; 0. 10/0.90 stimuli were used to elicit P300s (die
component of the ERP that occurs 300 msecs after the presentation of the stimulus). Control
subjects were also tested twice. Eleven channels of EEC as well as EOG were recorded and
digitized at the rate of 200 Hz over an 1 l(X)-msec recording interval (See Annual Report # ZOl
MH 00509-05 LPP). A subset of 6 patients and 2 normals were tested several times over the
course of light therapy, on days 2, 3 and 10 of treatment, in order to define the time course of
the enhancement of the P300 component in relation to the change in mood.
2. Neuroendocrine Challenge with m-Chlorophenylpiperazine (mCPP)
There are many clues that point to the possible pathophysiological importance of serotonergic
abnormalities in SAD. Such abnormalities have long been postulated to be an important
etiological factor in depression in general. Swedish researchers have found that hypothalamic
serotonin content in post-mortem human brains drops to its lowest in the winter months.
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Carbohydrate intake has been linked to brain serotonin metabolism in both animals and
humans, and we have suggested that the carbohydrate craving seen in SAD may represent a
behavioral attempt to enhance deficient serotonergic functioning. These lines of reasoning have
led us to challenge SAD patients and normals with serotonin agonists.
In a previous study we evaluated the effects of 5-Hydroxytiyptophan (5-HTP) in SAD patients
and normals but found no difference in their neuroendocrine responses. Both groups showed
no change in plasma Cortisol but a relative suppression of prolactin secretion following 5-HTP.
In order to discriminate more effectively between neuroendocrine responses in patients and
normals, we recently administered the more selective serotonin receptor agonist, m-
chlorophenylpiperazine (mCPP) before and after light treatment ie., in depressed and remitted
states.
10 depressed patients with SAD and 11 controls matched by age, sex, weight, and menstrual
history (ie pre- or post-menopausal) were given an intravenous bolus of mCPP (O.lmg/kg)
before and after treatment for a week with phototherapy. All subjects were drug-free for more
than 3 weeks, phototherapy-free for more than 2 weeks, and maintained a stricfly controlled
sleep schedule (6:00-6:30 a.m. wake-up) and enviroimiental light exposure beginning at least
one week prior to the first infusion day. On each infusion day two FVs were started at 8:30
a.m. and blood samples were drawn at -10, 0, 2, 5, 10, 20, 30, 40, 50, 60, and 90 minutes in
relation to an infusion of mCPP at 10:00 a.m. Blood pressure and pulse were recorded every
5 minutes and oral temperature was recorded at -15, 0, 30, 45, 60, 90 minutes. Subjects
completed the following self-rating scales at -15, 30, 60, and 90 minutes: NIMH 24-item scale
(6 composite items: activation-euphoria, depressed affect, anxiety, dysphoria, altered self,
functional deficit). Drug Effects Rating Scale, Stanford Sleepiness Scale, and 15 Visual Analog
Scale (VAS) 100 mm line items. Serum samples were assayed for prolactin and Cortisol by
RIA, and for mCPP blood level by HPLC. Prolactin, Cortisol, temperature recordings, and
self-rating scale scores were analysed by ANOVA, and baseline and Amax values were
compared by t-tests. During the week between the two mCPP infusions, phototherapy (2500
lux, 4 hours per day) was given at home or work.
3. Photo-Immune Studies in SAD and Normal Subjects
Mitogen stimulation was performed on lymphocytes by treating them with the mitogens,
Concanavolin A, phytohemagglutinin, or pokeweed mitogen (see Annual Report ZOl MH
02325-01 CP for further details).
Findings to Date:
1. Studv of the Psychobiological Effects of Light
Overnight studies of plasma hormones showed significantly reduced peak melatonin levels
(P<.05) and a non-significant tendency to delayed onset of melatonin secretion in patients with
SAD compared to normal controls. Light treatment delayed the nadir of Cortisol secretion by
approximately one hour (P<.05).
Sleep Studies were performed in 10 SAD patients and 6 normal controls both on and off light
treatment. Group differences were present in total sleep time, which was less in patients than in
normals; and REM time, REM percent and movement time, which were greater in patients than in
normals. Significant interactions between subject group and light condition were found for sleep
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efficiency (P=.05) and delta sleep and delta percent (P=.02). Light increased delta sleep and
sleep efficiency in SAD patients but decreased these parameters in normal controls.
Plasma norepinephrine levels in SAD patients were inversely related to their level of depression
off light treatment, as measured by the HDRS (N= 14; r = - 0.75; P<.05). The increase in
resting plasma norepinephrine during the "on-light' condition was directly proportional to the
improvement in depression level, as measured by the HDRS (N= 13; r = 0.59; P<.05).
Resting Metabolic Rate (RMR) in 10 SAD patients tended to be higher than in 9 normal
controls during the off-light condition (1648 versus 1357 Kcal/day; P<.10). Light treatment
significantly decreased RMR in patients by an average of 201 Kcal/day (P<.05) but had no
significant effect on RMR levels in normals.
The enhancement of the P300 component of the Event Related Brain Potentials (ERPs) in
response to visual stimuli, observed the previous year in a small number of patients (N=7) was
sustained in a larger number (N=17). This finding was observed in tracings from three
different scalp sites and while subjects were performing two different tasks. Correlations were
between 0.5 and 0.6, with significance values ranging between P<.05 and P<.001. Once
again, we observed no such correlation in the P300 change and mood in auditory ERPs. We
studied the time course of changes in the ERPs in response to light treatment in 6 SAD patients
and 2 normal controls, who were tested several times during light therapy as well as at
baseline. The enhancement of P300 was found to occur in SAD patients as early as 2 days
after light therapy was started, and increased with increasing antidepressant response to
treatment.
Lumbar punctures were performed on a total of 1 1 SAD patients on and off light treatment and
9 normal controls off light treatment. No differences between groups or conditions were
observed in the amine metabolites, MHPG, HVA and 5-HIAA, or in the peptides,
neuropeptide Y, peptide YY or Growth Hormone Releasing Hormone.
2. Administration of M-CPP before and after light treatment
Effects of Light Treatment on Mood: Patients' Hamilton Depression Rating Scale (HDRS)
scores (mean+S.D.) fell significantly over the course of phototherapy, from 17.3±3.7 on the
first infusion day (TDl) to 7.9±4.3 on the second infusion day (TD2) (t=6.33,P<0.001).
NIMH 24-item self-rating scale: ANOVAs of the mean composite NIMH-24 scale scores of
patients and controls revealed a significant group x day x time interaction for "activation-
euphoria" (p<0.01). Patients showed a significantly greater rise in "activation-euphoria"
following mCPP than controls, particularly on TDl (prior to phototherapy). Patients' mCPP-
stimulated "activation- euphoria" normalized on TD2 (following phototherapy and relief of
depressive symptoms).
Patients' ratings of depressed affect were significantly higher than controls' ratings (p<0.001)
on both TDl and TD2, and ANOVA failed to reveal significant interactions. "Depressed
affect" trended to be higher in both groups on TDl than TD2 (p<0. 1) and also varied
significantly over time for both groups (p<0.05). Both patients and controls showed
significantly higher mCPP-stimulated values on TDl than on TD2 on the following factors:
altered self, anxiety, and functional deficit . The mean time course of ratings (group x time
interaction) also differed significantly between the groups for "altered self' (p=0.(X)01),
"anxiety" (p<0.005), and "dysphoria" (p<0.005).
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ZOIMH 02206-03 CP
Drug effects rating scale: Patients' mean drug effects ratings differed significantiy from those
of controls over time on the two infusion days (group x day x time interaction) for "slowed
down" (p=0.0001) and "drowsy" (p=0.051), with patients starting from higher baselines and
having less mCPP- stimulated slowing and drowsiness than controls on TDl. On TD2, the
patients' mCPP-stimulated ratings of "slowed down" and "drowsy" were similar to those of
controls.
Patients and controls also reported significantly more of the following side-effects over time on
TDl than TD2 (day x time interaction): chilled(p=0.0001), light-headed (p<0.005), trouble
remembering (p<0.05), yawning (p<0.05), and hot/flushed (trend, p=0.06).
Stanford Sleepiness Scale: Patients were sleepier than controls on both days, but showed a
trend for less mCPP-stimulated sleepiness than controls on TDl (group x day x time interaction
p<0.06).
Temperature: no significant baseline differences were observed between patients and controls
and ANOVA revealed no significant interactions or group differences. TTiere was a trend
(p<0.1) for patients' Amax TD2 temperature to be greater than that of controls.
Hormone Studies
A. Cortisol: Patients showed a trend (p<0. 1) towards higher basal serum Cortisol levels than
controls on TDl and showed significantiy higher basal Cortisol levels than controls on TD2
(p<0.05). Both patients and controls showed robust stimulation of serum Cortisol in response
to IV mCPP. The mCPP-stimulated Cortisol rise was significantiy greater over time in patients
than in controls on both test days (p<0.0(X)5). mCPP-stimulated Cortisol levels were also
significantiy higher on TDl than on TD2 in both groups (p<0.005). Patients' Amax [peak-
baseUne] Cortisol levels showed a trend to be higher than controls' levels following the second
(p<0.06) but not the first infusion. Patients, but not controls, showed a significant decrease
^<0.01) in Amax Cortisol levels from TDl to TD2.
B. Prolactin: There were no differences in basal serum prolactin levels between SAD patients
and controls on either test day. Both patients and controls showed robust increases in serum
prolactin in response to mCPP. The mCPP-stimulated prolactin rise was significantiy greater
over time in SAD patients than in controls on both test days (p<0.(X)5). mCPP-stimulated
prolactin levels were significantly higher on TDl than on TD2 in both groups (p<0.005).
Controls, but not patients, showed a significant decrease in Amax prolactin levels from TDl to
TD2 (P<0.05). Patients' Amax prolactin levels were higher than controls' on TD2 (p<0.05) but
not on TDl.
Thus depressed SAD patients showed higher basal and mCPP-stimulated Cortisol levels, and
also higher mCPP-stimulated prolactin levels than matched controls. The higher Cortisol and
prolactin levels of patients were present in both depressed and remitted states, suggesting the
possibility that heightened sensitivity to changes in serotonergic function may be a trait marker
in SAD patients. We should note, however, in arguing against this idea, that patients did not
remit completely following light therapy. The current findings differ from those our earUer
study in which depressed SAD patients showed higher basal but equivalent 5-HTP-stimulated
Cortisol and prolactin levels than controls. The difference in the stimulated hormonal levels in
the two studies may reflect the greater serotonergic selectivity of mCPP.
As with the hormonal findings, SAD patients' subjective responses to mCPP were greater than
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controls' responses on many (but not all) items. The items "activation-euphoria", "slowed
down", and "drowsy" were of particular interest since patients' mCPP-stimulated ratings
tended to normalize with phototherapy and improvement in depression. These changes in
subjective response to mCPP may signify functional serotonergic differences between
depressed SAD patients and controls which are normalized by exposure to phototherapy. The
finding that mCPP produces less slowing and drowsiness in SAD patients than in normal
controls is reminiscent of our earUer finding that carbohydrate-rich meals also show a similar
differential subjective effect on these two groups. Both findings suggest a difference in the
serotonin system of SAD patients and normals.
Both patients and controls showed decreased hormonal and [in many cases] decreased
subjective responses to mCPP on the second infusion day compared with the first infusion
day. A second group of controls (N=6) given IV mCPP on two mornings a week apart
without exposure to phototherapy showed similar decreases in both hormonal and subjective
responses. Our preliminary data analysis levels suggests that these effects may represent a
down-regulation of serotonergic receptors present a week after a single exposure to IV mCPP.
The abnormal serotonergic function in SAD patients and its normalization by light, suggested
by the above study, is reinforced by recent findings from other groups, who have shown the
serotonin agonist, D-fenfluramine, and the serotonin precursor, L-tryptophan to be somewhat
effective in the treatment of SAD. Taken together, all these findings suggest that abnormalities
in serotonin metabolism may be an important etiological factor in SAD and that light therapy
may work, at least in part, by correcting this abnormahty.
3. Photo-Immune Studies in SAD and Normal Subjects
Peripheral blood lymphocytes (PBL) were stimulated to a greater degree in SAD patients than
in normals during both the winter and the summer. Seven to ten days of light treatment was
associated with a decrease of PBL stimulation to normal levels. In 10 normal subjects bright
(2500 lux) but not dimmer (3(X) lux) light was associated with significant increase in PBL
stimulation and since the skin was completely covered in this study, this effect was evidently
mediated via the eyes. For further details see Annual Report #Z01 MH 0235-02 CP.
Significance to Biomedical Research:
The definition of the syndrome of SAD and the recognition of its response to light have been
followed by new insights into the neurobiology of this subset of affective disorders and rapidly
accumulating information on the biological effects of light in humans. Light has emerged as a
valuable probe into the understanding of brain functioning.
Patients with SAD differ from commonly reported affective disorder patients in that they
typically overeat, oversleep and gain weight It appears as though their biological features may
also differ in some ways. For example, typical affective disorder patients have been shown to
have decreased peripheral blood lymphocyte (PBL) stimulation, whereas in SAD patients this
function appears to be increased. The inverse relationship between plasma norepinephrine
levels and severity of depression, reported here in SAD patients, has not been consistently
reported in more typical affective disorder patients. However, other biological findings
reported here, namely low nocturnal melatonin secretion and decreased total sleep time, have
been reported in classical depressives. We should note that we have previously reported total
sleep time to be increased during the winter in SAD, a confirmation of patients' clinical
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sleep time to be increased during the winter in SAD, a confirmation of patients' clinical
observation. It is only a minority who report decreased sleep time and it is conceivable that this
minority was overrepresented in the present sample of patients studied.
The many observed biological effects of light continue to add to our knowledge of this
environmental variable, only recenfly regarded as inert as far as human brain functioning was
concerned. The earlier observation diat light enhances the P300 component of the visual ERP
has been sustained in a larger sample size, as have the suppressing effects of light on PBL
function in patients with SAD. It appears that the antidepressant effects of light correlate with
improved processing of visual, but not auditory, information. This objective correlate of the
antidepressant response to light appears to hold promise for future studies, especially since it
can be observed as early as 48 hours after starting light treatment
An unexpected effect of bright light was the reduction of RMR in patients. Many of the
symptoms of SAD, such as increased sleeping, decreased activity, overeating and weight gain,
suggests energy-conserving strategies. Since light treatment reverses these symptoms, thus
decreasing energy conservation, we had predicted that it would increase RMR. The decrease in
RMR is difficult to explain but it may be secondary to the decreased appetite and weight loss
that occur in response to treatment We should note that antidepressant medications have been
shown to decrease RMR in non-seasonal depressives.
The significant reduction in RMR seen following light treatment in SAD patients was not
observed in normal subjects. Different effects of light in patients and normals were also seen
on PBL function, on sleep, and on stimulation of the serotonin system by M-CPP. Bright light
suppressed PBL function in SAD patients in the winter but increased it in normal subjects.
Similarly bright light increased sleep efficiency and delta sleep in patients but decreased these
parameters in normals. Such differential effects of light on SAD patients and normals may
provide a clue to the pathophysiology of SAD. The exaggerated sensitivity of the serotonin
system to stimultion with die post-synaptic serotonin agonist, M-CPP, provides one more
piece of evidence that serotonergic function may be abnormal in SAD and that alterations in this
system may partially explain the antidepressant effects of light
The phase-response theory as an explanation for how light therapy works, is not substantiated
by our data. Although there was a non-significant tendency for SAD patients to show a
delayed onset of melatonin secretion when compared to controls, there was no evidence from
hormonal or sleep data, that Ught treatment advances circadian rhythms, the effect that has been
hypothesized to underlie its antidepressant properties. On the contrary, effective light treatment
was seen to delay the Cortisol nadir in SAD patients.
Initially in the study of SAD and phototherapy, there appeared to be few biological markers of
the condition. The traditional biological abnormalities found in depression did not appear to be
present in SAD. Plasma Cortisol levels, the response to the dexamethasone suppression test,
and REM latency were normal. We have now shown several biochemical and physiological
abnormalities in SAD patients, both at baseline and in response to light. A problem for future
research efforts will be to understand better the nature of these abnormalities, to sort out which
will reveal most about the fundamental disturbance of this condition and, most important, to
attempt to weave a coherent story out of these many abnormal findings.
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Proposed Course:
The comparison of SAD patients and normals on and off light treatment has been an extremely
fruitful approach and several physiological systems appear to differentiate subjects and
conditions. Further studies are suggested to explore these earlier findings in greater depth.
How do patients and normals differ across the seasons? What are the effects of light on
norepinephrine levels in normals? Will challenges to the RMR elicit further patient-nomal and
on-light /off-light differences, for example a study of diet-induced thermogenesis? What is the
precise nature of the lymphocyte changes induced by light? Are there changes in phenotype of
B- or T-Cells, or are all lymphocytes affected similarly? Do the observed in vitro changes in
immunity correspond to any in vivo changes? Can the P300 enhancement provide information
about an immediate physiological effect of light? If so, does this effect show a circadian
variation, and is it a predictor of long-term antidepressant effects of Hght? How can we best
foUow up the abnormalities in the norepinephrine and serotonin systems and their apparent
normalization by bright light treatment? Many questions present themselves; the challenge will
be to choose those questions most likely to reveal the fundamental abnormality in SAD and best
elucidate the biological effects of light.
Publications:
Wehr TA, Sack DA, Jacobsen F, Tamarkin L, Arendt J, Rosenthal NE. Timing of
phototherapy and its effect on melatonin secretion are not critical for its antidepressant effect in
seasonal affective disorder. Archives of General Psychiatry 43:870-875, 1986.
James SP, Wehr TA, Sack DA, Parry BL, Rogers SL, Rosenthal NE. The dexamethasone
suppression test in seasonal affective disorder. Comprehensive Psychiatry 27:224-226, 1986.
Rosenthal NE, Sack DA, Jacobsen FM, James SP, Parry BL, Arendt J, Tamarkin L, Wehr
TA. The role of melatonin in seasonal affective disorder. Journal of Neural Transmission
(suppl) 21: 257-267, 1986.
Sack DA, Rosenthal NE. E>o changes in melatonin cause SAD? Commentary submitted to
Integrative Psychiatry, Oct. 1986.
172
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02222-01 CP
PERIOD COVERED
October 1, 1986 to September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between tt)e txjrders.)
The Treatment of Rapid-Cycling Manic-Depressive with Thyroxine
PRINCIPAL INVESTIGATOR (List other professional personnel betow the Principal Investigator.) (Name, title, latxiratory, and institute attillation)
PI: D. Sack Chief, Inpatient Services CPB/NIMH
Othere: T.Wehr Chief, Clinical Psychobiology Branch CPB/NIMH
N. Rosenthal Chief, OuQiatient Services CPB/NIMH
W. Mendelson Chief, Unit on Sleep Studies CPB/NIMH
COOPERATING UNITS (if any)
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
2_
PROFESSIONAL:
2
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
n (a1) Minors
n (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
There is some evidence that the hypothalamic-pituitary-thyroid axis plays a role in the
pathophysiology of affective illness, particularly in patients with rapid cycling forms of manic-
depressive illness, who have a very high incidence of lithium-induced hypothyroidism. Rapid
cycling patients are often refractory to standard types of mood-stabilizing treatments. Since the
1930s there have been three reports that hypermetabolic doses of thyrodxine suppress rapid
cycling. These reports were based on uncontoUed studies.
We treated seven rapid cycling patients with suppressive and hypermetabolic doses of thyroxine in
a placebo controlled, double-blind study. No patient improved on supressive doses. Two patients
remitted completely on hypermetabolic doses, but both eventually relapsed after many months of
treatment This result is interesting from a theoretical point of view, but does not seem to offer any
new approach to treatment for refractory rapid cycling patients. Therefore, the project has been
terminated.
173
PHS 6040 (Rev. 1/84)
SPO SI 4-9 It
ZOl MH 02222-01 CP
Project Description:
This project is designed to assess the hypothalamic-pituitary-thyroid axis (HPT) in rapid-cycling
manic-depressives and to test the therapeutic efficacy of exogenously administered thyroxine in
these patients. A complete description of the project and methods appears in ZOl MH 0222-03
CP.
Methods:
We have now evaluated the mood stabilizing effects of thyroxine in 7 patients in a double-blind
controlled trial. No patients improved on replacement doses of thyroxine. On hypermetabolic
doses of thyroxine 2 patients showed complete remission in their mood cycles. Both of these
patients relapsed when thyroxine was decreased to suppressive doses and remitted when thyroxine
was reinstated at the higher dose. Long term follow-up reveals that both patients eventually
relapsed into rapid-cycles despite continuing on hypermetabolic trreatment and showing elevated
thyroid values.
Significance to Biomedical Research:
1) Although rapid-cyclers differ from other groups of manic-depressive patients with respect to the
frequency of their episodes, they are the same with respect to clinical presentation, age of onset and
genetic predisposition. The overt thyroid disease that these patients manifest may be a particularly
useful model for understanding the more subtle HPT disturbvances seen in other affective patients.
2) While rapid-cyclers constitute approximately 15% of manic-depressive patients they are largely
refractory to conventional therapy and constitute a formidable problem in clinical practice.
Understanding the nature of the thyroid disturbance in these patients could lead new and better
therapies.
Proposed Course:
The results of this study are interesting from a theoretical point of view, but they do not seem to
offer any new approach to the treatment of refractory rapid cycling patients. Therefore, the project
has been terminated.
174
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01MH02223-04-CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between (he bordersj
Pentobarbital and Ethanol Toxicity: Relation to the Benzodiazepine Receptor
PRINCIPAL INVESTIGATOR (Ust other professional personnel tielow the Principal Investigator.) (Name, title, laboratory, and institute altilietion)
PI:
Others:
W. B. Mendelson Chief, Section on Sleep Studies
J.V. Martin
R. Wagner
Chemist
Guest Worker
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Psychobiology Branch
SECTION
Section on Sleep Studies
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0
PROFESSIONAL:
0
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In the coxirse of measurements of respiration, spontaneously occurring cessations of breathing,
analogous to human sleep apneas, were observed in rats. Subsequently, the apparatus for
monitoring respiration was dedicated to Project #Z01-MH 02382-01 CP in order to characterize an
animal model of sleep apnea. For this reason, and because the investigators left NIH in July,
1987, Project ZOl MH 02223-04-CP has been terminated.
175
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02225-04-CP
PERIOD COVERED
October 1, 1986 to September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Studies on the Role of Calcium Rux in the Sleepnlnducing Effects of Rurazepam
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Phncipal Investigator) (Name, title, laboratory, and institute afliliation)
PI: W. B. Mendelson Chief, Section on Sleep Studies CPB/NIMH
Others: J.V.Martin Chemist CPB/NIMH
R. Wagner Guest Worker CPB/NIMH
COOPERATING UNITS (if any)
S. Paul, Chief, Clinical Neuroscience Branch, NIMH
M. Majewska, Visiting Associate, Clinical Neuroscience Branch, NIMH
LAB/BRANCH
Clinical Psychobiology Branch
SECTION
Section on Sleep Studies
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.7
PROFESSIONAL:
0.5
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues S (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our previous studies have shown that there may be a functional relationship between
benzodiazepine (BZ) receptor stimulation and alterations in calcium flux. Nifedipine, a calcium
channel blocker, prevents the sleep-inducing effects of flurazepam in rats. Conversely, BAY K
8644, a calcium channel agonist, potentiates the effects of flurazepam on sleep. However, as we
reported last year, experimental models for the anticonvulsant and anxiolytic effects of BZ's are not
affected by calcium charmel antagonists, indicating that calcium channels may have a more
important role in sleep induction than in other actions of BZ's. The sleep-inducing effects of
pentobarbital were not sensitive to inhibition by nifedipine, indicating that barbiturates may cause
sleep through a mechanism that does not require changes in calcium flux. Since pentobarbital
causes changes in chloride ion flux, a second mechanism for sleep induction might involve a
chloride channel. To expand this line of investigation, this year we studied the effects on sleep of
two steriods. which enhance chloride uptake into synaptoneurosomes in a manner similar to
barbiturates.
The two steroids, 3alpha , Salpha -tetrahydrocorticosterone (THDOC) and 3alpha -hydroxy-
Salpha-dihydroprogesterone (OH-DHP), are endogenously occurring ring A metabolites of
corticosterone and progesterone. Since THDOC and OH-DHP not only alter chloride flux but also
enhance the binding of F^HIflunitrazepam to brain membranes, we studied the effects on sleep in
rats of 5 and 10 mg/kg of the steroids alone and in combination with a low dose of flurazepam.
THDOC, but not OH-DHP, had potent dose-dependent sleep-inducing properties and increased
nohREM sleep, but did not affect REM sleep. Flurazepam had a similar hypnotic effect but
additionally reduced REM sleep. There were no significant interactions between THDOC and
flurazepam.
This project was terminated in July, 1987 when the investigators in the Section on Sleep Studies
left NIH.
177
PHS 6040 (Rev 1/84)
GPO SI4-Sia
ZOl MH 02225-04-CP
Project Description:
Please refer to ZOl MH 02225-01-CP
MgthQ(Js:
Male rats were injected intraperitoneally (IP) with 5 or 10 mg/kg OH-DHP or THDOC or with
vehicle. Five minutes later the rats were given a second IP injection, of 20 mg/kg flurazepam or
vehicle. Standard two-hour EEG recordings were then performed.
Findings to Date:
In the present study, OH-DHP (5 and 10 mg/kg) had no significant effect on sleep, nor did it alter
the effects of flurazepam.
While the low dose of THDOC (5 mg/kg) had no significant effect on sleep, it tended to decrease
sleep latency. The high dose of THDOC (10 mg/kg) significantly reduced sleep latency from 14.6
± 2.4 min to 8.2 ±1.9 min (mean ± S.E.M; p < 0.01; n=15). As expected, flurazepam also
significandy decreased sleep latency, to 9.4 ± 2.0 min (p < 0.05). There was, however, no
significant interaction between THDOC and flurazepam; when both drugs were given together, the
sleep latency was 6.5 ± 1.2 min. Similarly, both THDOC (10 mg/kg) and flurazepam increased
nonREM sleep (p < 0.01 each) again with no significant interaction. Flurazepam, but not THDOC
reduced REM sleep (p < 0.05) without a significant interaction.
Significance to Biomedical Research:
Previous studies showed a parallel between effects of BZ's on sleep and a calcium ion flux related
to occupation of BZ receptors. However, the hypnotic effects of pentobarbital are not sensitive to
the calcium channel blocker nifedipine, implying that a second mechanism for sleep induction is
independent of the calcium channel. The present findings indicate that THDOC, a steroid
compound which stimulates chloride flux, is a potent sleep inducer. Both calcium and chloride ion
fluxes, therefore, deserve consideration as possible effector mechanisms for the actions of different
classes of hypnotic drugs.
The fmding that an endogenous corticosterone derivative has strong hypnotic qualities may have
important implications for investigations of the role of the adrenal gland in stress responses.
Furthermore, from a purely clinical viewpoint, the discovery of an endogenous compound which
induces nonREM sleep without detrimental effects on REM sleep is unusual and may have
therapeutic applications.
Publication:
Mendelson, WB, Martin, JV, Perils, M, Wagner, R, Majewska, MD and
Paul, SM: Sleep induction by an adrenal steroid in the rat. Psvchopharmacology.
in press.
178
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT MUMBER
ZOl MH 02290-03 CP
PERIOD COVERED
October 1. 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on orte line between the borders.)
Melatonin analysis of clinical samples
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
PI: L. Tamarkin Research Biologist CPB/NIMH
Others:
G. Paciotti
M. ColUns
Biologist
Biologist
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (It any)
W. Berretini and J. Numberger. Clinical Neurogenetics Branch, NIMH,
LAB/BRANCH
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH. NM. Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.2
PROFESSIONAL:
0.2
CHECK APPROPRIATE BOX(ES)
d (a) Human subjects
D (a1) Minors
D (a2) Interviews
G (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Melatonin has been measured in plasma and cerebrospinal fluid from human patients and normal
volunteers. The findings of the studies outlined in last year's report are currently being written for
submission to peer reviewed journals. The one major ongoing study is focused on the possible
heritability of the increased sensitivity to light at night of children of parents with major depression.
179
PHS 6040 (Rev. 1/84)
SPO SI4>SII
ZOl MH 02290-03 CP
Project Description:
The clinical study of the regulation of melatonin secretion is done in collaboration with Dr. W.
Berettini and is an acute exposure of human subjects to approximately 500 lux of light at night.
We have previously observed that the nocturnal secretion of melatonin from normal subjects is
not affected by exposure to this intensity of light at night. However, depressed patients either
during a depressive episode or euthymic are more sensitive to light at night and show a
suppression of plasma melatonin. Children of depressed parents were the focus of this
investigation and were asked to participate in a clinical study similar in design to the adult
study.
Methods:
Offspring of one bipolar parent (N=18) and offspring of an affectively ill couple (one bipolar,
N=7) were compared to 20 aged-matched controls with no famillial history of affective
disorder. Blood samples were drawn from lam to 2am in the dark and from 2am to 4am in the
light. Plasma was harvested for melatonin determination.
Findings to Date:
Significantly more subjects with one or two affected parents were sensitive to 500 lux of light
at night. Suppression of plasma melatonin was noted in 3/20 subjects in the 0 parent ill group,
6/18 in the 1 parent ill group, and 4/7 in the 2 parent ill group.
Significance to Biomedical Research:
These data would suggest that there is an association of increased sensitivity to light and
affective disorder, and further that this characteristic occurs more frequently in offspring of
affected parents. One hypothesis that needs to be explored is that this increased sensitivity to
light at night may also be predictive to increased vulnerability.
Proposed course:
Continued investigations of offspring are planned and further study of the specific mechanism
for the increased sensitivity to light are being discussed.
180
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 02292-03 CP
TITLE OF PROJECT (80 characters or less. Title must tit on one line between tt)e txirders.)
Melatonin Effect on Hormone-Stimulated Cell Growth
PRINCIPAL INVESTIGATOR (Ust other professional personnel tielow the Phncipal Investigator.) (Name, title, latxratory, and institute eHiliation)
PI: L. Tamarkin Research Biologist CPB/NIMH
COOPERATING UNITS (If any)
LAB/BRANCH
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH, Nm, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0
PROFESSIONAL:
0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues D (c) Neither
D (at) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Project held in abeyance
181
PHS 6040 (Rev. 1/84) gpobm-bh
Dr.tnher 1 J 986 to Sp.pfe.mhftr :^n, 1 QS7
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02294-03 CP
PERIOD COVERED
TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.)
Antidepressant Pharmacology of the Rodent Circadian System
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute ettiliation)
PI: W.Duncan Research Psychologist CPB/NIMH
Others: L.Tamarkin Research Biologist CPB/NIMH
T. Wehr Chief, Clinical Psychobiology Branch CPB/NIMH
COOPERATING UNITS (if any)
P. Sokolove, Professor of Biological Sciences
University of Maryland, Baltimore County (IJMBC)
LAB/BRANCH
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NTMH, NTH, Re.thesda, Maryland 20892
TOTAL MAN-YEARS:
_2_
PROFESSIONAL:
J
OTHER:
J
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues H (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The circadian system in patients with primary affective disorder is disorganized. A major symptom
of this disease includes a disturbed activity-rest cycle. Treatments which affect the circadian
system may correct a pathological state which underlies depression. Our goal is to understand how
antidepressant chemicals alter the dynamics of the mammalian circadian system. Results from these
studies will be valuable to understand the mechanism of chemical antidepressant treatments in
humans.
Previously we reported that in hamsters, clorgyline, a monoamine oxidase inhibitor with
antidepressant properties, increases the period of the circadian clock and decreases the rest
component of the daily activity-rest cycle. We also observed that this chemical alters the response
of the circadian clock to light These results indicate that clorgyline exhibits input properties to the
circadian clock: the location of its input has been unclear.
Processing of light information to the clock occurs at either the retina or the lateral geniculate
nucleus projection to the clock. Results from the past year indicate clorgy line's effects are not
mediated via the retinal projection to the clock. Our current hypothesis is that clorgyline alters the
hamster circadian system via a lateral geniculate nucleus projection to the circadian clock. We are
currently testing this hypothesis.
183
PHS 6040 (Rev 1/84) GPO 9i <!.»i»
ZOl MH 02294-03 CP
Project Description:
Disturbances of the activity-rest cycle which accompany depression may be due to
dysfunctional biological oscillators which populate the mammalian circadian system. Chemical
antidepressants which effect these abnormal oscillations may exert beneficial clinical properties
by correcting a pathological circadian process. We are interested in 1) describing the effects
and 2) identifying the input pathway, of antidepressant chemicals on the mammalian circadian
system.
Methods:
1) Experimental equipment
A description of the facility used to evaluate and monitor the rodent circadian system can be
found in project report ZOl MH 02294-01 CP.
2) Antidepressant chemical effects on the central pacemaker's period
We have extended our studies to include treatment with the selective MAOI deprenyl. High
dose deprenyl (25 mg kg-1 day-1) was chronically administered via Alzet osmotic mini-pumps.
Hamsters were maintained in constant conditions (constant darkness, temperature, ad lid food
and water) Wheel-running activity was continuously collected by laboratory computer.
3) Location of chemical input to the central pacemaker
There are four possible input pthways to the circadian pacemaker: 1. the suprachiasmatic
nucleus(SCN), 2. the retina and retinal hypothalamic path to the SCN, 3. the lateral geniculate
nucleus and its projection to the SCN, and 4. raphe complex and its projection to the SCN. In
order to assess the role of the retina in mediating clorgyllne's effects on the circadian clock,
hamsters received bilateral orbital enucleation and then were treated with chronic clorgyline as
described previously (see ZOl MH 02294-02 CP).
Findings to Date:
Previously we observed that chronic clorgyline treatment of Syrian hamsters 1. increased the
period of the circadian clock, 2. increased the activity-rest ratio and 3. altered the response of
the circadian system to light. Two observations emerge from our current investigations .
First, our studies suggest that chronic, high-dose deprenyl increased the period of the circadian
clock similar to chronic clorgyline treatment These results indicate the circadian effects are
probably related to a Type A MAO property rather than a non-pharmacological property of the
clorgyline molecule itself.
Second, we observed that bilateral enucleation failed to block the effect of clorgyline on
increasing the period of the circadian clock. Therefore, clorgyline appears to be altering the
period eitiier at the SCN itself, via the lateral geniculate nucleus (LGN) projection to the SCN,
or via the raphe projection to the SCN.
Our current hypothesis is that clorgyline's effect on the hamster circadian system is via the
LGN projection to the SCN. Several recent experiments conducted in the U.S., Canada and
Holland, support this hypothesis. LGN lesioned hamsters are functionally similar to
184
ZOIMH 02294-03 CP
1. an increased activity-rest ratio, 2. an increased clock period and 3. an altered response of
the circadian system to light
Significance to Biomedical Research:
Our experiments indicate that the MAOIs clorgyline and deprenyl, when administered at doses
which inhibit Type A MAO, alter the period of the circadian pacemaker in Syrian hamsters.
These results are consistent with our hypothesis that the antidepressant mechanism of these
compounds in humans may include effects on the circadian clock, and that Type A MAO
inhibition probably participates in this response.
Pharmacological interventions (chronic clorgyline) and non-pharmacological interventions
(light intensity, LGN lesions) which affect tiie retino=geniculohypothalamic (RGH) tract are
simiar in several respects. In Syrian hamsters, each of these treatments increase the activity-rest
ratio and the period of the central clock. Also, both LGN lesions and chronic clorgyline
treatment alter the processing of light signals by the central clock. A neuroanatomical nuclens
which regulates the expression of these circadian based processes has been postulated to be
abnormally expressed in some depressed patients.
Our current hypotheses are that since LGN lesions alter the activity-rest ratio, as do MAOI and
light treatments, a ) a dysfunctional LGN may contribute to the sleep disturbance which
accompanies primary depression and b ) a common mechanism of pharmacological and non-
pharmacological (i.e. bright Ught ) treatments of mood disorders may include functionally
similar effects on the LGN.
Proposed Course:
During the next year we plan on extending our studies to include tricyclic antidepressants and
lithium. These chemicals will be administered chronically to evaluate their effects on the period
of the central clock. Second, we plan on measuring the response of chronic clorgyline treated
hamsters to constant light in order to more fully evaluate the similarities between clorgyline
tijcatment and LGN lesions. Third, we will determine the circadian system response of LGN
lesioned hamsters to chronic clorgyline treatment.
185
PERIOD COVERED
Dctober 1, 1986 to September 30, 1987
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02303-02 CP
TITLE OF PROJECT (BO characters or less, nie must fit on one line between the bortiers.)
Studies of Sleep in Psychiatric Illness
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atliliatlon)
PI: W. Mendelson Chief, Section on Sleep Studies CPB/NIMH
COOPERATING UNITS (If any)
Clinical Psychobiology Branch
LAB/BRANCH
Section on Sleep Studies
SECTION
NflMH, NIH, Bethesda. Maryland 20892
INSTITUTE AND LOCATION
TOTAL MAN-YEARS:
1.5
PROFESSIONAL
0.3
1.2
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects D (b) Human tissues D (c) Neither
D (al) Minors
n (a2) Interviews
SUMMARY OF WORK (Use Standard unreduced type. Do not exceed ttie space provided.)
In a previous study patients with bipolar depression were compared to age and sex matched
controls, there were differences in sleep efficiency and total sleep, but no differences in REM
latency or power spectra. We have looked at the power spectra of insomniacs and compared it to
controls. There were no differences in power spectra of insomniacs when compared to controls.
Our previous studies of the frequency analysis of depressed bipolar patients compared to normal
controls slowed difference in sleep efficiency and total sleep but no difference in REM latency and
power spectra. We look at the power spectra of sleep in insomniacs and compared it with controls.
There were no differences found in any aspect of the frequency analysis.
187
PHS 6040 (Rev. 1/84) oPO bm-sk
ZOl MH 02303-02 CP
Project Descriprion:
Ten insomniacs and ten age and sex matched controls had sleep studies which were recorded
for analysis.
Methods:
Subjects EEG were recorded on FN tape for future analysis. Tapes were played back through
a Bruel and Kjaer frequency analyzer which produces plots of power. AU non REM epochs
were plotted and averaged over the night. The REM epochs were evaluated separately.
Findings to Date:
There were no differences between insomniacs and controls for REM or non REM power
spectra.
Proposed Course:
There is evidence that depressives have a high body temperature especially at night. Cooling at
night reduces REM and may have an antidepressant effect. We wish to study depressives
exposed to different temperature manipulations to see the effect of temperature on sleep in
depressives.
188
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02324-02 CP
PERIOD COVERED
October 1. 1986 to September 30. 1987
TITLE OF PROJECT (80 characters or less. We must fit on one line tjetweert the ttorders.)
Neuroendocrine Modulation of Cellular Immune Response
PRINCIPAL INVESTIGATOR (Ust other prolessionel personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfiliatlon)
PI:
Others:
L. Tamarkin
G. Paciotti
R. Skwerer
M. ColUns
Research Biologist
Biologist
Medical Staff Fellow
Biologist
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (H any)
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH. NIH. Bethesda. Maryland 20892
TOTAL MAN- YEARS:
0.2
PROFESSIONAL
0.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues (E (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The focus of this line of investigation is to compare short-term vs long-term neuroendocrine
challenges on the proliferation of splenic lymphocytes. This was accomplished by comparing the
in vitro proliferative response of lymphocytes to concanavalin A following an in vivo 4 hour
isoproterenol treatment compared to a 5 day constant infusion of isoproterenol. Following the
short-term isoproterenol treatment splenic lymphocyte proliferation was inhibited compared to
controls, while long-term exposure resulted in an increase in the proliferative response. These
results were not related to corticosterone levels which were elevated in both the acute and chronic
treatments, nor were they related to the presence of the adrenal gland. These studies suggest that
splenic lymphocytes become insensitive to the suppressive effect of chronically elevated
glucocorticoid.
189
PHS 6040 (Rsv. 1/84)
SPO SI4-«I(
ZOl MH 02324-02 CP
Project Description:
Factors that induce a stimulation of glucocorticoid secretion have been associated with a
suppression of immune function. To test this we have chosen a specific neurochemical
challenge, isoproterenol, that has direct sympathetic effects, particularly on the
cardiovascular system. A second chemical challenge that was undertaken was an insulin
tolerance test, in which blood glucose levels were dramatically reduced. Following either
or both treatments the rats were sacrificed and the spleens removed for processing in a
lymphocyte transformation test, using concanavalin A as the mitogen. Cellular
proliferation was determined by the amount of ^H-thymidine incorporated in 6 hours.
Methods:
The acute part of this study was previously described. The long-term isoproterenol
treatment was accompUshed by implanting rats with Alzet minipumps containing
isoproterenol. After 5 days of treatment the animals were given an injection of insulin and
after 2 hours the animals were sacrificed. Blood was collected for glucose and
corticosterone determination. Pineal glands were frozen for subsequent melatonin content
analysis and spleens were harvested for lymphocytes. The lymphocyte transformation test
has been previously described.
Findings to Date:
Acute in vivo treatment with isoproterenol resulted in a suppression in lymphocyte
proliferation. However, chronic isoproterenol treatment resulted in increased lymphocyte
proliferation compared to controls. Acutely, as well as chronically treated animals had
elevated serum glucocorticoid levels and increased pineal content of melatonin. The data
clearly indicate that lymphocytes become desensitized to the inhibitory effect of elevated
glucocorticoid.
Significance to Biomedical Research:
These data compare the effect of acute versus chronic systemic chemical challenges on the
cellular immune response and suggest that long-term challenges are compensated by a
resensitization of lymphocytes to mitogen. This may be critical in defining the range of
immune responses to physiologic and pathophysiologic events, and these data provide
insight into the sensitivity of the immune system to in vivo short-term and long-term
activation of the hypothalamic -pituitary-adrenal axis.
Proposed Course:
Assessment of the factor(s) that modulates the cellular immune response needs to be
investigated. Further exclusion of the adrenal axis involvement will be pursued and the
effect of other endogenous molecules will be studied.
190
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02325-02 CP
PERIOD COVERED
rwniv.r I mxn tr> spptpmnpr mi mx /
TITLE OF PROJECT (BO characnrs or less. Title must lit on one line between the borders.)
oht anH T ymnhoryfp Artivify- Rasir anH riiniral Stiirlips
llNCIPAL INVESTIGATOR (List other prolesslonal personnel t>elow the Principal Inves
Li
Investigator.) (Name, title, laboratory, and institute alfillatlon)
PI:
Others:
L. Tamarkin
R. Skwerer
G. Paciotti
M. ColUns
Rhyne.-Gre.y
'anyj
Research Biologist
Medical Staff Fellow
Biologist
Biologist
Biologist
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (If
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NMH. Nm, Bethesda, Maryland 20892
TOTAL MAN-YEARfe: PROFESSIONAL:
4J-
CHECK APPROPRIATE BOX(ES)
[^ (a) Human subjects
D (al) Minors
n (a2) Interviews
■ii-
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In last year's report we reported that peripheral blood lymphocytes from patients with winter
depression had different in vitro responses to mitogen following bright light treatment compared to
placebo treatment This year the same study was repeated with a different group of patients and the
observations were the same as that noted in the first study. To gain some further understanding of
the mechanism by which light affected the cellular immune response, a group of normal volunteers
was exposed to bright light or ordinary light restricted to their eyes. The data from this study were
quite clear, showing that light perceived through the eyes is transduced through the central nervous
system, impacting on the cellular immune response.
191
PHS 6040 (Rev. 1/84)
GPO SI4-91I
ZOl MH 02325-02 CP
Project Description:
A human physiologic study was undertaken to determine if the changes in peripheral blood
lymphocytes observed in patients also occurred in normal volunteers. Also it is possible that
the changes observed following Ught treatment might have been the result of light (uv) acting
directiy on the skin. To address this, normal volunteers were gowned so that tiie bright Ught
exposure could only be perceived by their eyes. Blood components, including lymphocytes,
were collected and analyzed.
Methods:
Normal volunteers were recruited and were asked to sit in front of a bank of bright lights
(approximately 3000 lux). The subjects were gowned so that the Ught would be restricted to
their eyes. This study was conducted twice, once where treatment occurred in the home and
once where it was administered on our clinical research ward. Blood samples were taken
before treatment, one day after treatment, and one week after treatment Peripheral blood
lymphocytes were isolated for mitogen testing and the plasma was stored for the analysis of
Cortisol and antibody titers.
Findings to Date:
Of the parameters studied, peripheral blood lymphocytes were consistently and significantly
stimulated after one week of bright Ught treatment. This occurred for both mitogens and
suggests that the ceUular immune system can be affected by light perceived through the eyes.
The results of this study are being written and wUl be presented for peer review.
Significance to Biomedical Research:
This study clearly demonstrates the range of physiologic responses of the cellular immune
system to environmental light and indicates that this system is responsive to changes in the
environment that can be transduced through the central nervous system to impact on the internal
milieu. This study advances our knowledge by suggesting one environmental factor, Ught
processed through the central nervous system, that may impact on the immune system's ability
to ward off various infectious diseases.
Proposed course:
More sophisticated analysis of the specific immune ceUs affected by bright Ught exposure will be the
focus of future studies. This wiU be accomplished by using monoclonal antibodies that can identify
specific immunologic cell sub- types and determining their distribution by flow cytometry. This
technology is readily available for human lymphocytes and for mouse lymphocytes. To pursue the
latter, we need to determine if mice also respond to bright Ught treatment
192
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02326-02 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECTJ80 characters or less. Title must fit on one line benvesn the borders.)
Behavioral Modulation of the Cellular Immune Response
PRINCIPAL INVESTIGATOR {List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
L. Tamarkin
M. Collins
R. Skwerer
G. Paciotti
Research Biologist
Biologist
Medical Staff Fellow
Biologist
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (if any)
S. Suomi, Laboratory of Comparative Ethology, NICHD, NIH
LAB/BRANCH
CHnical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.2
PROFESSIONAL
0.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (al) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Young rhesus monkeys were placed into groups of 4 by slowly introducing them to less and less
controlled environments. Blood samples were taken from aw^e animals in under 5 minutes and
peripheral blood lymphocytes were isolated and cultured. The data from three groups of 4 indicate
that the most dominant animal in the group has suppressive effect on the ability of lymphocytes to
proliferate in response to mitogen in vitro.
193
PHS 6040 (Rev. 1/84)
CPO ■14-SII
ZOl MH 02326-02 CP
Project Description:
Young rhesus monkeys were introduced to a novel cage setting. Dominance was
determined and blood samples were taken from awake animals. The goal of the study was
to determine if behavioral challenges rapidly affect the proliferative response of
lymphocytes.
Methods;
Unchanged from tiiat outline last year.
Findings to Date:
The behaviorally dominant animal consistendy had a more robust lymphocyte proliferation
response, while the more submissive animals had lymphocyte proliferation responses that
were clearly compromised and suppressed.
Significance to Biomedical Research:
These data suggest that social behavior impacts on the immune system's ability to recognize
an antigenic challenge. It is not clear at this time what the impact is on susceptibility to
systemic infections, and this needs to be the focus of future research.
Proposed course:
Further characterization of immune cells by flow cytometry may provide us with new
insight on the specific cell types affected by behavioral challenges. Also the time course of
these changes is critical in the evaluation of the range of responsiveness.
194
DEPARTMENT OF HEALTH AND HUMAN SEftVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02327-02 CP
&ao?)erTrf^86 to September 30, 1987
fiILE OF PBPJECT LBO characters Of less. Title must fit on one line tMtwean the tyordsrs.)
iirect effect of Lympnokines on Cultured Human Breast Cancer Cells
PRINCIPAL INVESTIGATOR (Ust Other professional personnel tjeiow the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: L. Tamarkin Research Biologist CPB/NIMH
Others: G. Paciotti Biologist CPB/NIMH
COOPERATING UNITS (If any)
Cunical Psychobiology Branch
^etii^esda, Maryland 20892
TOTAL MAN- YE,
PROFESSIONAL
:ars: PROFESSIOI
CHECK appropriate BOX(ES)
D (a) Human subjects 13 (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Interviews
summary of work (Use standard unreduced type. Do not exceed the space provided.)
Interleukin 1 and interleukin 2 are products of the immune system whose action has been almost
exclusively studied in the modulation of immune cell growth. Essentially these molecules are
considered paracrine hormones. Our hypothesis is that these proteins may be endocrine hormones,
whose action may be on cells of non-immune origin. We have seen that both IL-1 and IL-2 affect
human breast cancer ceU growth and further that this action occurs on hormone-dependent breast
cancer cells, but not on hormone-independent breast cancers. These studies have been conducted
in vitro using ^H-thymidine incorporation as an index of proliferation or actual cell number.
Additionally, an IL-1 receptor has been characterized on MCF-7 breast cancer cells. In vivo IL-2
has been shown to suppress tumor cell growth using athymic nude mice implanted with tumor cells
and IL-2 as the model system.
195
PHS 6040 (Rev. 1/84) OPOBi4.»ii
ZOl MH 02327-02 CP
Project Description:
In vitro: In last year's report 3H-thyniidine was the only measure of cell growth described.
This method is a standard technique, however, it is possible that it may not be valid and other
measures of cell growth should be used to confirm 3H-thymidine observations. The
unambiguous experiment to perform is to plate cells and count them at various days following
the initiation of treatment. By following cell growth a clear effect of both IL-1 and IL-2 on
MCF-7 cells was demonstrated. It should be noted that these cells are hormone dependent
cells, having a fuU complement of hormone receptors and are estrogen dependent when
implanted in vivo. To test whether or not the effect of IL-1 and IL-2 is related to the hormone-
dependency of the cell line, two other hormone independent breast cancer cell lines were used
in parallel experiments.
Current scientific knowledge concerning the mechanism of action of the proteins is that they
have specific cell surface receptors. Using the MCF-7 cells an DL- 1 receptor has been
demonstrated. This receptor is not measurable on the hormone independent cells.
In vivo: A critical test of the hypothesis that IL-2 may be acting as a hormone is to determine
if it has any growth inhibiting effect in an animal. The model system is the athymic nude
mouse. The strain is immunocompromised in that these animals have few T-cells, which are
not responsive to IL-2. Measurable tumors were observed by implanting ovariectomized nude
mice with MCF-7 cells and estrogen pellets. Once tumors were observed, the animals were
then implanted with IL-2 pellets.
Methods:
MCF-7 or ZR-75 breast cancer cells have both been shown to be hormone dependent tumor
ceU Unes. MDA-23 1 and HS-578-T have been shown to be hormone independent breast tumor
cell lines. Cells were plated, allowed to attach to the plates, and then treated with either
lymphokine. Individual wells were harvested every second or third day for 12 days and the
cells were counted in a particle counter. For the characterization of an IL-1 receptor on MCF-7
cells, iodinated IL-1 was incubated with plated cells. Cells were washed, detached and
radioactivity determined. This study was done four different ways: 1) hot only, 2) one dose
of hot and competing doses of cold, 3) various doses of hot and 200-fold excess of cold for
each dose, and 4) cross-linking of uncompeted and competed hot IL-1 followed by PAGE-
SDS analysis.
Findings to Date:
Both IL-1 and IL-2 affected hormone dependent breast cancer cell growth. This was observed
using either 3H-thymidine as an index of proliferation or cell number. Similar effects were not
noted for the hormone independent breast cancer cell lines. An IL-1 receptor has been
characterized on MCF-7 cells that has a Kd similar to that shown for T-cells. In vivo tumor
cell size was markedly inhibited by the IL-2 pellet compared to controls and this was not
associated with an increase in T-cell funtion nor with an increase in NK-cell activity. IL-2 had
no effect on the rapid growth of the hormone independent cells (MDA).
196
ZOIMH 02327-02 CP
Significance to Biomedical Research:
These data provide more evidence that these lymphokines may be acting as hormones. The
regulation of lymphokine secretion is not entirely clear and factors impinging on the CNS may
directly or indirecdy affect their secretion, which may have direct bearing on the etiology of
specific systemic diseases, such as cancers.
Proposed course:
Further characterization of the IL-1 receptor and characterization of the IL-2 receptor are
essential. The possible mechanism by which these lymphokines affect these cells may be
through a common growth factor, such as transforming growth factor-B, and the relationship
between lymphokines and TGF-B needs to be investigated. Finally, these lymphokines may be
affecting breast cancer cell cycles and characterization of cell cycles using flow cytometry
provides a new model for evaluating the effect of these lymphokines on these cells.
197
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02328-02 CP
'ac?ob'er l^'fQSe to September 30, 1987
UILE OF PROJECT (BQ-^iar^ters oUbss. We must fit on.one ipe, twfween the borders.)
Direct enects of lL-2 on Cultured Antenor Pitui tanes
PRINCIPAL INVESTIGATOR (Ust other professiorjal personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
L. Tamarkin
G. Paciotti
M. ColUns
K. Nyhus
Research Biologist
Biologist
Biologist
Summer Student
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (if any)
t^imicSTPsychobiology Branch
'mM,1^m°t'i^esda, Maryland 20892
TOTAL MAN-YE,
n
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The possibility that lymphokines affect other hormone dependent tumors, particularly tumor cells
from the pituitary is the focus to this study. AtT-20 is a mouse tumor cell Une that secrete ACTH
and we have seen that the growth of these cells and the secretion of ACTH is affected by both EL-l
and IL-2. When tumor cell growth is inhibited, ACTH secretion per ceU is increased. Conversely,
when tumor cell growth is increased ACTH secretion per cell is diminished.
199
PHS 6040 (Rev. 1/84)
CPO SI 4-9 II
ZOIMH 02328-02 CP
Project Description:
AtT-20 cells were grown in tissue culture plates and treated with various doses of EL- 1 or
IL-2. Cell growth was determined and the media was harvested for the determination of
ACTH concentration.
Methods:
Cells were plated and harvested for cell counting or for determination of ACTH
concentration in the media. This was done by radioimmunoassay for ACTH.
Findings to Date:
Data indicate that ACTH secretion into the media is increased when cell growth is inhibited,
and conversely, when cell growth is increased ACTH secretion is diminished.
Significance to Biomedical Research:
These data suggest that the effect of IL-1 or IL-2 is not restricted to breast cancers and may
include a variety of hormone dependent cells. The possibility exists that these lymphokines
play a role in cell differentiation and are critical for maintenance of the physiologic function
of the ceU.
Proposed course:
Evaluation of the genetic message for ACTH is being initiated and determination of changes
in the cell cycle with flow cytometry will also be undertaken in the next year.
200
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02382-01 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line befween (he borders.)
An Animal Model for Human Sleep Apnea
PRINCIPAL INVESTIGATOR (List other professional personnel be/oiv the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: W.B. Mendelson Chief, Section on Sleep Studies CPB/NIMH
Others:
J.V. Martin
R. Wagner
Chemist
Guest Worker
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (if any)
S.I. Rapoport Chief, Laboratory of Neurosciences, NIA
UB/BRANCH
CUnical Psychobiology Branch
SECTION
Section on Sleep Studies
INSTITUTE AND LOCATION
NIMH, NEH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.9
PROFESSIONAL:
0.5
0.4
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
"D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided )
Using a non-invasive technique which measures respiration as a function of chest and abdominal
movements, it was found that adult rats have periodic cessations in breathing analogous to human
apneas. In preliminary studies in Fischer-344 rats apneas tended to be more frequent in 22-month-
old than in 3-month-old rats. As in human apnea, apneas in Sprague-Dawley rats were
significantly more frequent and longer in duration in REM sleep than in nonREM sleep or waking.
The project was terminated in July, 1987 when the investigators in the Section on Sleep Studies
left NIH.
201
PHS 6040 (Rev 1/84)
ZOl-MH 02382-01 CP
Project Description:
The purpose of this study was to characterize spontaneously occurring cessations in breathing in
rats as an animal model of human sleep apnea. This model should be useful in studies of the neural
mechanisms underlying sleep apnea.
Methods:
Respiration was determined by a specially modified Columbus Instrument Respiration Monitor
Model RM 80 (Columbus Instrument Co., Columbus, Ohio). Rate and amplitude of respiration are
derived from the dynamic pressure change in a closed chamber due to movement of the chest and
abdominal wall of an unrestrained rat. In preliminary studies, the respiration of intact 3-month-old
and 22-month-old male Fischer-344 rats was characterized in 30 minute tests. In further studies,
male Sprague-Dawley rats were implanted with electrodes on the skull and in the neck musculature,
and after the rats recovered from surgery, 6-hour EEG and EMG recordings were made along with
the record of respiration (See Project ZOl MH 02225-01-CP). The EEG records were scored for
waking, nonREM sleep, or REM sleep and the occurrence of apneas lasting 2 seconds or longer
was determined within each of these defined stages of consciousness.
Findings to Date:
The respiratory rate of 22-month-old Fischer rats was found to be lower (p < 0.006) and the tidal
volume higher (p < 0.02) than in 3-month-old Fischer rats. Rats of both ages had multiple
cessations of respiratory effort lasting 2 seconds or longer. The number of apneas during the 30
minute test tended to be higher in the 22-month-old rats (3.9 ± 0.9 events; mean + S.E.M.) than in
the 3-month-old rats (2.5 ± 0.5 events) but this difference did not reach statistical significance (p <
0.07).
In a further six hour study in Sprague-Dawley rats, the apneas were characterized with greater
resolution and correlated with the EEG stage of consciousness. Cessations of breathing preceded
by large inspiratory movements were noted. These events were presumed to be due to lowered
pC02 after a deep breath and were not considered further. More importantly, all of the rats showed
instances of cessations of breathing which were not preceded by unusually large inspirations (19 +
1.5 events/6 hours). These apneas varied significantly (p < 0.03) in occurrence during the 6-hour
test according to EEG stage, with a predominance in REM sleep (waking = 3.5 ± 1.0 events;
nonREM sleep = 4.7 ± 1.2 events: REM sleep= 8.4 ± 2.1 events). The duration of the apneas also
varied significantly (p < 0.006) according to EEG stage (waking = 2.5 ± 0.1 sec/event; nonREM
sleep = 2. 1 ± 0. 1 sec/event; REM sleep = 2.8 ± 0. 1 sec/event). A bradycardia was apparent in the
EMG recording during many apneas.
Significance to Biomedical Research:
Sleep apnea syndrome is the most common diagnosis made in patients presenting with excessive
sleepiness at sleep disorder centers. Its complications, which include pulmonary hypertension, cor
pulmonale, systemic hypertension, and psychiatric symptomatology, make it a matter of substantial
public health concern. This research is the first development of an adult animal model for sleep
apnea. Such a model should be useful in elucidation of the neural mechanisms responsible for
202
ZOl-MH 02382-01 CP
sleep apnea. In addition, clinically oriented programs could benefit from an animal model of sleep
apnea, in for example, the evaluation of drugs which have the potential of exacerbating the
syndrome.
Publication:
Mendelson, WB, Martin, JV, Perlis, M, Giesen, H, Wagner, R.and Rapaport, S.I.: Apneas
during sleep in adult rats. Sleep, in press.
203
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
yni MM n9-^«^-ni rv
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit art one tine befween the borders.)
Effects on Sleep of a Microinjection of Triazolam to Discrete Brain Loci
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfiliation)
PI:
Others:
W.B. Mendelson Chief, Section on Sleep Studies
J.V. Martin
H. Stevens
R. Wagner
Chemist
Psychologist
Guest Worker
CPB/NIMH
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (it any)
LAB/BRANCH
Clinical Psychobiology Branch
SECTION
Section on Sleep Studies
INSTITUTE AND LOCATION
NIMH, Nm, Bethesda, Maryland 20982
TOTAL MAN-YEARS:
1.4
PROFESSIONAL:
0.5
0.9
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
As part of a systematic investigation of the possible brain sites at which benzodiazepines (BZ's)
alter sleep. 0.5 |ig of triazolam was injected into the dorsal raphe nucleus (DRN) of rats.
Injections of triazolam to the DRN significantly increased sleep latency and decreased nonREM
sleep as compared to injections of vehicle to the DRN or injections of triazolam to brain areas near
but outside the DRN.
This project was discontinued following departure of the investigators from NIMH in July, 1987.
205
PHS 6040 (Rev. 1/84)
SPO 814-916
ZOIMH 02383-01 CP
Project Description:
Although the hypnotic properties of parentally administered BZ's are well described, the
neuroanatomical sites responsible for the hypnotic action of these drugs are unknown. While our
previous work has provided evidence that the sleep-inducing effects of BZ's are mediated through
an action at the BZ receptor, these receptors are widely distributed throughout the brain. Through
lesion and electrical stimulation studies, numerous brain sites are implicated as functional centers
critical to regulation of specific aspects of sleep. The purpose of this project is to examine the
effects on sleep of the application of a BZ to a variety of brain areas, including those sites
classically associated with sleep regulation. In this way, we hope to characterize the
neuroanatomical sites of action of BZ's in relation to sleep.
Methods:
Adult male Sprague-Dawley rats are stereotaxically implanted with a 24 ga stainless steel cannula
whose tip rests 1.0 mm dorsal to the anatomical site of interest. During the same procedure,
stainless steel screws are implanted to serve as dural EEG electrodes, and teflon-coated stainless
steel wires are inserted into the nuchal musculature for EMG recording. After a one week recovery
period, a 31 ga stainless steel cannula is lowered through the outer cannula, extending 1.0 mm
beyond its tip, and vehicle or 0.5 |J.g triazolam in a volume of 0.5 |J.l is administered at a rate of
0.21 |il/min. Immediately after injection of vehicle or drug, an eight hour sleep recording is
performed. One week later, the alternative injection of vehicle or triazolam is given in the same
manner as the first injection, and a second EEG recording is performed.
Finally, the rats are injected with 0.1 |J.l of methylene blue dye, perfused, and histological
localization of the injection site is determined.
Findings to Date:
Triazolam injections into the DRN were found to have an alerting effect. Sleep latency was
increased from control values of 20.0 + 3.8 min (mean ± S.E.M.) to 41.4 + 4.2 min (n = 10; p <
0.002). In contrast there was no significant effect of triazolam on sleep latency when injected to
sites near but outside of the DRN (vehicle = 26.9 ± 3.6 min; triazolam = 23.8 + 5.6 min; n = 6).
Due to the proximity of the DRN to the cerebral aqueduct, the possibility existed that the drug
diffused into the cerebrospinal fluid and exerted its effects at a site remote to the DRN. For this
reason, we carried out a sleep study after injection of vehicle, 0.5 |ag or 1 .0 |J.g of triazolam to the
lateral ventricle of 10 rats. Sleep latencies for these conditions were 19.6 ± 1.6 min, 18.4 ± 4.0
min and 18.6 ± 3.2 min. respectively (difference not significant). The dose of triazolam employed
here, then, did not alter sleep induction when administered intraventricularly.
Injection of triazolam to the DRN also significantly decreased total sleep (p < 0.01) and nonREM
sleep (p < 0.02) and increased intermittent waking (p < 0.03), without a significant effect on REM
sleep. A drug x time period interaction (p < 0.01) indicated that the effect on total sleep was
greatest during the first two hours after administration of the drugs.
Triazolam, therefore, has a potent but transient alerting effect when injected to the DRN.
Significance to Biomedical Research:
The seemingly paradoxical finding that a drug which induces sleep when given systemically may
produce arousal when given locally may have a variety of implications for sleep research. It is
knovm that a GABAergic influence on the DRN has an inhibitory effect on cell firing in the DRN
and on serotonin turnover in these cells. Since BZ's increase the affinity of GAB A for its
206
ZOl MH 02383-01 CP
Project Description:
Although the hypnotic properties of parentally administered BZ's are well described, the
neuroanatomical sites responsible for the hypnotic action of these drugs are unknown. While our
previous work has provided evidence that the sleep-inducing effects of BZ's are mediated through
an action at the BZ receptor, these receptors are widely distributed throughout the brain. Through
lesion and electrical stimulation studies, numerous brain sites are implicated as functional centers
critical to regulation of specific aspects of sleep. The purpose of this project is to examine the
effects on sleep of the application of a BZ to a variety of brain areas, including those sites
classically associated with sleep regulation. In this way, we hope to characterize the neuro-
anatomical sites of action of BZ's in relation to sleep.
Methods:
Adult male Sprague-Dawley rats are stereotaxicaUy implanted with a 24 ga stainless steel cannula
whose tip rests 1.0 mm dorsal to the anatomical site of interest. During the same procedure,
stainless steel screws are implanted to serve as dural EEG electrodes, and teflon-coated stainless
steel wires are inserted into the nuchal musculature for EMG recording. After a one week recovery
period, a 31 ga stainless steel cannula is lowered through the outer cannula, extending 1.0 mm
beyond its tip, and vehicle or 0.5 |ig triazolam in a volume of 0.5 \i\ is administered at a rate of
0.21 |il/min. Immediately after injection of vehicle or drug, an eight hour sleep recording is
performed. One week later, the alternative injection of vehicle or triazolam is given in the same
manner as the first injection, and a second EEG recording is performed. Finally, the rats are
injected with 0.1 |ll of methylene blue dye, perfused, and histological localization of the injection
site is determined.
Findings to Date:
Triazolam injections into the DRN were found to have an alerting effect. Sleep latency was
increased from control values of 20.0 ± 3.8 min (mean ± S.E.M.) to 41.4 ± 4.2 min (n = 10; p <
0.002). In contrast there was no significant effect of triazolam on sleep latency when injected to
sites near but outside of the DRN (vehicle = 26.9 ± 3.6 min; triazolam = 23.8 ± 5.6 min; n = 6).
Due to the proximity of the DRN to the cerebral aqueduct, the possibility existed that the drug
diffused into the cerebrospinal fluid and exerted its effects at a site remote to the DRN. For ttiis
reason, we carried out a sleep study after injection of vehicle, 0.5 jxg or 1 .0 |lg of triazolam to the
lateral ventricle of 10 rats. Sleep latencies for these conditions were 19.6 ±1.6 min, 18.4 ± 4.0
min and 18.6 ± 3.2 min. respectively (difference not significant). The dose of triazolam employed
here, then, did not alter sleep induction when administered intraventricularly.
Injection of triazolam to the DRN also significantly decreased total sleep (p < 0.01) and nonREM
sleep (p < 0.02) and increased intermittent waking (p < 0.03), without a significant effect on REM
sleep. A drug x time period interaction (p < 0.01) indicated that the effect on total sleep was
greatest during the first two hours after administration of the drugs.
Triazolam, therefore, has a potent but transient alerting effect when injected to the DRN.
Significance to Biomedical Research:
The seemingly paradoxical fmding that a drug which induces sleep when given systemically may
produce arousal when given locally may have a variety of implications for sleep research. It is
207
ZOl MH 02383-01 CP
recognition site, it seems likely that local adminstration of a BZ will suppress dorsal raphe cell
function through a potentiation of the effects of GABA. The arousing effects of a possible
inhibition of the DRN by a BZ are in keeping with studies which have demonstrated an acute
waking effect of lesions of the DRN. These studies support the idea that the DRN is a brain site
important to the initiation of sleep, albeit not the site by which BZ's have their hypnotic effects
when given parenterally. The demonstration of a brain site which causes arousal in response to
BZ's might imply that not all of the BZ receptors in the central nervous system have a role in
mediating the hypnotic effects of BZ's. There may be a functional division of BZ receptors
according to neuroanatomical organization. Examination of the effects on sleep of microinjection of
BZ to a variety of brain nuclei wiU be necessary to clearly characterize the neuroanatomical sites
responsible for the hypnotic effects of these widely-prescribed drugs.
208
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02402-01 CP
OERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PR0JE9I f^^" characters Qrjsss. Title must lit on ongjirte txrween the Ixirders.)
Causes and Treatment of Summer Depression
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atfiliation)
PI: T. A. Wehr
Others: N. E. Rosenthal
P. Schulz
S. Kasper
K. Kelly
J.R. Joseph- Vanderpool
Chief, Clinical Psychobiology Branch CPB/NIMH
Chief, Unit on Outpatient Studies CPB/NIMH
Social Worker CPB/NIMH
Visiting Scientist CPB/NIMH
Medical Staff FeUow CPB/NIMH
Medical Staff FeUow CPB/NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN- YEARS;
2
PROFESSIONAL:
1
OTHER:
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Seasonal patterns of affective illness are of particular interest because they suggest that environmental
factors may be capable of causing and terminating affective episodes. We recently identified a group
of patients who become depressed in summer and normal, or hypomanic, in winter. This pattern is
opposite to one we previously described in patients who become depressed in winter, and who
respond to treatment with bright artificial light. Approximately 30 summer depressives have been
studied. Symptom profiles of winter and summer depression are similar in most respects, except that
winter depressives usually oversleep and overeat, while summer depressives sleep less and lose
weight. Both types of depression are much more common in women than in men. We tested the
hypothesis that seasonal changes in light or temperature might cause summer depression, in a
balanced randomization crossover study comparing possible therapeutic effects of darkness and cold.
The results of the study were encouraging, in that patients improved significantly after both types of
treatment. However, the results did not enable us to distinguish between the respective roles of light
and temperature. We are following up with a complementary study of the capacity of heat and light to
induce depressive symptoms in this population. Thyroid hormones decreased when patients became
depressed in the summer. Further research is necessary to determine whether these changes are
responsible for summer depressions. In nine patients, positron emission tomography (PET) scans
were obtained during their summer depressions. These will be repeated during fall/winter remissions
for comparison.
209
PHS 6040 (Rev. 1/84)
GPO a I 4-9 II
ZOIMH 02402-01 CP
Project Description:
Seasonal patterns of occurrence of some types of affective illness suggest that physical
environmental factors, such as temperature or light, may be capable of causing and
terminating episodes of depression and mania. Indeed this has been found to be the case
with winter depression, which responds to treatment with hght, and which therefore may be
caused by cyclic seasonal reductions in natural light. While most of our research on seasonal
affective disorder has focused on winter depression, we recently identified and have begun to
study a group of patients with recurrent summer depressions. Our aim was to validate
patients histories by prospectively ascertaining that they become depressed in the summer and
to investigate the possible role of changes in light and/or temperature as causes, and potential
treatments, of summer depression.
Methods:
Patients were recruited by referral by clinicians famiUar with our studies of seasonal
depression or by descriptions in the media of our research program. Patients' diagnoses
were based on the Revised Diagnostic and Statistical Manual of the American Psychiatric
Association (DSM-IDR) using data obtained with the Structured Clinical Interview (SCID).
Their clinical state was assessed with the Hamilton Depression Rating Scale (HDRS), the
Weekly Mood Inventory (WMI), and visual analogue rating scales (VAS).
Thyroid axis hormones (known to be influenced by temperature changes) were measured in
the spring (before the onset of depression) and summer (during depression).
Positron Emission Tomography (PET) scans were obtained during summer depressions and
after cold treatment
Patients were exposed to two treatment conditions: 1) isolation from bright light (with
special neutral density glasses) and exposure to darkness, and 2) isolation from heat (with
aircondtioning) and exposure to 40° F. The treatments were carried out during two different
five day periods, with time between the periods to allow for relapse if patients improved.
Dark and cold exposures lasted twenty minutes and were repeated four time a day. For an
additional twenty minutes four times a day patients were exposed to outdoor heat on the dark
condition and outdoor light on the cold conditon. Clinical state before and after treatment and
after withdrawal of treatment was assessed with blind raters using the HRSD and by the
patients using the VAS. Patients' expectations of the two treatments were assessed before
the treatments began.
Findings to Date:
Thirty patients with a history of summer depression were diagnosed with the SCID/DSM-
lUR. Most had a bipolar II pattern of illness, with major depression in the summer and
hypomania in the winter. The most frequent symptoms of depression were lethargy, social
withdrawal, loss of interest, insomnia, appetite and weight loss, loss of interest in sex,
sadness. Less freqent were guilt, hopelessness and suicidal thoughts.
Twenty-two patients were followed into the summer season and of these all but two became
significantly depressed (HRSD score >15), as predicted by their histories.
Thyroid hormone levels decreased in the summer compared with the previous spring.
210
ZOl MH 02402-01 CP
PET scans are not yet analyzed.
Eight patients agreed to participate in the experimental cold versus dark treatment study.
Patients significantly improved after both types of treatment with approximately 50%
reductions in HRSD scores. There are several possible explanations for this result.
a. Both temperature and light are capable of regulating mood
b. The effects of temperature and light were confounded in the study
c. Some other factor(s) that were not controlled in the study (e.g., hospitalization) were
responsible for the improvements.
The difficulty in interpreting this study can only be resolved by additional research. Many
unsystematic retrospective and prospective observations of the course of illness in these
patients suggest that changes in temperature (and humidity) play a major role in the
occurrence of summer depressions. However, these clinical insights must be subject to
experimental verification.
Significance to Biomedical Research:
Studies of seasonal depression provide a unique opportunity to explore the possible role of
physical environmental factors, such as light and temperature, as causes of and treatments for
depression. This type of research has already led to the development of a new type of
antidepressant treatment, phototherapy, for depression, and has stimulated a series of basic
studies of biological effects of occularly mediated light as possible mechanisms. This
research, in turn, has led to the discovery of eye-mediated effects of light on immune
function, effects that may prove to be relevant to a variety of seasonally occurring infectious
and autoimmune diseases. In the same way, proof of effects of temperature on clinical state
could be expected to lead to additional new types of treatment for depression and mania, and
to new knownledge about the biological effects of environmental temperature.
Proposed Course:
To further investigate the possible role of temperature and/or light as causes of depression
and mania, patients will be exposed to bright light and to heat during seasons other than
summer. The finding that thyroid hormone levels decline as patients become depressed in the
summer wUl be followed up by more detailed investigations of the thyroid axis in these
patients through the course of the year.
Publications:
Wehr TA, Sack DA, Rosenthal NE: Seasonal affective disorder with summer depression and
winter hypomania. American Journal of Psychiatry, in press.
Wehr TA, Sack DA, Rosenthal NE: Environmental and Behavioral Influences on Affective
Illness. Acta Psychiatrica Scandinavica, in press.
211
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT MUMBER
ZOl MH 02403-01 CP
PERIOD COVERED
October 1. 1986 to September 30. 1987
TITLE OF PROJECT (BO characters or less. Title must tit on one line between the borders.)
Mechanism of Action of Melatonin
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: L. Tamarkin Research Biologist CPB/NIMH
Others:
G. Paciotti
M. ColUns
J. Rhyne-Grey
Biologist
Biologist
Biologist
CPB/NIMH
CPB/NIMH
CPB/NIMH
COOPERATING UNITS (It any)
Steven Reppert, Associate Professor, Children's Service, Massachusetts General Hospital
Aravan Namboodiri, Assistant Professor. Department of Biology, Georgetown University
LAB/BRANCH
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH. NIH. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
0.3
PROFESSIONAL:
0.3
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interview/s
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The melatonin analog, iodo-melatonin, may provide us with an ideal agonist to identify,
characterize, and localize the melatonin receptor. We have made radioactive and "cold" iodo-
melatonin. This product has been characterized by HPLC and the "cold" iodo-melatonin has been
shown to be biologically active. Preliminary data indicate that melatonin can displace iodo-
melatonin binding to a crude supernatant of hamster brain, but no binding was observed with
similarly prepared rat brain homogenate.
213
PHS 6040 (Rev. 1/84)
SPO SI4-SII
ZOIMH 02403-01 CP
Project Etescription:
For testing the biological activity of iodo-melatonin, Siberian hamsters were chosen as the model
system. Previous studies have shown that melatonin infusions of specific durations will signal
gonadal development in the offspring or inhibit it. Exploiting this rapid response (a 4 day prenatal
infusion and 4 week postpartum observation before sacrifice), prenatal infusion of iodo-melatonin
was done on pregnant Siberian hamsters. For testing the binding activity ^^^I-iodo-melatonin
will be prepared and incubated with or without cold melatonin to investigate displacable binding in
membrane and/or cytosol fractions from a number of organs (including specific brain regions).
Methods:
Pregnant Siberian hamsters are infused with iodo-melatonin for 10 hours or 6 hours per day
on days 16 to 20 of pregnancy. The pups are maintained in a LD 14:10 lighting cycle until
30 days of age. Animals are sacrificed and testes weights are recored. For the
characterization of melatonin receptors l25j.io<jQ.meiatonin is prepared by iodination with
iodogen (a similar synthetic procedure is also done to prepare the cold iodo-melatonin) and
the material separated using HPLC. Crude tissue homogenates are prepared from rats or
hamsters and the assay is incubated in the cold for 4 hours. The separation of bound from
free is accomplished by using glass filters pre-soaked in polyethylenimmine.
Findings to Date:
Pups from pregnant dames infused with iodo-melatonin for 10 hours had large testes, similar
to that seen with a melatonin infusion. Pups from dames infused with iodo-melatonin for 6
hours had widely varied testes weights, different from that seen with melatonin. A melatonin
infusion for 6 hours results in consistently small testes in the pups. Displaceable binding
was observed in a crude brain preparation from the hamster, but not from the rat. Speciific
binding was also observed in a homogenate from hamster pituitary and ovary.
Significance to Biomedical Research:
The in vivo data suggest that iodo-melatonin is a melatonin agonists whose affinity for the
melatonin receptor is greater than that of melatonin. These data suggest that this hgand will
be ideal in the characterization of the site of action of the pineal hormone melatonin. The
preliminary binding data also argue that melatonin may be active in the hamster, but is not
active in the rat.
Proposed course:
For the in vivo study a clearance experiment would help us determine if iodo-melatonin is
metabolized to anotiier active compound. Second, in vivo injection of 1251-iodo-melatonin
win help us determine if specific uptake occurs in specific tissues. In vitro, further
characterization of a melatonin receptor and its subcellular localization needs to be done.
Second, CNS autoradiography needs to be done to determine if specific brain regions have
melatonin receptors.
214
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02405-01 CP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Chemical Antidepressant Effects on Body Mass and Body Composition in Hamsters
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: W.Duncan Research Psychologist CPB/NIMH
Others: T. Wehr Chief, Clinical Psychobiology Branch CPB/NIMH
COOPERATING UNITS (if any)
T.J. Bartness, Senior Research Associate,
Worcester Foundation Experimental Biology
Clinical Psychobiology Branch
INSTITUTE AND LOCATION
NIMH. Bethesda,
Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
_25_
_Q_
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
□ (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In humans, one of the side effects of chronic chemical antidepressant treatment is a change in body
mass. The mechanism of these induced changes is not clear but may include alterations in
metabolic rate, as well as effects on specific body components such as body lipid, protein or water.
We have previously noted chemical antidepressant induced alterations in the body mass of Syrian
hamsters being treated chronically with the monoamine oxidase inhibitor (MAOI) clorgyline. This
project was undertaken to more formally explore the behavioral and physiological mechanisms
responsible for these alterations in Syrian hamsters.
We have observed that initial clorgyline treatment decreases food intake. During chronic drug
administration, clorgyline administration reduces the rate of weight gain although the level of food
intake is not different from control. Thus, drug treated hamsters consume more food per unit of
body mass than do control animals, i.e. they exhibit a negative energy balance relative to control
animals.
Analysis of the carcass composition of clorgyline treated hamsters indicates that drug induced
changes are due to a decrease in body lipid content, rather than protein or water. Since seasonal
changes in body mass are due to fluctuations in body lipid content, our results indicate that
clorgyline may be affecting the same photoperiodic process which is responsible for photoperiodic-
induced changes of body mass in the Syrian hamster.
215
PHS6040 (Rev 1/84)
ZOIMH 02405-01 CP
Project Description:
Antidepressant chemicals produce changes in body mass when administered chronically to
humans. These side-effects may be due uniform changes in body component composition or to
selective effects on body lipid, protein or water content. Our objectives were to describe the effects
of chronic antidepressant drug treatment on body mass in Syrian hamsters, and then to identify the
specific body components affected by the drug treatment.
Methods:
1) Treatment
Hamsters were treated with the MAOI clorgyhne (2 mg kg-1 day^) administered via osmotic mini-
pumps implanted subcutaneously. Treatment continued for at least two months.
2) Clorgyline's effects on body mass and food intake in LD 14.5:9.5
Hamsters were group-housed in LD 14.5:9.5. Food intake of group-housed and body mass of
individual hamsters were measured at two-three day intervals.
3) Clorgyline's effects on body mass and carcass composition in constant darkness (DD).
Individually housed hamsters with running-wheel access were maintained in DD and were weighed
at least once per week. After two-months, hamsters were sacrificed and carcass composition
determined for lipid, protein and water content.
Findings t9 Datg;
We have observed two effects of chronic clorgyline on Syrian hamster body mass. First, the total
body mass of clorgyline treated hamsters was significantiy less than the total body mass of saUne
treated hamsters. This change in body mass was not due to a chronic decrease in food intake.
Clorgyline treated hamsters consumed more food per unit body mass than the control hamsters and
were therefore exhibiting a negative energy balance compared to control hamsters. Second,
analysis of carcass composition indicated that the change in body mass was not due to a uniform
change in protein, lipid and water compartments, but specifically due to a decrease in body Upid
content.
Significance to Biomedical Research:
Chemical antidepressants often induce undesirable changes in body mass in patients being treated
for depression. The mechanism(s) underlying these changes probably include drug- induced
effects in metabolic rate and differential effects on body compartment composition.
The physiological and metabolic effects of these compounds on body mass are not understood and
it would be useful develop an animal model for careful examination of these drug effects.
We have observed alterations of body mass and body lipid composition in Syrian hamsters treated
with chronic clorgyline. The Syrian hamster is a photoperiodic species which regulates seasonal
changes in body mass by altering body lipid content. These data indicate that clorgyline may alter
body mass in humans by affecting a circannual or photoperiodic process in weight regulation.
216
ZOl MH 02405-01 CP
Proposed Course:
During the next year, our studies will be directed in three areas. The first area of interest is to
examine the effects of tricyclic compounds on body mass and composition. The second area to
examine is the effect of antidepressant compounds on another seasonal rodent species, the Siberian
hamster, whose body mass response to chronic antidepressant treatment may more closely
resemble the human response. Third, we want investigate the effects of these compounds on
metabolic rate in order to understand the clorgyline-induced negative energy balance observed in
Syrian hamsters.
217
PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT 201 MH 00274-13 LCS
PERIOD COVERED
nctober 1. 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders )
Methods of Ionization in Mass Spectrometry
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Sanford P. Markey, Chief, Section on Analytical Biochemistry, LCS, NIMH
Others: Jeffrey P. Honovich, NRC Research Associate, SAB, LCS, NIMH
Tao-Chin Wang, Fogarty Fellow, IRP, NINCDS
COOPERATING UNITSJrf any)
Department of Pharmacology, George Washington University, Washington, DC
Oak Ridge National Laboratory, Analytical Chemistry Division, Oak Ridge, TN
LAB/BRANCH
Laboratory of Clinical Science
Analytiral Bi nchpmi" stry
^
INSTITUTE AND LOCATION
NIMH, ADAMHA, NTH, Rp.thp..qda , MP 7089?
TOTAL MAN-YEARS:
3.5
PROFESSIONAL:
1.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues Qc (c) Neither
n (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Mechanical and software improvements have been installed and tested on the
dual cell Fourier transform ion cyclotron resonance spectrometer (FT-ICR) which
have corrected many of the previously encountered problems. Laser desorption
of polyethyleneglycol mixtures has been used to optimize ion optics and test
ion trapping and transfer. Experiments to optimize sample sensitivity and
detectability of neuropeptides are in progress.
Collaborative studies on an organic ion microprobe to image the distribu-
tion of organic substances in tissue slices are continuing at Oak Ridge
National Laboratory. Collaborative studies on element and nuclide selective
analyses using a microwave reaction interface for gas chromatography -mass
spectrometry are in progress at George Washington University.
219
PHS 6040 (Rev. 1/84)
Project No. ZOl MH 00274-13 LCS
Other Professional Personnel Enpased on Project:
Fred P. Abramson Guest Worker Professor, Department of
Pharmacology, G.W. Univ.
Washington, DC
Peter J. Todd Collaborator Research Scientist, Oak
Ridge Nat'l Lab., Oak
Ridge, TN
Project Description
Obj ective :
Improvement in the specificity and detectability of complex organic
compounds in biological matrices requires new developments in mass spectro-
metric instrumentation. Surface ionization techniques (laser desorption, fast
atom bombardment) are being explored for the trace analysis of neuropeptides,
neurohormones, and drug metabolites. Fourier transform ion cyclotron resonance
spectroscopy (FT-ICR) is being explored as a high sensitivity and high perform-
ance mass analyzer. The objective of these studies is to develop and utilize
analytical procedures to resolve problems which cannot be solved with conven-
tional instrumentation.
Methods Employed:
Mass spectrometric instrumentation is designed, built, modified, or
purchased as required to meet the above objectives.
Major Findings:
During the past year major instrumentation changes were implemented on the
FT-ICR, correcting many of the previously detailed deficiences. In order to
optimize laser desorption (LD) -FT-ICR, the behavior of electron impact produced
ions was investigated in the dual cell ICR. Ions produced by electron impact
in the high pressure source cell can be transferred through a conductance limit
to a low pressure analyzer cell by grounding the conductance limit for control-
led time intervals. Mechanical alignment of the dual cell assembly in the
magnetic field was optimized by observing ion transfer efficiency during
ionization; this procedure influenced transfer efficiency by two orders of
magnitude. Alternatively, packets of ions stored in the source cell after
ionization could be transferred to the analyzer by grounding the conductance
limit for a specific time, thus simulating LD conditions. There is a mass
dependent transfer frequency because packets of ions of the same mass move with
a characteristic speed. At low pressures (10 - 10 torr) ion transfer was
very efficient (> 90%) , but at higher pressures the transfer efficiency was
very poor. Upon LD, high mass ions were transferred to the analyzer region in
a time -dependent fashion as was observed in EI, although LD produces ions with
a wider kinetic energy distribution than EI. LD-FT-ICR analyses of various
polyethyleneglycol mixtures demonstrated that (1) useful spectra could be
obtained on mixtures representing a wide range of molecular weights without
discrimination; (2) summed spectra are reproducible and compound dependent;
(3) and that high resolution mass analysis following LD may be possible
although not routine.
220
Project No. ZOl MH 00274-13 LCS
The transfer of the organic ion microprobe analyzer previously developed
as a joint effort between NHLBI and BEIB to Oak Ridge National Laboratory was
completed. Dr. Todd has restructured and reconfigured this instrument in order
to improve its performance, and is in the process of designing new ion lenses
to improve sensitivity.
The microwave discharge interface designed and built in SAB/LCS continues
to undergo evaluation and modification at George Washington University.
Planned studies with MPTP metabolites were not performed because C-MPTP
metabolites were identified by other means.
Significance to Biomedical Research:
Structure elucidation of unknown compounds in complex mixtures, or the
specific detection and quantification of known compounds and their isotopic
variants remain important areas of biomedical research. Polar, non- volatile
compounds are frequently encountered in neurochemistry , and the ionization
methods and instrumentation being tested are particularly relevant to the
analysis of neuropeptides and neurohormones.
Proposed Course:
Optimization of the LD-FT-ICR for neuropeptides will be persued, with
extensive evaluation of sensitivity parameters. Probe surfaces, materials, and
means of sample application and concentration will be evaluated. The ability
to measure isotopically- labelled neuropeptides will be studied for determining
the feasibility of in vivo turnover studies.
+ +
The ion microprobe system will be tested with MPP and MPP analogues for
its suitability for spatial analysis. The microwave discharge interface will
be tested with metabolites of labelled neurotoxins.
New instrumentation for routine high sensitivity GC-MS and fast atom
bombardment- tandem MS will be acquired and installed.
Publications :
Abramson, P.P. and Markey, S.P.: Mass spectrometric analysis of sulfur in
microgram quantities of biological macroraolecules using a reaction interface.
Biomed. Environ. Mass Spectrom. 13: 411-415, 1986.
Markey, S.P.: Mass spectrometry: Recent developments. J. Clin. Pharm.
26:406-411, 1986.
Markey, S.P.: Principles and applications of mass spectrometry in clinical
chemistry. Presented at XIII Int. Congress of Clin. Chem. , The Netherlands,
June 28-July 3, 1987.
221
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00276-08 LCS
PERIOD COVERED
rirr"h°r I, IQ"'^ <-V.-rr.i.rTV, gnp<-r^,i,iKr>T- T H -| O p ^
TITLE OF PROJECT (80 characters or less: Titte most fit on one line befween the
borders.)
PRINCIPAL INVESTIGATOR (Ust Other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
P.I.: Sanford P. Markey, Chief, Section on Analytical Biochemistry, LCS, NIMH
COOPERATING UNITS (if any)
Section on Neuroendocrinology , LDN, NICHD
Office of the Chief, LCS, NIMH
Department of Pediatrics , USUHS
Clin. Psychobiol. Branch; NIMH
UB/BRANCH
Laboratory of Clinical Science
Anfllyfi ral — Biochemistry
NSTiTUTE AND LOCATION
NIMH, ADAMHA, NTH, Rp.thp.c;da MO 7089?
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
1.0
CHECK APPROPRIATE BOX(ES)
[^ (a) Human subjects
Q (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Usa standard unreduced type. Do not exceed the space provided.)
This project was inactivated early in the year and subsequently
terminated.
223
PHS 6040 (Rev. 1/84)
SPO 9I4->1«
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PEHIOD COVERED
October 1 , 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less Title must tit on one line between ttte borders.)
Synthesis of Stable Isotope-Labeled Compounds
PROJECT NUMBER
ZOl MH 00277-08 LCS
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Sanford P. Markey, Chief, Section on Analytical Biochemistry, LCS, NIMH
Others: Adrian Weisz, Visiting Fellow, LCS, NIMH
Riccardo Boni , Visiting Fellow, LCS, NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Analytical Biochemistry
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS; PROFESSIONAL
1.5 1.2
OTHER:
.3
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues E (c) Neither
□ (a1) IVIinors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
Stable- and some radioisotope -labeled compounds have been synthesized to
support other laboratory projects. Structural analogues of 1 -methyl -4 -phenyl -
1,2,3,6-tetrahydropyridine (MPTP) were prepared; oxygen- 18- labelled- quinolinic
acid prepared by exchange for use as an internal standard; synthetic routes for
labelled tryptophan and its metabolites are under study.
225
PHS 6040 (Rev. 1/84)
Project No. ZOl MH 00277-08 LCS
Objectives :
The synthesis of labeled compounds is an integral program component in
the investigation of metabolism and distribution of endogenous and xenobiotic
compounds. Other projects in the SAB require labeled or structural analogues
in order to trace metabolic pathways, determine kinetics, or accurately
measure trace quantities with an internal mass standard.
Methods Employed:
Conventional routes of chemical synthesis employing isotopes and general
chemicals have been used. Sufficient l-methyl-4- (4-amino)phenyl- 1 , 2 , 3 , 6-
tetrahydropyridine (4' -araino-MPTP) was synthesized (8 gm) to continue both dog
and mouse testing of its properties (ZOl-MH-00279-05 LCS) . The synthetic
route used was nitration of 4-phenylpyridine and separation of the 2', 3', 4'
isomers by repeated fractional crystalization. The 4' -isomer was reduced with
SnCl„/HCl to the 4' -amino compound; acetylated with acetic anhydride; 1-meth-
ylated with iodomethane; hydrolyzed to remove the acetyl group; and then
reduced with sodium borohydride to 4' -amino-MPTP. Extensive purification was
effected at several stages to produce product with less than 0.1% impurities,
in contrast to a shorter synthetic route using a one-step Pd/C -borohydride
reduction of methylated nito compound.
1 8
Quinolinic acid-,oO, was prepared by acid catalyzed exchange of
quinolinic acid in H„ 0 and was found to be a suitable internal standard for
a negative ionization mass spectrometric assay when esterified under
non-acidic conditions (ZOl-MH-02384-01 LCS)
Major Findings:
The use of each of these synthetic products is described in other annual
reports (ZOl MH 00279-05 LCS, ZOl-MH-02384-01 LCS).
Significance to Biomedical Research:
The availability of labeled compounds is frequently the limiting step in
metabolism projects. A program in analytical biochemistry requires continuing
synthetic efforts to prepare stable and radioisotope analogues for the timely
and efficient solution to metabolism projects.
Proposed Course:
Additional structural and labelled analogues of MPTP will be prepared for
animal studies. Synthetic routes for labelled analogues of tryptophan and its
major metabolites kyurenine , anthranilic acid, kynurenic acid, 3-hydroxy-
kynurenine, and 3-hydroxyanthranilic acid will be investigated.
Publications :
Weisz, A., Markey, S.P., and Ito, Y.: Preparative separation of polar
unstable compounds (catecholamines) from a synthetic mixture by high-speed
counter-current chromatography. J. Chromatography 383: 132-136, 1986.
-|^2 Weisz, A. and Markey, S.P.: Synthesis of D/L-norepinephrine - (phenyl -
U- C) . J. Lab. Compounds & Radiopharm., in press, 1987.
226
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00279-05 LCS
PERIOD COVERED
October 1. 1986 through September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must fit on arte line befween the borders.)
Pharmacology of Neurotoxins
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Sanford P. Markey, Chief, Section on Analytical Biochemistry
Others: Jan Johannessen, Senior Staff Fellow, SAB, LCS, NIMH
Song-cheng Yang, Visiting Scientist, SAB, LCS, NIMH
Hiroya Ikeda, Guest Worker, SAB, LCS, NIMH
Mark Duncan, Visiting Fellow, IRP, NINCDS
Miles Herkenham, Senior Investigator, LNP, NIMH
Kris Bankiewicz, Visiting Fellow, IRP, NINCDS
COOPERATING UNITS (it any)
Laboratory of Neurophysiology, NIMH
Office of the Director, IRP, NINCDS
UB/BRANCH
Laboratory of Clinical Science
SECTION
Analytical Biochemistry
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda. MD 20892
TOTAL MAN-YEARS:
6
PROFESSIONAL:
4.5
OTHER:
1.5
CHECK APPROPRIATE BOX{ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The mechanism of action and the metabolism of the parkinsonian- syndrome
inducing neurotoxin MPTP (l-methyl-4-phenyl-l, 2, 3 ,6-tetrahydropyridine) has
been studied in mouse, dog, and monkey. The metabolism of radiolabelled MPTP
has shown that only the pyridinum metabolite, MPP+, is accumulated in monkey
brain. Distribution of that metabolite with respect to time (1, 3, or 10 days)
indicated that MPP-i- persists in the striatum and does not migrate toward or
co-exist with the dense neuromelanin deposits in the substantia nigral cells.
The conditions for the safe handling of animals treated with MPTP was
evaluated; MPTP and its metabolites are not vapor borne, and treated animals
can be handled safely with normal laboratory protective clothing.
The neurotoxic effects of MPTP in beagle dogs has been evaluated. MPTP
produces a profound and permanent loss of cells within the substantia nigra at
doses similar to those effective in primates. Dogs exhibit a transient
hypokinesia, but regain locomotor capability in spite of the extent of nigral
cell loss.
Immunoassay of MPTP and MPP+ has been improved and polyclonal rabbit
antibodies characterized for their binding characteristics. Brain extracts
contain substances with interfere with antigen-antibody recognition, necessi-
tating chemical separation. A metabolism study of another parkinsonian
producing neurotoxin, a-methyl- g-methylaminopropionic acid (BMAA) has been
initiated.
227
PHS 6040 (Rev. 1/84)
Project No. ZOl MH 00279-05 LCS
Objectives :
The neurotoxicity of l-methyl-4-phenyl-l , 2 , 3 , 6- tetrahydropyridine (MPTP)
in man and monkey result in a parkinsonian syndrome which is nearly indistin-
guishable from idiopathic Parkinson's disease. By determining the mechanism of
MPTP's action in destroying a specific sub-set of dopamine-rich cells in
primate brain, rationale therapy to slow or prevent the progressive idiopathic
disease process in man might be designed. Further, environmental toxins which
may have mechanisms similar to that of MPTP are being investigated. The
metabolism of a compound implicated in the high incidence of a parkinsonian
syndrome on Guam, a-amino- g-methylaminopropionic acid (BMAA) is being studied
to determine if there are characteristic urinary metabolites to indicate
dietary exposure .
Methods Employed:
MPTP toxicity is being studied by: qualitative and quantitative observa-
tion of animal behavior and locomotion; neurochemical determination of cate-
cholamines and their metabolites in specific brain regions by high pressure
liquid chromatography with electrochemical detection (HPLC-EC) ; determination
of the pattern of MPTP-distribution, metabolism, and excretion, using radio and
stable -labelled MPTP ( H, C, H) in mouse and monkey; autoradiography of
tissue exposed to labelled-MPTP (ZOl MH 01090-11); and mass spectrometry of
isolated metabolites. Enzyme-linked immunoassay and radioimmunoassay proce-
dures are being used to detect MPTP, MPP , and structurally related compounds.
Major Findings:
14
Studies on the metabolism of C,-MPTP in mice and monkeys revealed the
following:
(1) Metabolism of MPTP by monkey produces MPP as the only metabolite detect-
able after one day. Greater than 98% of the radioactivity persisting in monkey
brain after 24 hrs is MPP . Thus, previous observations of other metabolites
were due to label exchange and were artifacts of the experimental procedure.
(2) In the monkey, MPP is accumulated and stored in intact striatal axons, and
not in nigral cell bodies. This pattern of distribution after 24 hrs is
accentuated after 3 and 10 days survival. There is no retrograde transfer of
MPP from the axons to the cell bodies which are rich in neuromelanin. Thus,
it is unlikely that neuromelanin participates in the neurotoxic action of MPTP,
but is probably an indicator of susceptible cells with limited oxidation
capacity.
(3) The 2% of labelled MPTP metabolites which are bound to precipitable macro-
molecules in monkey brain are probably not relevant to MPTP neurotoxicity.
This interpretation is based upon mouse metabolism studies where the effects of
agents which block MPTP neurotoxicity (MAO-B inhibitors, dopamine uptake
inhibitors) were found to markedly increase the amount of bound metabolite.
The inverse correlation between the amount of bound metabolites and neuro-
toxicity is strong evidence that only MPP is relevant to neurotoxicity.
228
Project No. ZOl MH 00279-05 LCS
(4) Substantia nigra neurons die by slow retrograde degeneration, a fact which
may be particularly relevant to idiopathic Parkinson' s disease pathogenesis as
a striatal rather than nigral disease process.
(5) Procedures for the safe laboratory use of MPTP in animal studies have been
developed. This has been a major laboratory safety issue at NIH and many
institutions which utilize MPTP as a pharmacological tool. The distribution of
C,-MPTP administered to mice or monkey in environmental chambers permitted
the observations that MPTP is not vapor borne after i.v. administration, and
that unmetabolized MPTP on excreta or animal bedding may be safely contained
and destroyed.
Other studies have concentrated on determining the best conditions for
immunoassay of MPTP and MPP using the rabbit antibodies raised to amino-
analogues of MPTP and MPP cross -linked to bovine serum albumin. ELISA assay
based upon horseradish peroxidase linked to goat anti- rabbit antibody has been
the subject of intensive metholological investigation. The assay works well to
detect 'MPTP and MPP -like compounds, and many structural isomers have been
analyzed. However, aqueous or ethanol extracts of brain contain factors which
inhibit antigen- antibody recognition, and significantly confound the assay
sensitivity. Human brain tissue received from tissue banks was surveyed to
determine if extracts of Parkinsonian brains contain MPTP or MPP -like factors.
However, these studies were preliminary and not definitive due to the lack of
assay specificity. In order to correct these problems, radioimmunoassay rather
than colorimetric ELISA has been investigated. Results indicate that RIA is
both more sensitive and quantitative. Based upon characterization of various
rabbit antibody sera using RIA, those sera with highest titer and specificity
have been used to test whether an enzyme amplified ELISA might provide the
required sensitivity enhancement to preclude non-specific interferences from
tissue constituents. Preliminary data suggest that enzyme amplification works
to detect antibody at higher dilutions than possible with RIA or conventional
ELISA. Translating that sensitivity enhancement to greater sensitivity toward
MPP -like antigens has remained problematic.
The neurotoxicity of MPTP in primates and mice has been extensively
described. However, the effects of MPTP in other susceptible species has not
been well characterized. Previous work had demonstrated that beagle dogs were
susceptible to MPTP at doses similar to those effective in primates. These
observations have been extended in a recently completed study. Adult beagle
dogs of both sexes were injected with MPTP either alone or after pretreatment
with a monoamine oxidase inhibitor (pargyline) . Groups were sacrificed 2 h, 3
weeks, or 3 months after injection in order to determine acute and chronic
effects of MPTP on biogenic amines and their synthetic and degradative enzymes
in various brain regions. MPTP caused a massive and permanent loss of striatal
dopamine and the loss of cells within the substantia nigra pars compacta.
Despite a permanent loss of the nigrostriatal system, the dogs exhibited only a
transient hypokinesia lasting one to two weeks. This has important implica-
tions for preclinical neurotoxicity testing of drug safety, and suggests
reevaluation of the practice of using behavioural indices of neurotoxicity in
the dog and applying the findings to man. Further, the tremendous discrepancy
in the behavioral consequences of a loss of the nigrostriatal dopamine system
between the dog and primate forces a reexamination of the role of this system
in sub-primates. Pargyline pretreatment prevented the loss of striatal
dopamine and cells from the substantia nigra, but did not prevent a substantial
229
Project No. ZOl MH 00279-05 LCS
but reversible decrease in the concentration of dopamine metabolites. This
apparent inhibition of monoamine oxidase is due not to suicide inactivation of
the enzyme by MPTP, but to reversible inhibition by the pyridinium metabolite
MPP selectively in dopaminergic terminals.
The question of environmental factors which may be implicated as causative
agents of Parkinson's disease has drawn attention to a -amino-g-methylamino-
propionic acid (BMAA) as a neurotoxic constituent of the seeds of Cycas
circinalis, a plant used for food on the island of Guam. In former times when
natives on Guam used cycad flour as a basic food, a parkinsonian syndrome was
very common. Animal studies have implicated BMAA as the neurotoxic principle,
but little is known about its occurrence or metabolism. Studies have been
initiated to define whether there are unique urinary BMAA metabolites to mark
exposure to this amino acid.
Significance to Biomedical Research:
The MPTP-lesioned primate has been firmly established as a useful animal
model of idiopathic Parkinson's disease in man. The mechanism of action of
MPTP may be relevant to the human disease process , and attempts to identify
neurotoxic environmental agents may lead to preventative measures for a very
common disease of aging.
Proposed Course:
Questions regarding the subcellular localization of MPTP are most impor-
tant in determining whether MPP is working as a mitochondrial toxin. The
metabolism of 4' -amino-MPTP w^ill be further characterized and studies of its
action in dogs completed. Procedures to separate compounds related to MPP
will be used in conjunction with RIA and ELISA procedures to re-examine human
brain tissue.
Publications :
Johannessen, J.N., Savitt, J.M., Markey , C.J., Bacon, J. P., Weisz, A.,
Hanselman, D.S., and Markey, S.P.: The development of amine substituted
analogues of MPTP as unique tools for the study of MPTP toxicity and
Parkinson's disease. Life Sci. 40: 697-704, 1987.
Markey, S.P., Weisz, A., and Bacon, J. P.: Reduced paraquat does not
exhibit MPTP-like neurotoxicity. J. Anal. Toxicol. 10: 257, 1986.
Markey, S.P. and Schmuff, N.R.: The pharmacology of the parkinsonian
syndrome producing neurotoxin MPTP (l-methyl-4-phenyl-l , 2 , 3 , 6- tetrahydropyri-
dine) and structurally related compounds. Medicinal Chem. Res. 6- 389-429
1986.
Kopin, I.J., Burns, R.S., Chiueh, C.C, and Markey, S.P.: MPTP-induced
parkinsonian syndrome in humans and animals . Alzheimer's and Other Age Related
Disorders: Strategies in Research and Development (Eds. A. Fisher, C.
Lachmann, and I. Hamin) , Plenum Press, pp. 519-530, 1986.
230
Project No. ZOl MH 00279-05 LCS
Markey, S.P., Yang, S.-C, Johannessen, J.N., Burns, R.S., Herkenham, M. ,
and Bankiewicz , K. : Mechanisms of MPTP toxicity. Submitted to the 6th
International Symposium on Catecholamines, Jerusalem, Israel, June 14-19,
1987.
Kopin, I.J. and Markey, S.P.: MPTP toxicity: Implications for research
in Parkinson's disease. Ann. Rev. Neurosci., in press, 1987.
Markey, S.P.: MPTP - A new tool to understand Parkinson's disease.
Discussions in Neurosciences (FESN) , Vol. 3, No. 4, 1986.
231
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02384-01 LCS
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Brain Quinolinic Acid Metabolism: Role in Neuropathology
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Melvyn P. Heyes , Visiting Associate, LNP , NIMH
Other: Sanford P. Markey, Chief, Section on Analytical Biochemistry, LCS, NIMH
''°eKmim6^^S'r^europhysiology
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Analytical Biochemistry
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL;
■ 0.5
1.0
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Humans are afflicted with a wide variety of neurodegenerative diseases,
especially of the cerbral cortex and striatum, which result in neuropsychiatric
disturbances. Key anatomical and neurochemical characteristics of certain
neuropathologic conditions can be replicated in experimental animals by local
injections of quinolinic acid (QUIN) , an endogenous metabolite of tryptophan
(TRP) . QUIN has proved to be a potent neurotoxin and convulsant and may
therefore be involved in human neuropathology. To investigate such a role, we
developed a sensitive, accurate and specific assay for QUIN in brain and
cerebrospinal fluid (CSF) . We found that brain QUIN is increased by systemic
TRP and 3 -hydroxyanthranilic acid (3-OHAA) loads. Inhibition of TRP metabolism
by pyrazinamide (PYR) facilitates increases in QUIN following TRP administra-
tion. These findings are consistent with an etiologic role of QUIN in glutaric
aciduria type I, a pediatric disorder associated with impaired metabolism of
TRP.
233
PHS 6040 (Rev. 1/84)
Project No. ZOl MH 02384-01 LCS
Obi ective :
One mechanism that may cause nerve cell death in human neurodegenerative
disorders is the presence of neurotoxins, derived either from the environment
or present as intermediates of metabolism. One group of compounds have been
identified as potent excitants of neuronal activity, convulsants and neuro-
toxins. The first of these ' excitotoxins ' to be identified include the weakly
toxic endogenous amino acid glutamic acid and the potent, exogenous glutamate
analog, kainic acid. Recently quinolinic acid (QUIN) , an endogenous metabolite
of tryptophan (TRP) , was shown to be a potent neurotoxin and convulsant. QUIN
may be responsible for neural degeneration and may also have importance in
convulsant disorders .
An intregral part of investigating a possible role for QUIN in neuropathol-
ogy is the development of a specific, sensitive, and accurate assay for QUIN in
brain. Two previous assays for brain QUIN had been described, both gas chroma-
tography /mass spectrometry (GC/MS) methods, but sensitivity was low and
required 100-300 mg of brain tissue for analysis.
A more sensitive mode of mass spectrometric analysis is electron capture
negative chemical ionization (CI) which both efficiently forms a molecular
anion and fragments QUIN to a lesser degree than previous approaches. Conse-
quently, negative CI GC/MS requires smaller samples and offer more specific
detection of QUIN in the sample. We used this technique, and prepared, „
[ 0]-QUIN for use as internal standard. Extensive tests showed the [ 0] -QUIN
was the most suitable internal standard and substantially increased the
accuracy of QUIN measurement.
This technique was used to determine the effects of systemic administra-
tion of QUIN precursors: L-tryptophan (L-TRP) , L-kynurenine (L-KYN) , and
3-hydroxyanthranilic acid (3-OHAA) on brain QUIN content. In addition, the
effects of impairing the catabolism of L-TRP through the glutaryl-CoA pathway
was investigated by administration of pyrazinamide (PYR) . This drug results in
the inhibition of 2-amino-3-carboxymuconic semialdehyde dehydrogenase, the
first enzyme in the glutaryl-CoA pathway.
Methods :
[ OJ-QUIN was prepared by heating 6 mol of QUIN in 1 ml of [0] -water/3
N HCL for 48 hours at 80 C. QUIN was quantitated as the dihexaf luoroisopro-
panol ester (mass 467 daltons) .
Samples were analyzed following GC separation using a Finnigan 3200
chemical ionization quadrupole mass filter with Extrel electronics and a
Teknivent data system 1050. The carrier gas was methane. The minimum
detectable quantity at a signal to noise ratio of 10:1 was 6 fmol, a 1000-fold
improvement over previous assays.
Sample collection and preparation.
Studies were done on male Sprague-Dawley rats weighing 300-350 g. Brain
P^gts weighing 10 to 120 mg were sonnicated in perchloric acid containing
[ 0]-QUIN (60 or 300 pmol/ml)^g For the standard curve, 1 ml of the 6% PCA
containing 60 or 300 pmol of [ 0] -QUIN was added to known quantities of QUIN
234
Project No. ZOl MH 02384-01 LCS
(6-1200 pmol in duplicate or triplicate) dissolved in water. All samples were
centrifuged at 12,000 g for 15 min at 0 C. Supernatants were neutralized with
10 M potassium hydroxide. Following centrifugation the aqueous layer was
applied to the Dowex columns. Columns were washed with 4 ml water and 4 ml 0.1
N formic acid. QUIN and [ 0] -QUIN were eluted with 5 ml of 6.0 N formic acid,
collected into screw capped tubes and immediately frozen in solid C0„ . Samples
were lyophylized overnight or evaporated using rotary evaporator.
Major Findings:
QUIN was identified in samples from brain and CSF, as shown by the
presence of QUIN-specific ions at mass -to -charge ratio (m/z) 467 and 316
eluting at the correct time from the GC column. Importantly, no ion currents
atgm/z 471 and 320 were observed at the time of QUIN elution, establishing that
[ 0] -QUIN can be used as internal standard. Extensive tests established that
quantitation of QUIN using [ 0]-QUIN was over 30 times more accurate than
previous assays.
In saline-treated rats, cortical QUIN content was similar in the three
regions measured. QUIN content was increased to 1107%, 977%, and 1235% of
control in frontal, parietal, and occipital cortex, respectively, 2 h after a
systemic L-TRP load. In a second group of rats, L-TRP increased QUIN content
in frontal and parietal cortex by 252% and 519% respectively. Cortical QUIN
content was unchanged by PYR- treatment. However, co-administration of PYR and
L-TRP resulted in a 1.88 -fold and a 1.80 -fold potentiation of the increase in
QUIN in frontal and parietal cortex respectively. L-TRP increased QUIN content
of parietal cortex, striatum, and thalamus by 1065%, 1150%, and 1467% respec-
tively. QUIN content was alsa increased following 3-OHAA administration, but
to a greater extent than by L-TRP loading. L-KYN administration increased QUIN
to a lesser extent than did either L-TRP or 3-OHAA.
Significance to Biomedical Research:
Quinolinic acid has proved to be a potent neurotoxin and convulsant when
injected into the central nervous systems of experimental animals. Quinolinic
acid is present in brain and clearly may have etiologic importance in human
brain dysfunction. A sensitive, specific and accurate assay for brain and CSF
QUIN is therefore a prerequisite to investigating such a role. Considerable
time and effort were expended at this stage of the project to establish the
necessary assay. We have hypothesized that an increase in brain QUIN may cause
the neuropathological characteristics of glutaric aciduria. Our observation
that PYR, which blocks the first step in the glutaryl-CoA pathway, increases
brain QUIN is consistent with this hypothesis. Experimental animals treated
with PYR may serve as a model for this inborn error of metabolism.
Proposed Course:
Four linked approaches will be pursued over the coming year.
1) Cerebral Metabolism.
Continuation of studies in rodents on the metabolism of QUIN in brain.
These will include the effects of diet, circulating amino acid concentrations
and the effect of drugs and agents already known to influence L-TRP metabolism.
235
Project No. ZOl MH 02384-01 LCS
2) Animal Models of Human Neuropathology.
Both ischemia and hypoglycemia induce neurodegeneration which can be
blocked by antagonists of NMDA-type excitatory amino acid receptors, suggesting
that activation of these receptors mediate nerve cell damage in these situa-
tions. QUIN is a potent agonist of these receptors and mediates at least some
of its neurotoxic effects by their activation. Perhaps QUIN is involved in the
neuropathology of these conditions. This hypothesis is directly testable using
the assay for QUIN we have developed. Studies of PYR will continue, particu-
larly with respect to the effects of diet.
3) QUIN Concentrations in CSF.
Measurement of QUIN in the CSF in humans may open a window into QUIN
metabolism in brain. If so, it may be possible to determine ongoing QUIN
metabolism in human neuropathological states. To investigate how well QUIN
concentrations in CSF reflect brain QUIN levels, rhesus monkeys will be given
treatments which have been shown to either increase or decrease QUIN concentra-
tions in brain of rodents, and the concentrations of QUIN in CSF will be
determined. Later, postmortem studies will be done to fully characterize the
relationship between QUIN in brain and CSF.
4) QUIN Concentrations in Human Neuropathology.
QUIN concentrations will be measured in samples of CSF and brain in
patients suffering from and who have died from a variety of neurodegenerative,
convulsant, and motor disorders, including AIDS. This result will be pursued
in the next fiscal year.
Publications :
Heyes, M.P.: Hypothesis: A role for quinolinic acid in the neuropathology of
glutaric aciduria type I. Can. J. Neurol. Sci., in press, 1987.
236
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00351-13-LCS
PERIOD COVERED
October 1, 1986 - September 30, 1<
TITLE OF PROJECT (BO characters or less. We must fit on arte line between the tmrders.)
Clinical Pharmacology of the Central Nervous System
PRINCIPAL INVESTIGATOR (List other prolessionei personnel t>elow the Principal Investigator) (Name, title, laboratory, and institute attiliation)
David C. Jimerson, M.D., Chief, Section of Biomedical Psychiatry
COOPERATING UNITS (H any)
SAB, LCS, NIMH; SCBB, LCS, NIMH
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Biomedical Psychiatry
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, MD
20892
TOTAL MAN-YEARS:
0.6
PROFESSIONAL:
0.3
0.3
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
B (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies in the laboratory focused on the effects of altered feeding
patterns, stress, and pharmacologic treatments on the regulation of appetitive
behavior, satiety responses and body weight, with particular focus on the
influence of central catecholamine and endogenous opiate systems. In a series
of studies on the behavioral pharmacology of feeding in laboratory rodents
begun during the past year, we showed that naloxone decreased sham feeding of
sucrose in a dose dependent pattern similar to that produced by the dopamine
antagonist pimozide. Preliminary results of a study neonatal treatment of
laboratory rodents with opiate agonists demonstrated that these animals have
long-lasting alterations in opiate-mediated feeding behaviors. These
preclinical studies may assist in the development of animal models pertinent
to the clinical disorders of bulimia and anorexia nervosa.
237
PHS 6040 (Rev. 1/84)
GPO 914-9II
ZOl MH 00351-13-LCS
Other collaborative personnel engaged on the project;
S.P. Markey Chief, SAB LCS, NIMH
J. A. Stuckey Guest Researcher LCS, NIMH
T.R. Insel Staff Psychiatrist LCS, NIMH
Project Description:
Objectives:
The purpose of this study is to develop techniques for the assessment
of central and peripheral monoamine function, and to assess the relationship
of these measures to activity in other physiological and behavioral systems.
Behavioral studies in animals have been Initiated to examine the role of
central monoamines and other neuromodulators, especially endogenous opiate
systems, in preclinical models of eating disorder syndromes.
Methods:
Biochemical methods for assay of endogenous monoamine metabolites in
tissues and body fluids include gas chromatography-mass spectrometry (GCMS)
and high pressure liquid chromatography. Radioimmunoassay techniques are used
for assay of hormone levels regulated by monoamine neurotransmitters in vivo.
Effects of pharmacologic treatments and behavioral paradigms on
neurotransmitter and neuropeptide receptor activity in laboratory rodents is
assessed in vitro using selective radioreceptor binding techniques.
Behavioral studies in rodents include measurement of food and water
intake and longitudinal changes in body weight, and ratings of appetitive and
satiety behaviors. These measures are recorded in response to acute
pharmacological challenge, or as longitudinal changes in response to drug
treatments or to alterations in diet or housing conditions.
Major Findings:
Over the past year we have begun studies on preclinical models for
altered patterns of appetite and satiety observed in patients with eating
disorders. Sham feeding provides one possible model for binge-purge
episodes in bulimic patients, since in this paradigm animals do not absorb
nutrients from ingested food. Clinical studies on neurotransmitter and
neuropeptide function in patients with anorexia nervosa and bulimic disorder
suggest that central opiate systems may play a prominent role in the
pathophysiology of these syndromes. Thus, anorexic patients at low weight
have reduced CSF concentrations of beta-endorphln and related peptides.
During the past year, we completed a study with rodents showing that
naloxone produced dose-dependent attenuation of sham feeding of sucrose
solutions with a similar time course to that produced by the dopamine
antagonist naloxone. This finding shows that naloxone-lnduced satiety is
Independent of gut absorption of nutrients consumed.
Previous studies have shown that neonatal treatments of rats with opiates
238
ZOl MH 00351-13-LCS
produce changes in opiate receptors that persist into adulthood, with some
evidence for changes in behaviors which are thought to be mediated in part
by endogenous opiates. In a preliminary study of neonatal treatment of
laboratory rats with the opiate agonists morphine and trifluodom, we have
observed a long term potentiation of naloxone-induced inhibition of feeding
behavior in the adult animals. These initial data suggest that factors
altering activity of endogenous opiate systems early in life could have
residual effects resulting in altered eating behaviors in adulthood.
Significance to Biomedical Research and the Program of the Institute
Investigation of the effects of neurotransmitters, endogenous opiates,
and other neuromodulators on feeding behavior may help in the development of
animal models for anorexia nervosa and bulimia. This work is important in
evaluating the possible role of pharmacological paradigms or specific
behavioral experiences, such as chronic stress or chronic dieting, in the
development of neurochemical vulnerability to altered eating patterns, and
could ultimately help in the development of new pharmacological treatment
strategies for patients with eating disorders.
Proposed Course:
Preclinical studies of feeding behavior will further assess the effects
of altered dietary patterns, stress, and pharmacologic treatments on natural
feeding and on sham feeding, with particular focus on opiate, catecholamine
and serotonergic systems. Behavioral results from current studies will be
correlated with post-mortem analyses of brain monoamine metabolites, and
receptor binding studies involving central opiate and catecholamine pathways.
Publications:
Pohl, R. , Ettedgui, E., Bridges, M. , Lycaki, H. , Jimerson, D.C., Kopin, I.J.,
Rainey, J.M. : Plasma MHPG levels in lactate and isoproterenol anxiety states.
Biol. Psychiatry 22: 1127-1136, 1987.
239
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02289-03-LCS
PERIOD COVERED
October 1. 1986 - September 30, i;
TITLE OF PROJECT (80 characters or less. Title must fit on one line tietween the borders.)
Psychobiology of Eating Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel tielow the Principal Investigator) (Name, title, laboratory, and institute affiliation)
David C. Jimerson, M.D., Chief, Section on Biomedical Psychiatry
COOPERATING UNITS (if any)
SCN, LCS, NIMH; SCP, LCS, NIMH; SCN, BPB, NIMH; CNG, NIMH; SCS, NSB, NIMH
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Biomedical Psychiatry
INSTITUTE AND LOCATION
NIMH. ADAMHA. NIH. Bethesda. MD 20892
TOTAL MAN-YEARS:
5.7
PROFESSIONAL:
2.7
1.0
CHECK APPROPRIATE 80X(ES)
E (a) Human subjects
n (a1) Minors
n (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Clinical investigations involving the syndromes of bulimia and anorexia
nervosa during the past year included neurotransmitter, neuropeptide,
neuroendocrine, metabolic, pharmacologic and related behavioral studies on
neurobiologic factors thought to contribute to the etiology of these
disorders, and to their variable responsiveness to available treatments.
Consistent with preclinical data implicating hypothalamic serotonin
dysregulation in impaired post-prandial satiety, administration of the
serotonin agonist m-chlorophenylpiperazine (m-CPP) decreased caloric intake in
test meal studies. Prolactin responses to m-CPP were significantly blunted in
eating disorder patients and in bulimic anorexic patients. Analysis of
pharmacokinetic data showed that m-CPP-induced migraine-like headaches in
eating disorder patients were related to blood level of the drug, as well as
to personal and family history of migraine headache. Analysis of samples from
a newly completed lumbar puncture study demonstrated that cerebrospinal fluid
levels of homovanillic acid were significantly reduced in patients with severe
bulimia, consistent with an alteration in central reward pathways involving
dopamine. In preliminary results of a collaborative study of behavioral and
neurochemical responses to 2-deoxyglucose, bulimic patients had blunted
responses as reflected in hunger ratings and caloric consumption during a test
meal. Follow up on our previous findings of dysregulation of opiate and
hypothalamic-pitultary-adrenal function in anorexic patients included
collaborative studies utilizing the opiate antagonist naloxone and a
glucocorticoid antagonist. Interviews continued for the family study of
bulimia. Studies on energy metabolism showed that resting metabolic rate was
significantly reduced in weight stable bulimic patients, suggesting increased
efficiency of energy utilization. Preliminary results Indicated a substantial
role of the thyroid axis in elevation of resting metabolic rate observed in
anorexic patients during weight gain.
241
PHS 6040 (Rev. 1/84)
:po bi 4.91a
ZOl MH 02289-03-LCS
Other collaborative personnel engaged on the project:
T.D.
Brewerton
M.D,
Lesem
H.A.
Brandt
J. A.
Kassett
E.
Obaraznek
A.
Hegg
W.H.
Kaye
D.L.
Murphy
P.W.
Gold
W.Z.
Potter
D.
Pickar
D.
Weinberger
E.S.
Gershon
W.H.
Berrettini
C.
Duncan
Medical Staff Fellow
Medical Staff Fellow
Medical Staff Fellow
Clinical Social Worker
Guest Researcher
Guest Researcher
Associate Professor
Chief
Chief, SCN
Chief, SCP
Chief, SCS
Chief,
Chief
Staff Psychiatrist
Senior Staff Fellow
LCS,
NIMH
LCS,
NIMH
LCS,
NIMH
LCS,
NIMH
LCS,
NIMH
LCS,
NIMH
Univ
. of Pittsburgh
LCS,
NIMH
BPB,
NIMH
LCS,
NIMH
NSB,
NIMH
CBDB
, NIMH
CNG,
NIMH
CNG,
NIMH
LPP,
NIMH
Project Description:
Objectives:
The purpose of this project is to study the psychobiology of major eating
disorders from the standpoint of neurotransmitter metabolism, neuroendocrine
and neuropeptide function, cognitive and psychological function, and
pharmacologic treatment. The major focus of this project is presently
directed toward the syndromes of anorexia nervosa and bulimia.
Methods:
1. Behavioral and psychological assessment: Baseline evaluation of
patients and control subjects entails clinical and structured diagnostic
interviews; subjective and objective mood, attitude and behavioral ratings;
and psychometric approaches. Baseline measures and treatment-related
alterations are assessed by research psychiatrists, social worker,
psychologist, clinical nutritionist and nursing staff.
2. Neurobiologic assessment: Studies of presynaptic neurotransmitter
activity include measurement of the biogenic amines and their major
metabolites in blood, urine and cerebrospinal fluid. These studies focus on
norepinephrine and 3-methoxy-A-hydroxyphenylethylene glycol (MHPG) , dopamine
and homovanillic acid (HVA) , and serotonin and 5-hydroxyindole acetic acid
(5-HIAA) . Neurotransmitter receptor activity is assessed through challenge
studies using selective pharmacologic probes, with measurement of physiologic,
metabolic, neuroendocrine and behavioral responses. Receptor sensitivity is
measured in vitro using cellular elements from blood.
Methods used in neuroendocrine protocols are established techniques of
measuring baseline hormone levels in blood (Cortisol, corticotropin (ACTH) ,
prolactin, growth hormone, triiodothyronine, and thyroid stimulating hormone)
242
ZOl MH 02289-03-LCS
or urine (free Cortisol) , as well as responses to hypothalamic releasing
factors, other pharmacologic probes, and physiologic challenges. These
techniques test the integrity of feedback regulation in specific endocrine
systems, as well as the sensitivity of neurohormonal and neurotransmitter
receptors. Activity of neuropeptide systems in the central nervous system is
evaluated by measurement of cerebrospinal fluid peptide concentrations in a
baseline state and during treatment.
Nutritional and metabolic state is evaluated using standard biochemical
measures in blood and urine, anthropometry, potassium-40 scanning and indirect
calorimetry. Cerebral electrophysiologic responses are measured in
collaboration with investigators in other branches. Cerebral anatomic and
functional imaging studies utilize computed tomography, positron emission
tomography, and nuclear magnetic resonance techniques.
Major Findings:
A. Serotonin Function in Relation to Eating Disorder Sjnnptomatology
Preclinical studies in laboratory animals and pharmacologic studies in
human subjects indicate that dysregulation of hypothalamic serotonin function
can result in impaired post-prandial satiety responses. We have postulated
that the bingeing episodes characteristic of bulimia may reflect, at least in
part, such dysregulation of hypothalamic serotonin activity. Similarly, this
serotonin abnormality is postulated to contribute to bingeing episodes
observed in approximately fifty percent of low weight anorexic patients.
Moreover, alterations in central nervous system (CNS) serotonin function could
play a role in the symptoms of major depression and behavioral impulsivity
frequently reported in association with bulimic disorder and anorexia nervosa.
To compare serotonin function in eating disorder patients and healthy
controls, we have measured neuroendocrine and behavioral responses to the
serotonin receptor agonist m-chlorophenylpiperazine (m-CPP) (in collaboration
with Dr. D. Murphy) and to the serotonin precursor L-tryptophan. In results
from standardized test meal sessions following drug administration, we have
demonstrated that acute administration of m-CPP decreases caloric consumption,
similar to results previously reported in laboratory animals. Neuroendocrine
results from the completed m-CPP study demonstrated blunted plasma prolactin
responses in 26 female bulimic patients at normal weight, in comparison to
responses in 15 age-matched healthy volunteers. Prolactin responses
following intravenous L-tryptophan administration were significantly blunted
only in the subgroup of bulimic patients with major depression. It appeared
that patient - control differences in these studies did not result from
pharmacokinetic factors, since both subject groups had similar peak
post-infusion plasma levels of L-tryptophan (assayed in the Section
neuropharmacology laboratory) and of m-CPP (measured in collaboration with
Dr. D. Murphy). Neuroendocrine results are also being correlated with such
other clinical variables as severity and duration of bulimic s3anptoms.
Initial neuroendocrine results in twelve low weight anorexic patients (ten of
whom also had bulimic symptoms) also showed significantly blunted plasma
prolactin responses both to m-CPP and to L-tryptophan administration. These
243
ZOl MH 02289-03-LCS
data are consistent with our proposal for a role for altered hypothalamic
serotonin function as a predisposing factor toward binge episodes in bulimic
patients. In a related study, we completed double-blind controlled
therapeutic trials of the serotonin active drug fenfluramine in seven normal
weight outpatients with bulimic disorder. Subjects showed decreased
frequency of bulimic episodes during drug treatment. Pharmacologic challenge
studies in outpatients treated with fenfluramine or with imipramine are being
analyzed to evaluate the possible role of increased efficiency of central
nervous system serotonin neurotransmission in the therapeutic effects of
these drugs in bulimia.
Ratings of somatic symptoms/side effects following administration of
m-CPP in a group of 37 eating disorder patients and 15 healthy controls were
notable for a significant incidence of migraine-like headaches. Tryptophan
infusions did not produce this pattern of headaches. Analysis of the data
suggested that the migraine-like headaches were most common in subjects with a
history of migraine headaches in first degree relatives. Analysis of
pharmacokinetic data showed that headache severity was correlated with peak
m-CPP blood level measured during the three hours after drug administration.
From the standpoint of migraine, these results indicate that m-CPP may be a
valuable pharmacologic probe for extending previous studies linking migrainous
symptoms with altered serotonin activity.
B. Central Dopamine Function and Bulimia
While previous neurotransmitter studies in bulimia have focused largely
on norepinephrine and serotonin function, the altered eating patterns in
bulimia are also consistent with possible involvement of central dopamine
systems. Thus, pharmacological studies in laboratory animals have shown that
decreased dopamine activity in the lateral hypothalamus leads to hyperphagia
and weight gain. Moreover, dopamine appears to be a major link in mesolimbic
pathways important in the hedonic aspects of food intake. Increased
responsivity in this pathway could accentuate the reward properties of eating,
possibly consistent with a pattern of alternate phobic avoidance of food, and
periodic over indulgence in a binge meal. Conversely, diminished responsivity
in this pathway could lead to episodic consumption of large meals as a person
attempted to achieve the usual satisfaction expected from a meal.
Dysregulation of mesolimbic dopamine could also contribute to the depressive
sjnnptoms common in bulimic and anorexic patients.
Meaurement of levels of the major dopamine metabolite HVA in CSF
provides the most direct index of the rate of presynaptic dopamine release
available for clinical studies. Results from our recently completed CSF
studies showed that eight severely symptomatic bulimic patients, who reported
a history of bingeing at least twice daily, had significantly lower levels of
HVA than did the thirteen less severely symptomatic patients or the eleven
healthy controls. Although the patients weighed less than the healthy
controls, these results appear not to be related to body weight per se, since
body weight was not different for the patient groups with high and low binge
frequencies. When patients were restudied after three weeks of abstinence
from bingeing and vomiting during the hospital treatment program, CSF HVA
244
ZOl MH 02289-03-LCS
values for the severely symptomatic group had increased significantly toward
control values.
These results provide preliminary evidence for altered turnover of
central dopamine in patients with severe symptoms of bulimia. Follow-up
studies should include measures of dopamine receptor activity, along with
metabolite measures. It is presently unclear which factors related to the
symptom patterns of bulimia, such as recent fluctuations in body weight or
chronic alterations in dietary composition, could result in relatively
persistent changes in central neurotransmitter activity. Preclinical studies
in laboratory animals have shown that acute changes in eating pattern may in
fact affect dopamine turnover in hypothalamic and mesolimbic pathways.
Additional analysis of the current data is necessary to compare other clinical
variables such as depression, family psychiatric history, and menstrual phase
at the time of lumbar pucture for the two patient subgroups.
C. Pharmacological Probes of Appetite Regulation in Bulimia and Anorexia
Nervosa.
Preclinical studies and pharmacologic investigations in human subjects
indicate that endogenous opiates play an important role in the regulation of
feeding behavior, as indicated by decreased meal size resulting from
pretreatment with opiate antagonists. We previously demonstrated
significantly low levels of CSF beta-endorphin and other pro-opiomelanocortin
related peptides in low weight anorexic patients. These observations have
prompted us to investigate whether opiate dysregulation, either as a
preexisting condition or as a result of persistently abnormal eating paterns,
could play a role in the preoccupation with food and binge-eating patterns
observed in bulimics and in many anorexic patients. Over the past year we
completed collection of additional CSF samples in a new group of anorexic
patients and in bulimic patients, to evaluate clinical correlates of the CSF
measures. In collaboration with Dr. D. Pickar, we have continued behavioral
and neuroendocrine studies with high dose infusion of naloxone in eating
disorder patients. In a preliminary group of seven anorexic patients studied
following weight restoration, naloxone pretreatment resulted in a paradoxical
increase in the amount of food consumed during a test meal, in contrast to
effects previously described in volunteers and obese subjects. The anorexic
effects of naloxone also appear to be blunted in a group of 15 bulimic
patients studied during a phase of abstinence from bingeing and vomiting in
the research program. Results of neuroendocrine responses to naloxone
infusion in the eating disorder patients are currently being analyzed. The
behavioral results obtained to date suggest that modulation of eating
behaviors by central opiate pathways may be altered in bulimia and anorexia,
and may be pertinent in evaluating other evidence for therapeutic responses to
opiate antagonists reported in some severe cases of bulimia and anorexia.
In a related study on the possible therapeutic effects of opiate
antagonist treatment in obese patients, we have studied the first eight
subjects in a double-blind, placebo-controlled outpatient trial with
nalmefene in collaboration with Dr. D. Pickar. Behavioral ratings, dietary
information and neuroendocrine responses will be compared for the two week
245
ZOl MH 02289-03-LCS
periods of active drug and placebo treatment. This study will provide new
information on the question of possible rapid development of tolerance to the
effect of opiate antagonist drugs on eating behavior.
As another probe of appetite regulation in bulimia, we have studied the
behavioral and neuroendocrine responses to 2-deoxyglucose in patients and
controls (in collaboration with Drs. S, Paul and D. Pickar) . Preclinical
studies have shown that administration of 2-deoxyglucose results in an
intracellular glucopenia, with increased appetite and feeding being prominent
behavioral effects. These behavioral effects appear to include an interaction
with hypothalamic opiate pathways, since pretreatment with naloxone
antagonizes the increased eating (but not the increased hunger) resulting
from 2-deoxyglucose administration. In initial results, 13 bulimic patients,
in comparison to eleven healthy controls, had blunted responses on hunger
ratings following adminstration of the drug, and attenuated 2-deoxyglucse
induced increases in caloric consumption on a test meal. These preliminary
results provide the first evidence that bulimic patients may develop
dysregulation of glucose sensitive hypothalamic pathways involved in the
modulation of appetite and food intake.
D. Studies of the HPA Axis in Anorexia Nervosa.
Collaborative studies with Dr. P. Gold on the regulation of the
hypothalamic-pituitary-adrenal (HPA) axis in eating disorder patients have
continued over the past year. Previous studies from this collaboration
demonstrated blunted ACTH responses to corticotropin releasing hormone (CRH)
and elevated levels of CRH in CSF in anorexic patients, suggesting that
hyperactivity of the HPA axis in anorexic patients originates in the
hypothalamus or more rostral brain regions. Demonstration of elevated CRH in
low weight anorexic patients is of particular interest because of the
anorectic effects of this peptide when administered to laboratory animals.
During the past year, we have completed a collaborative pilot study on the
neuroendocrine and behavioral effects of the glucocorticoid antagonist
RU-38486 in six anorexic patients studied at low weight and following weight
restoration. Data from this study are currently being analyzed. The results
may help clarify the site of HPA dysregulation in anorexia nervosa.
E. Regulation of Energy Metabolism Eating Disorder Patients
From a clinical perspective, alterations in metabolic control of energy
metabolism and weight regulation could contribute to the difficulties in
weight stabilization commonly reported by patients with eating disorders.
Patients with the syndrome of bulimia frequently complain of a tendency to
gain weight, in spite of apparently modest caloric intake. During this past
year, we demonstrated that a group of 15 bulimic patients at normal weight had
lower resting metabolic rate (as assessed by indirect calorimetry) than did
age and sex matched healthy controls. This finding is an extension of our
previous observations that bulimic patients are able to maintain a constant
body weight with significantly smaller daily caloric intake than healthy
controls, and that anorexic patients with bulimic symptoms require fewer
calories to maintain their weight than do non-bulimic anorexics. These
246
ZOl MH 02289-03-LCS
findings are of importance because they provide a physiological explanation
for the complaint of many bulimic patients that they find themselves
particulary prone to gain weight on a typical diet. These results will be
compared to clinical variables (including present deviation from expected body
weight, duration and severity of bulimic symptoms) and to activity level in
physiological systems (including the sympathetic nervous system and the
hypothalamic-pituitary-thyroid axis) involved in the regulation of metabolic
rate.
Clinical observations suggest that the caloric requirements for weight
gain are unexpectedly large for low weight anorexic patients participating in
non-pharmacological, behaviorally oriented weight restoration treatment
programs. Over the past year we have extended our series of anorexic patients
studied longitudinally, assessing body composition using anthropometric
measures and metabolic rate using indirect calorimetry. In comparison to
results for healthy controls, data from ten anorexic patients showed that
during the weight restoration, refeeding phase of the treatment program, the
anorexic patients had significantly increased rates of energy metabolism.
This data suggested that anorexic patients differ from other low weight
subjects in terms of their inability to maintain efficient utilization of
caloric intake during weight restoration. Preliminary results of
neuroendocrine assays indicate that substantial activation of the
hypothalamic-pitiutary-adrenal axis plays a significant role in the
non-adaptive metabolic response observed during weight gain in anorexic
patients.
E. Family Study of Bulimic Disorder
As in anorexia nervosa, there is substantial evidence implicating a
relationship between the syndrome of bulimia and affective Illness. Bulimic
patients have a high frequency of major and minor depressive episodes, and the
limited data available from family history studies indicate an elevated
frequency of major affective illness in first degree relatives of bulimic
probands. In an effort to replicate results from previous family studies, and
to extend them to the more reliable family interview method, we are conducting
a family study of bulimia in collaboration with Dr. E. Gershon. Data
collection continued actively over the past year, with 28 bulimic probands and
130 first degree relatives having been interviewed to date.
In a related study, we examined the time course of onset of bulimic
symptoms in anorexic patients admitted to this program between 1976 and 1987.
Between 30 and 50 per cent of anorexic patients typically develop symptoms of
bulimia during the course of the disorder. Reports in the literature show
that bulimic symptoms generally develop within one and one half years after a
patient has started restricting food Intake and losing weight. This pattern
suggests that a low weight episode of anorexia nervosa might be a major
predisposing factor in the development of bulimic symptoms in these patients.
In our retrospective study of 74 patients diagnosed by research criteria, we
observed a progressive increase in the relative proportion of patients who
developed bulimic symptoms prior to the onset of their first low weight
anorexic episode. Although preliminary because of the limited sample size,
247
ZOl MH 02289-03-LCS
the present study suggests emergence of a new symptom pattern different from
that previously reported in the literature. From a psychobiologic
perspective, the present data suggest that bulimic anorexic patients may have
predisposition to the onset of bulimia not dependent on the prior expression
of a low weight anorexic episode.
Significance to Biomedical Research and the Program of the Institute:
The substantial prevalence of bulimic disorder and anorexia nervosa,
particularly in the high risk population of high school and college age women,
is a significant public health concern. The studies outlined above reflect a
targeted focus on neurobiologic systems currently implicated in altered
eating patterns and associated psychiatric symptoms observed in these
disorders. Further studies on the role of serotonin, dopamine,
norepinephrine, endogenous opiates and related neuromodulators may help to
identify biological and behavioral vulnerability factors contributing to the
onset and persistence of bulimic and anorexic symptoms, and may contribute to
the development of new treatment approaches.
Proposed Course:
Along with continued study of behavioral and neuroendocrine responses to
pharmacological challenge with serotonin active drugs in anorexic patients, we
will be analyzing data from patients and controls to assess the
interrelationship within subjects of behavioral, neuroendocrine and CSF
metabolite measures. The studies of behavioral and neuroendocrine responses
to challenge testing with an opiate antagonist will be completed, as will the
collaborative study responses to nalmefene in obesity. Collaborative studies
on HPA axis activity in anorexia will continue with neuroendocrine challenge
tests utilizing a glucocorticoid antagonist. Data collection will continue
for energy metabolism studies in a comparison group of patients with affective
illness and for the family study of bulimia.
Publications:
George, D.T., Weiss, S., Gwirtsman, H.E., Blazer, D.: Hospital treatment of
anorexia nervosa: a 25 year retrospective study from 1958 to 1982. Int. J.
Eating Disorders 6: 321-330, 1987.
Kaye, W.H., Gwirtsman, H.E., Obarzanek, E., George, D.T., Jimerson, D.C.,
Ebert, M.H.: Caloric intake necessary for weight maintenance
in anorexia nervosa: Nonbulimics require greater caloric intake
than bulimics. ^lu J^ Clin. Nutr. 44: 435-443, 1986.
Petersen, R. , Kaye, W.H. , Gwirtsman, H.E.: Comparison of calculated estimates
and laboratory analysis of food offered to hospitalized eating disorder
patients. J. P^. Diet. Assoc. 86: 490-492, 1986
Jimerson, D.C., George, D.T. , Brewerton, T.D. and Kaye, W.H.: Anxiety in
bulimic disorder: Behavioral responses to lactate and isoproterenol
infusions. In Disorders of Eating Behavior: A Psychoneuroendocrine
248
ZOl MH 02289-03-LCS
Approach, Ferrari, E, and Brambilla, F. (Eds.)» New York, Pergamon Press,
1986, 319-323.
Jimerson, D.C. and Docherty, J.P (Eds), Psychopharmacology
Consultation, Washington, D.C, American Psychiatric Press, 1986.
Jimerson, D.C: Psychopharmacology consultation for bulimia andanorexia
nervosa: A clinical overview. In Jimerson, D.C and Docherty, J.P. (Eds),
Psychopharmacology Consultation, Washington, D.C, American Psychiatric Press,
1986, 49-70.
Kaye, W.H., Gwirtsman, H.E., George, D.T., Weiss, S.R., Jimerson, D.C:
Relationship of mood alterations to bingeing behaviour in bulimia. Brit. J.
Psychiatry 149: 479-485, 1986.
George, D.T., Jimerson, D.C: Changes in serum chloride concentration
following sodium lactate infusion (Itr.). Am. J. Psychiatry 143: 1499, 1986.
Kaye, W.H., Rubinow, D. Gwirtsman, H.E., George, D.T., Jimerson, D.C, Gold,
P.W. : CSF somatostatin in anorexia nervosa and bulimia: relationship to the
hypothalamic-pituitary-adrenal axis. Psychoneuroendocrinol. , in press, 1987.
Kaye, W.H. , Gwirtsman, H.E., Brewerton, T.D., George, D.T., Jimerson, D.C.
Ebert, M.H.: Serotonin regulation in bulimia. In Psychobiology ofBulimia,
Hudson, J.I. and Pope, H.G. (Eds.), Washington, D.C, American Psychiatric
Press, in press, 1987.
Jimerson, D.C, George, D.T., Kaye, W.H., Brewerton, T.D., Goldstein, D.S.:
Norepinephrine regulation in bulimia. In Psychobiology of Bulimia, Hudson,
J.I. and Pope, H.G. (Eds.), Washington, D.C, American Psychiatric Press, in
press, 1987.
George, D.T., Brewerton, T.D., Jimerson, D.C: Comparison of lactate-induced
anxiety in bulimic patients and healthy controls. Psychiatry Res. , in press,
1987.
Kaye, W.H. , Gwirtsman, H.E., George, D.T., Jimerson, D.C, Ebert, M.H.: CSF
5-HIAA concentrations in anorexia nervosa: reduced values in underweight
subjects normalize after weight gain, Biol. Psychiatry, in press, 1987.
Kaye, W.H., Berrettini, W.H,, Gwirtsman, H.E., Chretien, M. , Gold, P.W.,
George, D.T., Jimerson, D.C, Ebert, M.H.: Reduced cerebrospinal fluid levels
of immunoreactive pro-opiomelanocortin related peptides (including
beta-endorphin) in anorexia nervosa. Life Sci. , in press, 1987.
Kaye, W.H. , Gwirtsman, H. , Jimerson, D.C, George, D.T,, Karoum. F., Ebert,
M.H.: Catecholamine function in anorexia nervosa at low weight and after
weight restoration. In Proceedings of the Sixth International Catecholamine
Symposium, Dahlstrom, A. (Ed.), New York, Alan R. Liss, in press, 1987.
Jimerson, D.C, Kaye, W.H., Lesem, M.D. : Preliminary evidence for low CSF
249
ZOl ^fH 02289-03-LCS
homovanilllc acid in patients with severe sjnnptoms of bulimia. In
Proceedings of the Sixth International Catecholamine Symposium, Dahlstrom, A.
(Ed.), New York, Alan R. Liss, in press, 1987.
Jimerson, D.C., Brandt, H.A., Brewerton, T.D.: Evidence for altered serotonin
function in bulimia and anorexia nervosa: behavioral implications. In
Proceedings of the Max Planck Institute Symposium on Bulimia Nervosa, Plrke,
K.M. and Ploog, D. (Eds.), Berlin, Springer-Verlag, in press, 1987.
250
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1. 1986 to September 30. 1987
PROJECT NUMBER
ZOl MH 00326-14 LCS
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Clinical Neuropharmacology and Psychobioloav of Depression and Mania
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfiliation)
Dennis L. Murphy, M.D. Chief Section on Clinical Neuropharmacology LCS NIMH
COOPERATING UNITS (11 any)
BP, CBP, NIMH; VA Medical Center, Bronx, NY
LAB/BRANCH
Laboratory of Clinical Science
Section on Clinical Neuropharmacology
INSTITUTE AND LOCATION
NIMH. NTH. Rethesda. Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
ILJL
OTHER:
Q.I
CHECK APPROPRIATE BOX(ES)
((D (a) Human subjects
D (a1) Minors
n (a2) Interviews
P (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This is the final, termination report on this project. No new data have
been collected in the past year. The publications listed in this report were
discussed in last year's annual report. Our Section's wori< in the area of the
neuropharmacology and psychobiology of depression has been refocused during the
past several years on affective symptomatology as it occurs in the context of
obsessive-compulsive disorder (see annual report ZOl MH 00336) and in a
comparative study of Alzheimer's disease and depression in the elderly (see
annual report ZOl MH 00339).
251
PHS 6040 (Rev. 1/84)
CPO SI4-SII
ZOl MH 00326-14 LCS
Other collaborative professional personnel engaged on the project:
C.S. Aulakh Staff Fellow LCS NIMH
L.J. Siever Staff Psychiatrist VA Med Ctr, Bronx NY
E.A. Mueller Staff Physician LCS NIMH
T. Sunderland Staff Physician LCS NIMH
R.M. Cohen Section Chief LCM NIMH
N.A. Garrick Biologist LCS NIMH
Project Description:
Objectives: Individuals with depression, mania, and related disorders
with affective components, including individuals with depression secondary
to other psychiatric disorders (e.g. personality disorders; panic disorder,
obsessive-compulsive disorder) and neuropsychiatric disorders (e.g.
Parkinson's disease, Alzheimer's dementia), have been studied in attempts to
understand the psychological and biological mechanisms involved in therapeutic
drug effects in these disorders. As individual differences in psychoactive
drug responsiveness appear to depend upon many psychological and biological
factors, a variety of study approaches are utilized.
Methods Employed:
Several strategies are currently used to approach an understanding of
the mechanism of antidepressant, antimanic and antianxiety drugs in neuro-
psychiatric patient populations. Baseline, pretreatment biological, psycho-
logical and demographic characteristics are assessed to evaluate possible
markers of potential patient subgroups or individual patient differences which
may be predictive of drug response.
The principal emphasis in our studies over the past several years has
involved (a) detailed investigations of the clinical and biochemical effects
of monoamine oxi dase-i nhibiting antidepressants, particularly newer agents
with substrate selective actions; (b) the contributions of changes in the
brain serotonergic system to the mechanisms of action of psychoactive
drugs; and (c) the contributions of interactions between some less-studied
neuromodulators and/or neurotransmitters (e.g., the neuropeptides and the
trace amines) and the more "classical" neurotransmitters including the
catecholamines and serotonin. These approaches have required the develop-
ment of new in vitro assay systems as well as animal model studies, both
of which have most frequently been accomplished via joint efforts with
collaborators outside the section.
Major Findings:
Because this section's principal clinical efforts both on our 6D
inpatient unit and our outpatient space in the past two years have been
directed towards two specialized patient populations (Alzheimer's disease
patients with neuropsychiatric problems, including depression, and obsessive
compulsive disorder) our current findings relevant to this affective disorders
project report are mostly from several areas involving data which required
252
ZOl MH 00326-14 LCS
extensive analysis or which was incorporated in reviews, as listed in the
publications section of this report. The findings from antidepressant drug
investigations in the specialized populations are included in separate project
reports (ZOl MH 00336) and (ZOl MH 00339).
Significance to Biomedical Research and the Program of the Institute:
An important cautionary corollary to the intertwined neurochemical conse-
quences of antidepressant drug administration is the recently clarified evi-
dence that antidepressants are therapeutically active in a wide variety of
non-affective disorders such as obsessive-compulsive disorder, panic disorder,
anxiety disorder, attention deficit disorder, bulimia, enuresis, migraine and
the chronic pain syndrome. This therapeutic responsiveness may sometimes be
related to improvement in secondary depressive symptoms, but may also clearly
occur in the absence of secondary depression. In particular, improvement in
the core symptoms of at least some of these disorders (e.g. obsessive-
compulsive disorder) may occur without a change in mood.
Many patients with these disorders display psychobiological anbormalities
that show many similarities to but also some differences from those observed
in patients with affective disorders, despite the frequent absence of affec-
tive symptoms. Although an improvement in subclinical or "masked" depression
remains one hypothesis linking antidepressant responsiveness to some shared
biological abnormalities in this diverse group of diagnostic entities, an
alternative hypothesis suggests that patients responding to antidepressants
have a core disorder with common psychobiological abnormalities but different
clinical and diagnostic presentations. A third hypothesis suggests that the
multiple actions of antidepressant drugs (e.g. on adrenoceptors, muscarinic
receptors or the serotonin system) may each be differently important in the
therapeutic outcome in depressed or other patients with specific or predomi-
nant dysfunctions responsive to alterations in one or another of these trans-
mitter systems. An examination of both the similarities and the differences
among the affective and non-affective antidepressant-responsi ve disorders may
provide further clues about the mode of action of antidepressant agents across
the spectrum of psychiatric disorders, and possibly about psychobiological
elements in depression and the other disorders responsive to antidepressant
treatment.
Proposed Course:
As indicated above, over the past several years our Section began to
investigate the affective features of obsessive-compulsive disorder and of
Alzheimer's disease. We gradually began to focus our efforts on other aspects
of these disorders as well, as reviewed in other annual reports from our
section, and hence we are terminating this project.
Publications:
Murphy, D.L.: Serotonin neurochemistry : A commentary on some of its
quandaries. Neurochem. Int. 8: 161-163, 1986.
253
ZOl MH 00336-14 LCS
Murphy, D.L., Aulakh, C.S., and Garrick, N.A.: How antidepressants work:
cautionary conclusions based on clinical and laboratory studies of the longer-
term consequences of antidepressant drug treatment. In Antidepressants and
Receptor Function, from CIBA Foundation Symposium 123. Sussex, UK, John Wiley
and Sons, Ltd., 1986, pp. 106-125.
Siever, L.J., Uhde, T.W., Jimerson, D.C., Lake, C.R., Kopin, I. J., and Murphy,
D.L.: Indices of noradrenergic output in depression. Psychiatry Res. 19:
59-73, 1986.
Murphy, D.L., Sunderland, T., Garrick, N.A., Aulakh, C.S., and Cohen, R.M.:
Selective amine oxidase inhibitors: basic to clinical studies and back. In
Dahl, S.G., Gram, L.F., Paul, S.M., and Potter, W.Z. (eds.): Clinical
Pharmacology in Psychiatry, Series III. Selectivity in Psychotropic Drug
Action - Promises or Problems? Heidelberg, FRG, Spri nger-Verlag, 1987,
pp. 135-146.
Murphy, D.L., Aulakh, C.S., Garrick, N.A., and Sunderland, T.: Monoamine
oxidase inhibitors as antidepressants: implications for the mechanism of
action of antidepressants and the psychobiology of the affective disorders and
some related disorders. In Meltzer, H.Y. et al . (eds.): Psychopharmacol ogy :
The Third Generation of Progress. New York, Raven Press, 1987, pp. 545-552.
Murphy, D.L., Cohen, R.M., Sunderland, T., and Mueller, E.A.: Selective
monoamine oxidase inhibitors in treatment-resistant depression. In Zohar, J.
and Belmaker, R.H. (eds.): Special Treatment for Resistant Depression. New
York, PMA Publishing Corp., 1987, pp. 257-268.
254
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30 ^
PROJECT NUMBER
ZOl MH 00332-09 LCs
1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line tietween the borders.)
Animal Models for the Study of Neuropharmacologic Effects
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Charanjit S. Aulakh Staff Fellow, LCS, NIMH
COOPERATING UNITS (if any)
LCM, NIMH
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Neuropharmacology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
1.6
PROFESSIONAL
1.3
0.3
CHECK APPROPRIATE BOX{ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Intravenous administration of the 5-HTj^g receptor agonist
m-chlorophenylpiperazine (m-CPP) to rats produced increases in plasma prolactin
and corticosterone concentration and a decrease in plasma growth hormone.
Short-term (2-4 days) or long-term (3 weel<s) treatment with either the tricylic
antidepressant imlpramine or the monoamine oxidase (MAO) type A inhibiting
antidepressant clorgyline did not have any significant effect on the baseline
levels of either of these hormones. However, long-term but not short-term
imipramine treatment potentiated m-CPP's effect on plasma prolactin, while
long-term but not short-term clorgyline treatment attenuated m-CPP's prolactin
effect. Neither antidepressant altered m-CPP's effects on corticosterone or
growth hormone. These results indicate that various agents effective in
different types of affective disorders exert differential modulatory influences
on serotonergic mechanisms mediating neuroendocrine responses in vivo.
255
PHS 6040 (Rev. 1/84)
SPO >l4-«lt
ZOl MH 00332-09 LCS
Other collaborative professional personnel engaged on the project:
D.L. Murphy Chief, LCS, NIMH
R.M. Cohen Section Chief, LCS, NIMH
J. Zohar Visiting Associate LCS, NIMH
K.M. Wozniak Visiting Associate LCS, NIMH
G. Bagdy Visiting Fellow LCS, NIMH
J.L. Hill Biostatistician LCS, NIMH
Project Description:
The serotonergic neurotransmitter system is considered to play a pivotal
role in the etiology of a number if neuropsychiatric disorders. In recent
years, radioligand studies have demonstrated the existence of various
subtypes of 5-HT receptors. We have conducted a series of experiments to
correlate these various 5-HT receptors subtypes with different behavioral,
neuroendocrine and other physiologic functions. Furthermore, we have used
selective 5-HT subtype agonists as challenge agents to explore functional
adaptational changes in the serotonergic neurotransmitter system following
the long-term administration of antidepressant drugs. These adaptive changes
might help in understanding the molecular mechanisms responsible for both the
therapeutic and side effects of these drugs.
Methods Employed:
Under halothane anesthesia, the left femoral artery and vein were
cannulated in each animal and the catheters were exteriorized subcutaneously
at the back of the neck. Saline or various doses of m-CPP were injected
intravenously at least 48 hours after the surgery. In the antidepressant
studies, clorgyline (Img/kg/day) or imipramine (5mg/kg/day) or saline was
subcutaneously administrated continuously by means of osmotic minipumps for
28 days. Blood samples were drawn from the femoral artery and collected in
heparinized tubes. Following centrifugation, plasma samples were collected.
The plasma concentrations of prolactin, corticosterone and growth hormone
were measured by radioimmunoassay.
Major Findings:
Intravenous administration of the 5-HTj^g receptor agonist
m-chlorophenylpiperazine (m-CPP) to rats produced increases in plasma
prolactin (peak effect at 15 minutes), corticosterone (peak effect at 30
minutes) and a decrease in plasma growth hormone (peak effect at 15 minutes)
concentrations. Administration of m-CPP produced dose-related changes only
in prolactin levels but not in corticosterone or growth hormone levels.
Long-term but not short-term treatment with the tricyclic antidepressant
imipramine potentiated m-CPP's effect on prolactin but not on
corticosterone or growth hormone. These findings suggest the development of
functional supersensitivity of 5-HTib receptors involved in prolactin
secretion following long-term imipramine treatment.
256
ZOl MH 00332-09 LCS
In another study, long-term but not short-term treatment with the
MAO-type A inhibiting antidepressant clorgyline attenuated m-CPP's effect on
prolactin but not on corticosterone or growth hormone. These findings
suggest the development of functional subsensiti vity of 5-HTib receptors
involved in prolactin secretion following long-term clorgyline treatment.
The demonstration of dose-related changes in prolactin but not in corticos-
terone or growth hormone following m-CPP administration on the one hand and
potentiation or attenuation of m-CPP's effect on prolactin but not corticos-
terone or growth hormone following long-term imipramine or clorgyline treat-
ment respectively, on the other hand suggests either a differential regula-
tion of these hormones by serotonergic mechanisms or different 5-HT receptors
subtypes mediating different neuroendocrine functions.
Significance to Biomedical Research and Programs of the Institute:
Since the serotonergic neurotransmitter system is considered to play a
pivotal role in the etiology of the affective illness, the demonstration of
functional adaptational changes in this neurotransmitter mechanism following
long-term antidepressant drug treatment are important. The net sensitivity
changes of m-CPP following long-term antidepressant drug treatment observed
in the present study are of particular interest since m-CPP is a metabolite
of the antidepressant, trazodone and, therefore, may contribute to the
pharmacologic and therapeutic effects of trazodone. The present as well as
our previous findings with m-CPP in various animal models help validate the
use of m-CPP as an index of central serotonergic function in investigations
in humans. The present findings also indicate that 5-HT agonist-induced
changes in prolactin levels may be a better neuroendocrine measure for
assessing serotonergic function following long-term antidepressant drug
treatment.
Proposed Course:
Several papers describing these findings have been submitted for
publication. During the next year, we will continue to explore functional
adaptational changes in the serotonergic system using other 5-HT subtype
agonists as challenge agents as well as other animal behavioral models. In a
separate series of experiments with fawn-hooded rats, a rat strain with a
peripheral platelet storage pool disease, we will examine whether this rat
strain also possesses altered central serotonergic function.
Publications
Aulakh, C.S., Cohen, R.M., Hill, J.L., Murphy, D.L. and Zohar, J: Long-term
imipramine treatment enhances the locomoter and food intake suppressant
effects of m-chlorophenylpiperazine in rats. Br. J. Pharmacol., in press.
257
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00336-08 LCS
PERIOD COVERED
October 1. 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must lit on one line tietween the borders,;
The Phenomenology and Treatment of Obsessive-Compulsive Disorder in Adults
PRINCIPAL INVESTIGATOR (List other professional personnel t>elow the Principal Investigator) (Name, title, laboratory, and institute atliliation)
PI: T. R. Insel Staff Physician LCS NIMH
Others:
D.
1.. Murphy
Chief
LCS
NIMH
J.
Zohar
Visiting Associate
LCS
NIMH
R.
Zohar-Kadouch
Visiti ng Fel 1 ow
LCS
NIMH
C.
Benkelfat
Visiting Fellow
LCS
NIMH
M.
Pato
Medical Staff Fellow
LCE
NICHD
COOPERATING UNITS (If any)
LAB/BRANCH
Laboratory of Clinical Science, NIMH
SECTION
Section on Comparative Studies of Brain and Behavior
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda. Maryland 20892
TOTAL MAN-YEARS:
3.0
PROFESSIONAL:
2.7
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
G (a) Human subjects
D (a1) Minors
K (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Obsessive-compulsive disorder has been studied from several different
perspectives since this project began in 1980. During the past year, we have
focused primarily on the treatment of this disorder. Previously we showed that
the tricyclic antidepressant clomipramine was specifically anti -obsessional , in
contrast to several other antidepressants. As clomipramine is considerably more
potent than other tricyclic antidepressants in its serotonergic effects, we
hypothesized that these effects might be important for its anti-obsessional
properties. To test this hypothesis, we extended our earlier findings that the
selective serotonin postsynaptic receptor agonist m-chlorophenylpiperazi ne (m-CPP)
increased obsessional symptoms by retesti ng obsessive-compulsive disorder patients
after chronic treatment with clomipramine. Following treatment, patients showed
less of a response to m-CPP, suggesting that clomipramine was associated with
postsynaptic subsensitivi ty. Furthermore, metergol ine, a serotonin receptor
antagonist, appeared to partly reverse the effects of chronic clomipramine
treatment. These results, tai<en together with recent findings from other
investigators correlating clinical improvement on clomipramine with changes in
serotonin function, strongly suggest that the drug's serotonergic effects are
integral to its clinical efficacy for obsessive-compulsive disorder.
Although we have learned much about the biochemical mechanism of clomipramine's
action, results from another study reminded us of the limitations of this treatment,
pf 18 patients discontinued from chronic clomipramine treatment, 17 relapsed
iwithin 7 weeks. Clearly, the drug is a treatment not a cure. Our continued
follow-up studies of these patients have revealed the importance of nonpharmacol ogi ([:
factors in outcome.
259
PHS 6040 (Rev. 1/84)
SPO S14-9I1
ZOl MH 00336-08 LCS
Objectives: In this, the eighth year of this project investigating the
psychobi ology of obsessive-compulsive disorder, we addressed two major
questions.
Question 1: Are clomipramine's anti-obsessional effects mediated by its
actions on serotonergic receptors? We knew from previous studies that
(a) clomipramine was more potent than other tricyclic antidepressants
for the relief of the symptoms of obsessive-compulsive disorder and
(b) clomipramine was more potent than other tricyclic antidepressants
for the inhibition of serotonin reuptake. By defining a pharmacologic
mechanism for anti-obsessional effects, new compounds might be devel-
oped for the treatment of this complex illness.
Question 2: How long do obsessive-compulsive disorder patients need to
remain on clomipramine? As this disorder is considered chronic, it is
important to determine whether treatment should continue for 6-12 months,
as with the treatment of depression, or for extended maintenance as
with the treatment of schizophrenia.
Methods Employed: Treatment studies are conducted in the NIMH
Outpatient Clinic at the Clinical Center. Local patients with obsessive-
compulsive disorder are accepted if they have been ill for at least 1 year,
are willing to stop all psychotropic medication, and do not require hospitali-
zation. Clomipramine is given orally in doses up to 300 mg/day. To test for
changes in response to a serotonergic challenge, the serotonin postsynaptic
agonist, m-chl orophenylpiperazi ne (m-CPP), is given orally in a single dose
(0.5 mg/kg) in a double-blind, placebo controlled trial prior to and at least
8 weeks following the onset of clomipramine treatment. Behavioral measures,
including self and observer ratings, are completed following m-CPP and placebo
trials--both before and after clomipramine treatment. In addition, blood is
collected vi a an in-dwelling cannula to assess changes in plasma prolactin
and corti sol .
In a separate study, patients receive the serotonin receptor antagonist
metergoline following chronic clomipramine administration. Metergoline
(4.0 mg) is given orally for 4 consecutive days, again under double-blind
placebo-controlled conditions. Behavioral and endocrine changes are monitored
through botti the 4 days of metergoline and 4 days of placebo admi ni stration--
with the two treatments at least 2 weeks apart. Clomipramine is taken daily
throughout the study.
To determine the appropriate length of treatment, 18 obsessional patients
who have been treated with clomipramine between 6 and 36 months are being
switched from their medication to placebo under double-blind conditions.
Ratings of behavior and mood are collected for 6 weeks prior to the switch
and for 7 weeks subsequently.
Finally, we are continuing our follow-up studies of previously treated
patients including face-to-face interviews, administration of the SADS-anxiety
structured interview, self and observer ratings, and the MMPI.
260
ZOl MH 00336-08 LCS
Major Findings:
Clomipramine and serotoni n--m-CPP. Although previous studies with m-CPP
revealed minimal behavioral effects in healthy volunteers, we noted highly
significant increases in ratings of obsessions and anxiety in 12 obsessional
patients prior to clomipramine treatment. Endocrine (i.e. prolactin and
Cortisol) responses to m-CPP were not different between untreated patients
and controls. Following chronic clomipramine treatment, the nine patients
who were rechallenged with m-CPP did not show significant increases in
ratings of obsessions. This hypo-responsiveness to m-CPP was not correlated
with clinical Improvement. Endocrine responses to m-CPP were not signifi-
cantly different on .clomipramine, although baseline plasma prolactin was
nearly doubled from its pretreatment value.
Clomipramine and serotoni n--metergol i ne. In our previous studies, we
determined that metergoline did not increase obsessive-compulsive symptoms
when given as a single dose. Following chronic clomipramine treatment
however, 10 patients showed an overall slight but significant Increase in
obsessive-compulsive symptoms during the metergoline trial compared to
placebo. Metergoline administration was also associated with a decrease In
prolactin in the patients on chronic clomipramine treatment.
Length of treatment. Discontinuation of clomipramine was associated
with relapse in 17 of 18 obsessive-compulsive patients, even after 2-3 years
of treatment. Increases in depression and obsessions were observed within 7
weeks of discontinuation. Lei^gth of treatment was not related to relapse.
Curiously, follow-up of 20 patients from our 1980-1983 cohort revealed
only one on clomipramine. In general, these ex-patients continued to have
significant obsessive-compulsive symptoms, but in most cases the symptoms
were not disabling. As an exception, one patient from the original cohort
suicided due to a persistent obsessional symptom. In the remainder of the
group, improvement was associated with work, interpersonal relationships,
and higher levels of premorbid functioning.
Significance to Biomedical Research and the Program of the Institute:
With the recent finding that obsessive-compulsive disorder is 40-60 times more
common than previously reported--with a higher prevalence than schizophrenia,
panic disorder, and anorexia nervosa--the importance of a safe and effective
treatment has been recognized. Our research program since 1980 has demon-
strated the effectiveness of clomipramine (1980-1982), the relative ineffec-
tiveness of structurally related compounds (1983-1984), and the role of
serotonin in the mediation of these effects (1985-1987). Results from this
year's research not only describe a potential mechanism for the drug's
anti-obsessional effects, they reveal the practical difficulty of relapse
when the drug is stopped and the importance of nonpharmacol ogi c factors in
long-term outcome.
Proposed Course. In the coming year, we will be returning to our earlier
focus on the psychopathol ogy of this disorder. Specifically, our earlier studies
(1986) of cortical blood flow in obsessional patients will soon be completed.
261
ZOl MH 00336-08 LCS
We hope to extend this study to the ^^0 technique to allow an analysis of the
role of subcortical structures, such as the striatum, in the pathophysiology
of obsessions andd anxiety. In addition, we plan to use the PET scan to
visualize ^^¥-2\)C-, uptake in this same region. Ultimately, by combining these
novel imaging techniques with clomipramine treatment, we hope to better
understand both the pathophysiology and the pharmacotherapy of this intriguing
di sorder.
Pub! i cations :
Insel, T.R. , and Akiskal, H.: Obsessive-compusl i ve disorder with psychotic
features: A phenomenologic analysis. Amer. J. Psychiatry 143: 1527-1533,
1986.
Insel, T.R., and Zohar, J.: Psychopharmacologi c approaches to obsessive-
compulsive disorder. In Meltzer, H.Y. (Eds.): ACNP: A Generation of
Progress. New York, Raven Press, 1987, pp. 1205-1210.
Zohar, J., and Insel, T.R.: Obsessive-compulsive disorder: Psychobiolo-
gical approaches to diagnosis, treatment, and pathophysiology [A.E. Bennett
Award paper]. Biologic Psychiatry 22:667-687, 1987.
Zohar, J., Foa, E.B., and Insel, T.R.: The treatment of obsessive-
compulsive disorder. APA Manual for Psychiatric Treatments (in press).
Zohar, J., and Insel, T.R.: Diagnosis and treatment of obsessive-compulsive
disorder. Psychiatric Annals (in press).
Zohar, J., and Insel, T.R.: Psychopharmacologic treatment of OCD.
J. Affective Disord, (in press).
Zohar, J., and Insel, T.R.: Biologic approaches to the diagnosis and
treatment of obsessive-compulsive disorder. In Risch, S.C. and Janowsky, D.
(Ed.): The Art of Psychopharmacol ogy. New York, Guilford Press (in press).
Zohar, J., Insel, T.R., Foa, E.R., Skeketee, G., Berman, K., Weinberger, D.,
and Cohen, R.M.: Physiological and psychologic changes during in vivo
exposure and imaginal flooding of obsessive-compulsive disorder patients.
Proceedings of 1985 World Congress of Psychiatry (in press).
Zohar, J., Mueller, E.A., Insel, T.R., Zohar-Kadouch, R., and Murphy,
D.L.: Serotonin receptor sensitivity in obsessive-compulsive disorder:
comparison of patients and healthy controls. Arch. Gen. Psychiatry, in
press.
Zohar, J., Insel, T.R., Zohar-Kadouch, R.C., Hill, J.L., and Murphy,
D.L.: Serotonergic responsivity in obsessive-compulsive disorder:
effects of chronic clomipramine treatment. Arch. Gen. Psychiatry, in
press.
262
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30. 1987
PROJECT MUMBER
ZOl MH 00337-
LCS
TITLE OF PROJECT (80 characters or less. Title rrtust fit on one line between the borders.)
Neuropharmacology of Neuroendocrine and Neurotransmitter Regulatory Mechanisms
PRINCIPAL INVESTIGATOR (List other professional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and institute affiliation)
Dennis L. Murphy, M.D. Chief Section on Clinical Neuropharmacology LCS NIMH
COOPERATING UNITS (if any)
Centre for Reproductive Biology, Edinburgh, Scotland; BP and CP, NIMH;
NIB and LNRI, NINCDS
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Clinical Neuropharmacology
INSTITUTE AND LOCATION
NIMH, NIH. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
3.1
PROFESSIONAL:
2.0-
OTHER:
1.1
CHECK APPROPRIATE BOX(ES)
O (a) Human subjects
D (a1) Minors
D {a2) Interviews
Q (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
m-Chlorophenylpiperazine (m-CPP) , a direct serotonin receptor agonist, has
been studied extensively this year by our group in rodents, monkeys, and humans.
Neuroendocrine, temperature, cardiovascular, and behavioral effects of m-CPP
have been characterized. Studies using m-CPP as a probe of serotonin CNS
function in different psychiatric patient groups and during psychoactive drug
treatment conditions are underway.
A distinct circadian rhythm in corticotropin releasing hormone was found in
cerebrospinal fluid in a study in rhesus moni<eys. This rhythm was over twelve
hours out of phase with that of Cortisol in cerebrospinal fluid or plasma, and
is hypothesized to reflect non-hypophysiotropi c functions of this peptide, which
is well-known to be widely distributed in many brain areas outside of the
hypothalamic-pi tuitary system.
Marked effects of antidepressant drugs, especially MAO-i nhibitors, have
been observed on plasma and cerebrospinal fluid melatonin in monkeys and humans;
changes in N-acetylserotonin and serotonin accompanying diurnal melatonin
rhythms have also been observed in monkey cerebrospinal fluid.
Antibodies to beta-endorphin, somatostati n and other neuropeptides have
been identified and characterized in human plasma.
263
PHS 6040 (Rev. 1/84)
SPO S14-VIB
ZOl MH 00337-08 LCS
Other collaborative professional personnel engaged on the project:
G. Bagdy, Ph.D. Visiting Fellow LCS NIMH
N.A. Garrick, Ph.D. Biologist LCS NIMH
P.W. Gold, M.D. Section Chief BP NIMH
J.L. Hill, Ph.D. Biostatistician LCS NIMH
S.P. Markey, Ph.D. Section Chief LCS NIMH
H.F. McFarland, M.D. Section Chief NIB NINCDS
D.E. McFarlin, M.D. Lab Chief NIB NINCDS
J.W. Rose, M.D. Staff Physician VA Salt Lake City, Utah
B.F. Roy, M.D. Staff Physician VA Washington, D.C.
K. Szemeredi, Ph.D. Visiting Fellow LNRI NINCDS
L. Tamarkin, Ph.D. Staff Fellow CPB NIMH
P. Taylor, Ph.D. Chemist Centre for Reproductive
Biology, Edinburgh
T.P. Tomai Chemist BP NIMH
Z. Zukowska-Grojec, Ph.D. Guest Researcher LNRI NINCDS
Project Description:
Objectives: The discovery that many newly-characterized peptides and
hormones are present in high concentrations in the brain, cerebrospinal fluid
(CSF), and plasma has led to an entire field of inquiry into the interactions
among peptides, hormones, and both the classical monoamine neurotransmitters as
well as trace amines in brain. All of these substances may function as
modulators of neurotransmission. This project has focused on the measurement
of various peptides, hormones, and several monoamines and their metabolites in
cerebrospinal fluid and plasma in an attempt to evaluate (a) CNS and other
physiologic influences on hormones and monoamines; (b) the relationship between
peripheral, brain, and CSF peptide levels, and (c) in particular, to assess the
effects of drugs (especially agents with selective actions), as well as stress
and other stimuli on monoamines, peptides, and hormones using biochemical,
behavioral, neuroendocrine, and other physiologic response measures.
Methods Employed:
Human plasma is obtained from blood samples collected via indwelling
venous catheters. Cerebrospinal fluid from non-human primates is collected by
means of indwelling lumbar or lateral ventricular cannulae for continuous flow
through a refrigerated line into a fraction collector housed in a freezer.
Plasma from the non-human primates and rodents is obtained by use of indwelling
venous catheters which are usually implanted 15 to 24 hours prior to our
studies to permit investigations under non-stressful, basal conditions. Some
examples of hormones measured by radioimmunoassay include Cortisol, prolactin,
growth hormone, beta-endorphin, melatonin, ACTH, and vasopressin. Antibodies
to beta-endorphin, ACTH, somatostatin and other peptides are determined by
enzyme- linked immunoabsorbent assay (ELISA). Serotonin and N-acetyl -serotonin
are measured by capillary mass spectrometry. Monoamines and monoamine
metabolites are measured by high performance liquid chromatography with
electrochemical detection.
264
ZOl MH 00337-08 LCS
Major Fi ndings:
In studies investigating possible modulatory function of serotonin on
neuroendocrine mechanisms, behavior, and other physiologic functions, dose-
dependent responses to the serotonin agonist, m-chlorophenylpiperazine (m-CPP),
in humans, monkeys, and rodents have been documented. In monkeys, m-CPP
induced neuroendocrine and behavioral changes accompanied by alterations in
cerebrospinal fluid 5-hydroxyi ndoleacetic acid concentrations, without changes
in other monoamine metabolites, confirming data obtained with serotonin
antagonists in all species indicating that m-CPP acts primarily through brain
serotonin mechanisms.
In addition to increasing plasma prolactin, ACTH, Cortisol and
vasopressin, and increasing temperature, m-CPP was found to have unexpected
cardiovascular effects. In rats, blood pressure and heart rate were increased
in a dose- dependent fashion by m-CPP. These effects were also observed in
pithed rats and in adrenal demedullated rats, indicating that m-CPP was acting
directly on the peripheral cardiovascular system. Mediation of m-CPP's effects
via serotonin receptors was suggested by studies with antagonists, since
metergoline (and, in the case of the pressor responses, ritanserin) blocked
m-CPP's effects, while noradrenergic and other antagonists were ineffective.
Further studies are needed on m-CPP's neuroendocrine effects, but in
initial experiments to evaluate whether corti cotropi n-releasing hormone (CRH)
might be affected by m-CPP, baseline experiments revealed a marked circadian
rhythm in CRH in rhesus monkey CSF. Peak CRH values of 80 pg/ml occurred in
the evening at 7:30 pm, while" the CRH nadir of 30 pg/ml occurred at
approximately 8:00 am. This rhythm was inverse to that of CSF and plasma
Cortisol, which peak in the morning at 8-9 am.
Antibodies to two peptides, beta-endorphin and somatostatin, were
identified and characterized for the first time in studies using human plasma.
Among the more than fifty subjects studied to date, the concentrations of the
immunoglobulin G specific to beta-endorphin were highest in plasma from
individuals with major depression.
Significance to Biomedical Research and the Program of the Institute:
Serotonin, melatonin, and related indoleamines participate in the
regulation of behavior, sleep, locomotor activity, reproductive function, and
influence several different hormones, including ACTH, CRH, Cortisol, prolactin,
and vasopressin. Abnormalities in these functions are found in depression and
some other psychiatric disorders. The neuroendocrine, temperature, and
behavioral responses found using the serotonin receptor agonist, m-CPP, further
advance our hopes that this agent may be of value as a probe of the status of
central serotonin receptors in various psychiatric disorders and during
treatment with antidepressant and other drugs thought to act, in part, via
serotonergic mechanisms. In fact, evidence obtained this year from patients
with depression, bulimia, and obsessive-compulsive disorder suggests that
neuroendocrine and/or behavioral responses to m-CPP are different from those in
normal controls.
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ZOl MH 00337-08 LCS
The findings of a marked circadian rhythm in corticotropin releasing
hormone (CRH) are of special interest. While hypothalamic CRH is regarded as a
major physiologic regulator of pituitary ACTH secretion and, thereby, of the
circadian and stress-related release of Cortisol from the adrenal gland, CRH
and CRH receptors are also widely distributed in other brain areas of primates
and rodents. The marked difference in the circadian rhythm of CRH versus
Cortisol suggests that CRH in CSF reflects or mediates some
non-hypophysiotropic brain functions of this peptide.
The studies of melatonin, n-acetyl-serotonin , and serotonin in CSF suggest
that MAO-inhibiting antidepressants (especially the MAO-A selective inhibitor,
clorgyline) markedly alter pineal gland function via a mechanism different from
that of the tricyclic antidepressants, i.e. by increasing the availability of
melatonin's indoleamine precursors. This interesting difference is of
relevance to theories regarding the mechanism of action of antidepressants in
rodent brain, as a reduction in melatonin release during chronic tricyclic
treatment in rodents had been suggested to be a reflection of the functional
importance of beta-receptor down-regulation. The fact that net output of
melatonin is higher during chronic MAO-i nhibitor treatment suggests that the
beta-receptor down-regulation is superceded by the increased precursor
availability in the case of MAO inhibitors. This constitutes a valuable lesson
in emphasizing the need to focus on the final net consequences of drugs to
properly assess their mechanism of action.
Many peptides function as neuromodulators in brain, and the more complete
delineation of their localization will be of help in defining their function.
Antibodies to neuropeptides in human plasma have not previously been
identified, and further study in larger patient and control populations is
needed to evaluate their significance.
Proposed Course:
Based on our studies with m-CPP in rodents, monkeys, and humans, we have
begun to use this central serotonin agonist as a probe to evaluate possible
abnormalities in serotonin function in various psychiatric disorders.
Collaborations employing m-CPP have been established with other groups within
NIMH to evaluate patients with eating disorders, panic disorders, and
borderline personalities, and in other NIH institutes and elsewhere to evaluate
possible antinociocepti ve and cardiovascular effects of m-CPP. In regard to
the neuropeptides, explorations by Dr. Benajmin Roy are underway regarding the
occurrence of antibodies to other peptides in human plasma and their
relationship to such neuropsychiatric disorders as depression and Alzheimer's
di sease.
Publ ications:
Roy, B.F., Rose, J.W., McFarland, H.F., McFarlin, D.E., and Murphy, D.L.:
Anti-beta-endorphin immunoglobulin G in humans. Proc. Natl. Acad. Sci . USA 83:
8739-8743, 1986.
Murphy, D.L., Garrick, N.A., Tamarkin, L., Taylor, P.L., and Markey, S.P.:
Effects of antidepressants and other psychotropic drugs on melatonin release
and pineal gland function. J. Neural Trans. ?.l: 291-310, 1986.
266
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Murphy, D.L., Garrick, N.A. , Hill, J.L., and Tamarkin, L.: Marked enhancement
of clorgyline of nocturnal and daytime melatonin release in rhesus monkeys.
Psychopharmacology 92: 382-387, 1987.
Bagdy, G., Szemeredi , K., Zukowska-Grojec, Z., Hill, J., and Murphy, D.L.:
m-Chlorophenylpiperazine increases blood pressure and heart rate in pithed and
conscious rats. Life Sci . 41: 775-782, 1987.
Garrick, N.A. , Hill, J.L., Szele, F.G., Tomai , T.P., Gold, P.W., and Murphy,
D.L.: Corticotropin releasing hormone: A marked circadian rhythm in primate
cerebrospinal fluid peaks in the evening and is inversely related to the
Cortisol circadian rhythm. Endocrinology, in press.
267
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00339-06 LCS
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one lirte betweery the Ixrders.)
Neuropharmacology of Cognition and Mood in Geriatric Neuropsychiatry
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Trey Sunderland Chief Unit on Geriatric Psychopharmacology LCS NIMH
COOPERATING UNITS (if any)
LCM, NIMH; NIDA; Enzor Research Foundation
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Clinical Neuropharmacology
INSTITUTE AND LOCATION
IMH, NIH, Bethesda, Maryland 20892
TOTAL MAN- YEARS;
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
P (a) Human subjects
n (a1) Minors
n (a2) Intervievi/s
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A pharpiacologic challenge strategy in Alzheimer pati
and non-chol inergic agents has been the focus of this uni
years. We have previously documented that Alzheimer pati
and cognitively more sensitive to acute anticholinergic b
nous scopolamine, suggesting increased functional sensiti
matched controls. This year, we have shown that this inc
not seen in geriatric depressed patients, another populat
presenting with cognitive deficits but without documented
pathology. This functional sensitivity to anticholinergi
patients has potential therapeutic implications, and we a
sensitivity of this population to a series of cholinergic
arecoline and nicotine.
ents using chol inergic
t for a number of
ents are behavioral ly
lockade with intrave-
vity compared to age-
reased sensitivity is
ion frequently
central cholinergic
c agents in dementia
re now exploring the
agonists, including
Another major thrust of our un
selective monoamine oxidase inhibit
Work from this last year has demons
and behavioral effects in the Alzhe
In addition, study of the biochemic
drug has been helpful in characteri
action. While the therapeutic bene
overall devastation of the illness,
we are currently in the process of
in demented patients.
it has been the therapeutic usefulness of the
or (MAOI), L-deprenyl , in dementia patients,
trated that deprenyl has beneficial cognitive
imer population without serious side effects,
al changes which accompany the use of this
zing some of the possible mechanisms of
fit was relatively small compared to the
the positive effects were encouraging, and
studying the longer-term effects of this drug
269
PHS 6040 (Rev. 1/84)
CPO SI 4-9 II
ZOl MH 00339-06 LCS
Other collaborative professional personnel engaged on the project:
D.L. Murphy, M.D. Chief LCS NIMH
H. Weingartner, Ph.D. Guest Researcher BPB NIMH
P.N. Tariot, M.D. Guest Researcher LCS NIMH
P. A. Newhouse, M.D. Guest Researcher LCS NIMH
R.M. Cohen, M.D., Ph.D. Section Chief LCM NIMH
M. Gross, M.D. Staff Psychiatrist LCM NIMH
E.A. Mueller, M.D. Guest Researcher LCS NIMH
A.M. Mellow, M.D., Ph.D. Staff Psychiatrist LCS NIMH
B. Lawlor, M.D. Staff Psychiatrist LCS NIMH
S. Molchan, M.D. Staff Psychiatrist LCS NIMH
J. Grafman, Ph.D. Staff Psychologist ETB NINCDS
Project Description:
Objectives:
ubjj
ITT
To better characterize pharmacologically the cholinergic system in
Alzheimer s disease: Deficits in the central cholinergic system
remain the strongest therapeutic lead in the study of Alzheimer's
disease; yet, replacement cholinergic therapies have not reversed
the cognitive impairments associated with the illness. To better
understand this apparent paradox, Alzheimer patients and age-matched
normal controls are tested in a series of studies with cholinergic
agonists and antagonists. Changes in sensitivity to these
pharmacologic agents may help further the understanding of residual
cholinergic function in Alzheimer's disease and provide leads for
more specific therapeutic treatments in the future.
(2) To investigate biologic markers at baseline and longitudinally in
dementia and geriatric depression: Because dementia and depression
are the two single biggest problems in geriatric psychiatry and
because of the tremendous clinical overlap between these two
syndromes, we are investigating the possibility that biologic
similarities exist as well. Already, we have discovered several
biologic markers which display this overlap, and we are now
following these markers under acute treatment conditions and over
time to discover if they provide prognostic or therapeutic
usefulness.
(3) To explore new therapeutic modalities in Alzheimer's disease and
geriatric depression: An ultimate goal in our investigations is to
develop and extend new treatment approaches to these two illnesses.
We have already shown that careful administration of monoamine
oxidase inhibitors, specifically L-deprenyl, can provide some
benefit to Alzheimer patients and are now currently involved in a
long-term comparison study of deprenyl. We continue to study the
therapeutic usefulness of cholinergic agonists in dementia and will
also be expanding those studies to include non-choli nergi c agents
(i.e., peptidergic: thyrotropi n-releasing hormone, TRH, or
serotonergic: m-chlorophenylpiperazi ne, m-CPP).
270
ZOl MH 00339-06 LCS
Methods Employed:
Clinical Assessment: The clinical diagnosis of Alzheimer disease is
based on the DSM-III and the NINCDS-ADRDA criteria as well as the Dementia
Rating Scale of Hughes and coworkers in St. Louis. The latter scale is an
amalgam of multiple scales including the Blessed Dementia Scale, the Face-Hand
test, the Pfeiffer short portable mental status questionnaire and others, and
provides a measure of severity of illness. The clinical diagnosis is made
only after thorough evaluation with the exclusion of any patients suspected of
having multi-i nfarct or other forms of non-Alzheimer dementia. For those
patients followed longitudinally until death, the clinical diagnosis will be
confirmed by autopsy. The diagnosis of major affective disorder in the
elderly is made on the basis of DSM-III criteria.
Behavioral and Psychological Assessment: Mood and other behavioral
characteristics are measured with global (15-point) ratings scales, the Brief
Psychiatric Rating Scale, and the Hamilton depression rating scale where
appropriate. A dementia mood assessment scale (DMAS) has been developed
specifically for dementia patients on our unit because of the difficulty
encountered with self-rating forms or other mood scales designed for general
depressed patients. Activities of daily living are assessed by family and
staff throughout the hospitalization with a measurement tool also developed
specifically for our geriatric populations as part of this project.
For the evaluation of cognitive skills, a large number of tests are
employed. There are several routine psychometric measures including the
Wechsler memory quotient in addition to a series of recently designed or
modified tasks for this population. These tests assess effortful and semantic
memory and include measures of attention, free recall and recognition memory.
Though primarily evaluating verbal memory, some of the tests do measure visual
memory and sustained motor attention.
Biological Assessment: Plasma, platelets, urine, and cerebrospinal fluid
are collected for measurement of enzymes, hormones, levels of biogenic amines
and their metabolites. The dexamethasone suppression test and the TRH stimu-
lation tests are also used. Some patients and controls are asked to undergo a
skin biopsy for culturing and subsequent biochemical testing of the skin
fibroblasts. A major portion of the biologic testing involves pharmacologic
challenge studies. Patients and normal volunteers are given intravenous or
oral medications (i.e. scopolamine, nicotine, arecoline, TRH, or m-CPP) and
followed over the next several hours for physiologic, behavioral, neuroendo-
crine, or cognitive changes which are then compared to placebo conditions.
Major Findings:
Alzheimer patients have been shown to be more sensitive to central
cholinergic blockade than age and sex-matched normal controls along cognitive
and behavioral but not physiologic parameters. This increased functional
sensitivity was not observed in elderly depressed patients when they were
tested over the last year by Dr. Paul Newhouse. In addition, the mostly
elderly controls showed evidence, at least briefly, of significant cognitive
impairment when given the highest dose of scopolamine (0.5 mg i.v.). By
271
ZUl MH 00339-06 LCS
transiently mimicking the cognitive profile for mild dementia, the scopolamine
test therefore provides for a possible pharmacologic modelling of dementia in
otherwise normal aged humans.
Continued investigation of the biologic links between depression and
dementia has led to the finding of other areas of significant overlap.
Cerebrospinal fluid measurement of somatostatin-1 ike-immunoreacti vity has been
shown to be decreased in both elderly depressives and Alzheimer patients.
Following the previously-reported blunted response of thyroid stimulating
hormone (TSH) to thyrotropin releasing hormone (TRH) in Alzheimer and elderly
depressed patients, we have now also discovered and reported a blunted
prolactin response to TRH in both groups compared to normals.
Therapeutically, there has been modest improvement noted in the Alzheimer
patients with low but not high doses of L-deprenyl . Ratings of mood and
behavior as well as performance on effort-demanding cognitive tasks showed
significant change on 10 mg/day of deprenyl (a dose previously shown to be
relatively MAO-B selective) over a three-week trial. These behavioral changes
were accompanied by slight changes in cerebrospinal fluid (CSF) monoamine
metabolites. When the dosage was increased, the CSF metabolites revealed
changes more characteristic of a non-selective MAGI, and the behavioral and
cognitive improvements were lost. Whether this improvement at low, MAO-B
selective doses of deprenyl is specific to Alzheimer patients or might also be
seen in elderly depressed patients is currently being investigated.
Significance to Biomedical Research and the Program of the Institute:
Alzheimer's disease still remains a clinical diagnosis of exclusion which
is in doubt until autopsy or biopsy confirmation. The ability to establish an
antemortem biologic marker for Alzheimer's disease would therefore be of great
value for clinicians and researchers alike to establish earlier diagnoses and
to reduce diagnostic heterogeneity in studies. While the scopolamine
challenge test is not yet such a marker, this pharmacologic probe has revealed
significant differences between normals and dementia patients, and now between
dementia patients and elderly depressed subjects. This latter comparison is
potentially significant clinically because of the frequent overlap between the
presenting symptoms of dementia and elderly depression (i.e., pseudodementia).
For future investigations, the response to a challenge dose of 0.25 mg of
scopolamine might help determine a patient's underlying cholinergic sensi-
tivity and help provide a predictive guide to the individual responsiveness to
therapeutic agents, including cholinergic agents such as nicotine or areco-
line. In addition, the transient, mild dementia-like picture created by
scopolamine in elderly normal controls provides a pharmacologic model of
Alzheimer's disease in humans which may serve as a platform for new drug
development.
The increasing documentation of biologic links between depression and
dementia has already led to significant research and clinical results. By
treating Alzheimer patients with an antidepressant such as the monoamine
oxidase inhibitor L-deprenyl, we are simultaneously learning more about the
underlying biochemistry of the illness and providing at least some benefit to
this otherwise unrelenting, progressive illness. The fact that
272
ZOl m 00339-06 LCS
antidepressants may actually improve some of the symptoms of dementia also
opens the door for a whole host of non-cholinergi c agents to be tested in what
has for years been considered primarily a disease of cholinergic neurons. It
may well be that drugs or combinations of drugs that alter neurotransmitter
systems other than the cholinergic system have an important role in future
treatment strategies.
Proposed Course:
We have already shown that the increased anticholinergic sensitivity in
Alzheimer patients is not found in age-matched controls and depressed
patients. We must now go on to see if this increased sensitivity is found
with other non-cholinergic central nervous system drugs (i.e. benzodiaze-
pines). We must also investigate other demented populations (i.e. Korsakoff's
or Parkinson's patients) to see if this sensitivity is specific to Alzheimer's
dementia. If the selectivity remains, then exploratory testing with the off-
spring of demented subjects, in twins, or within families with a high genetic
loading of Alzheimer's disease would be valuable. While the ethical consider-
ations of a diagnostic challenge test must be addressed, the potential thera-
peutic benefit of a more definitive diagnosis and earlier initiation of treat-
ment could be tremendous. We must also test the relationship between anti-
cholinergic sensitivity and response to cholinergic agonists. It is possible,
for instance, that previous studies have tested potentially helpful medica-
tions too far out on the dose-response curve and have therefore missed the
"therapeutic window."
Our study of biologic links between depression and dementia will be
enlarged to include family history studies and comparisons of mapping electro-
encephalograms across diagnostic groups. We will also be attempting to
carefully quantify the severity of depression in our demented subjects.
Therapeutically, the short-term gains identified in Alzheimer patients with
low-dose deprenyl will be compared to other clinically available medications
in a longitudinal outpatient study. Acute challenge studies with other poten-
tially therapeutic agents such as TRH, m-CPP, nicotine, and arecoline will
also be continued.
Up until now, our projects have been limited to relatively small drug and
biologic studies. Over the past five years, however, we have accumulated in
depth behavioral, neuropsychological, and biologic data on scores of Alzheimer
patients. in all stages of the illness. Over the next two years, we plan to
combine these expanding datasets to establish a profiling system for
Alzheimer's disease. As our project continues and the brain bank inevitably
expands, this profiling system will include final pathologic diagnoses. The
ultimate biologic fingerprint of baseline and longitudinal information may
eventually be quite valuable for both diagnostic and prognostic purposes.
Publications:
iot, P.N., Sunderland, T., Murphy, D.L., Cohen, M.R., Weingartner, H.,
house, P. A., and Cohen, R.M.: Design and interpretation of opiate
agonist trials in dementia. Prog. Neuro. Psychopharmacol . Biol. Psychi.
273
ZOl MH 00339-06 LCS
Sunderland, T., Tariot, P.N., Weingartner, H., Murphy, D.L., Newhouse, P. A.,
Mueller, E.A., and Cohen, R.M.: Pharmacologic modelling of Alzheimer's
disease. Prog. Neuro. Psychopharmacol . Biol. Psychiatry 10: 599-610, 1986.
Tariot, P.N., Sunderland, T., Weingartner, H., Murphy, D.L., Cohen, M.R., and
Cohen, R.M.: Naloxone and Alzheimer's disease: Cognitive and behavioral
effects of a range of doses. Arch. Gen. Psychiatry 43: 727-732, 1986.
Newhouse, P. A., Sunderland, T. , Tariot, P.N., Mueller, E.A., Murphy, D.L., and
Cohen, R.M.: Prolactin response to TRH in Alzheimer's disease and elderly
controls. Psychiatry Res. 21: 963-967, 1986.
Sunderland, T., Mellow, A.M., Gross, M., Cohen, R.M., Tariot, P.N., Newhouse,
P. A., and Murphy, D.L.: TRH in Alzheimer's disease (letter). Am. J.
Psychiatry 143: 1318, 1986.
Newhouse, P. A., Sunderland, T. , Thompson, K., Tariot, P.N., Weingartner, H. ,
Mueller, E.A., Cohen, R.M., and Murphy, D.L.: The effects of intravenous
nicotine on cognitive and psychologic functioning in DAT patients and
controls. Am. J. Psychiatry 143: 1494-1495, 1986.
Tariot, P.N., Murphy, D.L., Sunderland, T., Mueller, E.A., and Cohen, R.M.:
Rapid antidepressant effect of addition of lithium to tranylcypromine. J.
Clin. Psychopharmacol. 6: 165-167, 1986.
Barbaccia, M.L., Costa, E., Fgrrero, P., Guidotti, A., Roy, A., Sunderland,
T., Pickar, D., Paul, S.M., and Goodwin, F.K.: Di azepam-binding inhibitor: A
neuropeptide present in human spinal fluid: studies in depression, schizo-
phrenia and Alzheimer's disease. Arch. Gen. Psychiatry 43: 1143-1147, 1986.
Eisenhofer, G., Goldstein, D.S., Stull, R., Keiser, H.R., Sunderland, T.,
Murphy, D.L., and Kopin, I.: Simultaneous 1 iquid-chromatographic determina-
tion of 3,4-dihydroxyphenyl glycol , catecholamines, and 3, 4-di hydroxy-phenyl -
alanine in human plasma and their responses to inhibition of monoamine
oxidase. Clin. Chem. 32: 2030-2033, 1986.
Tariot, P.N., Cohen, R.M., Sunderland, T., Newhouse, P. A., Yount, D., Mellow,
A.M., Weingartner, H., Mueller, E.A., and Murphy, D.L.: L-deprenyl in
Alzheimer's disease: Preliminary evidence of behavioral change with monoamine
B inhibition. Arch. Gen. Psychiatry 44: 429-433, 1987.
Nee, L.E., Eldridge, R., Sunderland, T., Thomas, C.B., Katz, D., Thompson,
K.E., Weingartner, H. , Weiss, H., Julian, C, and Cohen, R.M.: Dementia of
the Alzheimer type: clinical and family study of 22 twin pairs. Neurology
37: 359-363, 1987.
Sunderland, T. , Tariot, P.N., Cohen, R.M., Newhouse, P. A., Mellow, A.M.,
Meuller, E.A., and Murphy, D.L.: Dose-dependent effects of deprenyl on CSF
monoamine metabolites in patients with Alzheimer's disease.
Psychopharmacol ogy 91: 293-296, 1987.
274
ZOl MH 00339-06 LCS
Tariot, P.N., Sunderland, T., Weingartner, H., Murphy, D.L., Welkowitz, J. A.,
Thompson, K., and Cohen, R.M.: Cognitive effects of L-deprenyl in Alzheimer's
disease. Psychopharmacology 91: 489-495, 1987.
Sunderland, T., Tariot, P.N., Cohen, R.M., Weingartner, H., Mueller, E.A., and
Murphy, D.L.: Anticholinergic sensitivity in patients with dementia of the
Alzheimer type and age-matched controls: A dose-response study. Arch. Gen.
Psychiatry 44: 418-426, 1987.
Newhouse, P. A., Sunderland, T., Tariot, P.M., Mueller, E.A., Murphy, D.L., and
Cohen, R.M.: TRH stimulation in Alzheimer's disease (letter). Acta
Psychiatr. Scand. , in press.
Risby, E., Hsiao, J., Sunderland, T., Agren, H., and Rudorfer, M.: Effects of
antidepressants on the cerebrospinal fluid HVA/5HIAA ratio. Clin. Pharmacol.
Ther. , in press.
Skurla, E., Rogers, J.C., and Sunderland, T.: Direct assessment of activities
of daily living in Alzheimer's disease: A controlled study. J. Am.
Geriatrics Soc, in press.
Sunderland, T., Rubinow, R.D., Tariot, P.N., Cohen, R.M., Newhouse, P. A.,
Mellow, A.M., Mueller, E.A., and Murphy, D.L.: CSF somatostatin in dementia,
elderly depression and age-matched controls. Am. J. Psychiatry, in press.
Tariot, P.N., Sunderland, T., Cohen, R.M., Newhouse, P. A., Mueller, E.A., and
Murphy, D.L.: Tranylcypromine compared with L-deprenyl in Alzheimer's
disease. J. Clin. Psychopharmacol . , in press.
Weingartner, H., Cohen, R.M., Sunderland, T., and Tariot, P.N.: Diagnosis and
assessment of cognitive dysfunction in the elderly. In Meltzer, H.Y. (Ed.):
Psychopharmacology: Third Generation of Progress. New York, Raven Press, in
press.
275
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl tm 00433-07 LCS
PERIOD COVERED
October 1, 1986 throu^ September 30, 1987
TITLE OF PROJECT (80 charactars or less. Tith must tit on one line between the txirders.)
Role of Neuropeptides and Biogenic Amines in Neuroendocxine Regulation
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfiliation)
J.M. Saavedra, Qiief , Unit on Preclinical Neuropharmacology/LCS/NIMH
PI:
Others: M. Aiso
E. Castren
R. Cruciani
M. Kurihara
K. Saito
C. Gonzalez
VF/LCS/NIMH F. Correa
VF/LCS/NIMH S. Guillaume
VF/LCS/NIMH J.S. Gutkind
VF/KS/NIMH D. IfcKenna
VF/LCS/NIMH L. Fochtmann
VF/HI/NHLBI A.J. Nazarali Alberta Found. Fellow
Guggenheim Fellow
Guest Researcher
Int. Fogarty Fellow
PRAT Fellow/NIOyiS
PE^T Fellow/NIGJyiS
COOPERATING UNITS (If any)
Laboratory of Clinical Science
SECTION
Section on Clinical Pharmacology
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
9.0
PROFESSIONAL:
7.5
1.5
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
n (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
With the use of quantitative autoradiography, we studied the role of
neuropeptides (angiotensin II. atrial natriuretic peptide) and biogenic amines
(dopamine) in the central regulation of autonomic nervous system and pituitary
gland. We also studied the role of neuropeptides in the regulation of the
immune response by localizing neuropeptide receptors in immune organs and
immune cells and by investigating the second messenger response.
We demonstrated a role for central angiotensin II and atricil natriuretic
peptide in the control of cardiovascular function. These neuropeptides may
also be involved in regulation of the immune response. Specific angiotensin II
and B-adrenoceptors were quantified in the rat heart conduction system. New
methods were developed to study localization and transport of D^ and D2
dopamine receptors in brain, and to study localization of malignant melanomas.
Psychotomimetic phenyl isopropylamine receptors were localized and quantified in
selective brain areas.
277
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CPO 9I4.SII
ZOl MH 00433-07 ICS
ERQIECr DESCRIPTION
Obiec±ives
To study the role of centrcil and peripheral biogenic amines and
neuropeptides in restricted brain areas, and in peripheral tissues involved
in cardiovascular function, pituitary control and regulation of immune
response. Ihis study includes the continual development of new quantitative
autoradiograpiiic methods for determination and quantification of receptors,
peptides and related enzymes in rat brain nuclei.
To develop quantitative autoradiographic methods for the study of biogenic
amine and neuropeptide receptors in human blood cells, and to apply these
methods to clinical studies.
Methods Employed
Neuroanatomical , surgical, biochemical (RIA, gel electrophoresis,
radioenzymatic assays) and autoradiography with image analysis combined with
conputerized microdensitometry.
Major Findings
1. We determined the presence of angiotensin II receptors in a forebrain
band, vAiich continues from the subfornical organ to the organon vasculosum
laminae terminalis and the paraventricular nucleus. This band may represent
the link between peripheral and central angiotensin systems.
2. Selective areas corresponding to this forebrain band (nucleus
preopticus medianus, organon vasculosum laminae terminalis, and
paraventricular nucleus, as well as previously described subfornical organ)
show hi(^er number of angiotensin II receptors in strains of spontaneously
hypertensive rats. Activation of the forebrain band angiotensin system may
play a role in the development and maintenance of genetic hypertension.
3. After immobilization stress, rats have a selective increase in the
number of angiotensin receptors in the paraventricular nucleus, with no
change in anterior pituitary angiotensin receptors. This result suggests
that brain angiotensin receptors have a role in the central regulation of the
stress response.
4. In the DOCA-salt hypertensive rat, another model of hypertension, an
increase in number of angiotensin II receptors occurs in specific brain areas
(subfornical organ, paraventricular nucleus, nucleus of the solitary tract,
median preoptic nucleus) , indicating that participation of central
angiotensin systems in the control of blood pressure is not restricted to
genetically hypertensive animals, and may be a more general phenomenon.
5. Angiotensin II receptors are present in the human adrenal medulla and
zona glomerulosa.
6. Receptors for atrial natriuretic peptide were studied in brain and in
several peripheral organs of the rat. High receptor concentration was
278
ZOl MH 00433-07 LCS
detected in syirpathetic ganglia (superior cervical, stellate and celiac),
pituitary gland (anterior and posterior lobes) , kidney glomeruli and medulla,
adrenal cortex, and immune organs (thymus and spleen) , as well as in isolated
thymocytes and spleen cells. Incubation of isolated thymocytes and spleen
cells with atrial natriuretic peptide results in a concentration-dependent
increase in cyclic (MP. Similar results were obtained in isolated
syitpathetic ganglia.
7. More than an 90% decrease was noted in the number of atrial
natriuretic peptide binding sites in syirpathetic ganglia, pituitary, thymus
and spleen from spontaneously hypertensive rats vdien compared to that of
normotensive control rats. The cyclic GJyiP increase obtained after incubation
of thymocytes and spleen cells with atrial natriuretic peptide, however, was
no different in hypertensive than in control rats. This result indicates a
possible heterogeneity of the atrial natriuretic peptide binding sites, and
suggests that changes in hypertensive rats probably reflect the loss of
binding sites not linked to guanylate cyclase.
8. A new method was developed, vAiich allows application of quantitative
autoradiography of neuropeptide and biogenic amine receptors to saitples
prepared from isolated vdiole cell preparations. This method has wide
application to both animal and clinical research, and is ten times more
sensitive than classical membrane binding techniques. Adrenergic B-receptors
and VIP receptors were quantified with this method in human blood
lyirphocytes.
9. Several neuropeptide -receptors, other than those for atrial
natriuretic peptide (including angiotensin II and substance P) , were
localized in the thymus and spleen of the rat.
10. We developed a dissecting procedure to localize the conduction system
of the rat heart. This method is used for quantitative autoradiographic
techniques. Specific areas such as the sinoatrial node, atrioventricular
node, and intrinsic heart parasyitpathetic ganglia, can be identified and
studied. We localized B^ and B2 adrenoceptors and quantified their ratio
(about 50/50%) in the sinoatrial and atrioventricular nodes. In addition, we
identified angiotensin II receptors in all parts of the rat heart conduction
system.
11. The number of both D^ and D2 dopamine receptors was quantified in
brain areas and in pituitary gland by autoradiography and the use of new,
■•■^^I-ligands. We showed that D^ receptors are present in the caudate-nigral
pathway, and demonstrated their bidirectional transport.
12. A specific D-i dopamine receptor antagonist binds specifically to
melanin, and may prove to be a diagnostic agent capable of detecting
pigmented melanomas.
13. There is a significant increase, as determined by quantitative
autoradiography, in D^ receptors in the substantia nigra of rats submitted to
chronic electroconvulsive treatment.
279
ZOl MH 00433-07 KS
14. Insulin-like growth factor I (IGF I) receptors were localized in human
brain cortex and pituitary gland. The number of these receptors is hi^er in
gliciblastamas .
15. As determined by autoradiography, the iodinated 5HT2 agonist, with
psychotomimetic properties, (4-iodo-2,5-diinethoxy-phenylisopropylamine)
(l25j_Qoi) binds specifically to rat cortex (layer IV) , claustrum, and
olfactory tracts. This indicates anatomical selectivity for the
psychotomimetic phenyl isopropylamines.
16. Specific NPY receptors were located in the zona glomerulosa of the
bovine adrenal cortex, suggesting a role for this peptide in control of
aldosterone secretion.
Significance to Biomedical Research and to the Institute
implication of quantitative autoradiographic metliods to basic research is
beginning to yield a significant number of new findings v*iich will help to
clarify the role of neuropeptides and biogenic amines in central regulation
of cardiovascular function, pituitary control, stress, and iiranune response.
These methods will also help to clarify iitportant anatomical and
physiological aspects of the action of psychotomimetic compounds. In
addition, quantitative autoradiographic methods may now be applied to
clinical studies for the correlation of multiple neuropeptide and amine
receptors in human blood sanples.
Proposed Course
With the use of quantitative autoradiography, we plan to study further the
interactions between neuropeptides and biogenic amines in brain areas
involved in the control of pituitairy and autonomic function, with eitphasis on
correlation between binding sites and corresponding second messenger
responses. New methods for the quantification of neuropeptides in restricted
brain areas will be developed using similar techniques. We will focus these
studies on the regulatory mechanisms for cardiovascular, pituitary and immune
system function. In addition, we plan to apply our new autoradiographic
methods for the study of neuropeptide and biogenic amine receptors in human
peripheral blood lynphocytes, under a variety of physiological and
pathophysiological conditions, with special enphasis in neuropsychiatric
disorders.
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ZOl MH 00433-07 LCS
Publications
Also, M. , Shigematsu, K. , Kebabian, J.W. , Potter, W.Z., Cruciani, R.A. , and
Saavedra, J.M. : Dopamine D^ receptor in rat brain: a quantitative
autoradiographic study with ^^^1-SCtl 23982, Brain Res. 408: 281-285, 1987.
Agren, H. , Koiilu, M. , Saavedra, J.M. , Potter, W.Z., and Linnoila, M. :
Circadian covariation of norepinephrine and serotonin in the locus coeruleus
and dorsal raphe nucleus in the rat. Brain Res. 397: 353-358, 1986.
Agui, T., Bryant, G. , Kebabian, J.W. , Larson, S., Saavedra, J.M. , Shigematsu,
K. , Yamamoto, T. , and Yokoyama, K. : ^^^I-Iodinated benzazepines bind to
melanin: inplications for the noninvasive localization of pigmented
melanomas. Nucl. Med. Biol. 14: 133-141, 1987.
Correa, F.M.A. , Plunkett, L.M. , and Saavedra, J.M. : Quantitative
distribution of angiotensin-converting enzyme (kininase II) in discrete areas
of the rat brain by autoradiography with coiiputerized microdensitometry.
Brain Res. 375: 259-266, 1986.
Fuchs, E., Shigematsu, K. , and Saavedra, J.M. : Binding sites of atrial
natriuretic peptide in tree shrew adrenal gland. Peptides 7: 873-876, 1986
Fuchs, E. , Shigematsu, K. , and Saavedra, J.M. : Localization of atrial
natriuretic peptide 6-33 (ANP) binding sites in tree shrew (Tupaia belangeri)
adrenal gland by in vitro receptor autoradiography. Acta Endocrinol. rSupp. 1
fCopenh.) 283: 97, 1987.
Israel, A., Barbella, Y., and Saavedra, J.M. : Coirpensatory increase in
adrenomeduilary angiotensin-converting enzyme activity (kininase II) after
unilateral adrenalectomy. Recoil. Pept. 16: 97-105, 1986.
Koulu, M. , Saavedra, J.M. , Bjelogrlic, N. , Niwa, M. , Agren, H, , and Linnoila,
M. : Serotonin turnover in discrete hypothalamic nuclei and mesencephalic
raphe nuclei of young and adult spontaneously hypertensive rats. Brain Res.
379: 257-263, 1986.
Kurihara, M. , Katamine, S., and Saavedra, J.M. : Atrial natriuretic peptide,
ANP(99-126) , receptors in rat thymocytes and spleen cells. Biochem. Biophys.
Res. Commun. 145: 789-796, 1987.
Kurihara, M. , Saavedra, J.M. , and Shigematsu, K. : Localization and
characterization of atrial natriuretic peptide binding sites in discrete
areas of rat brain and pituitary gland by quantitative autoradiography.
Brain Res. 408: 31-39, 1987
Kurihara, M. , Shigematsu, K. , and Saavedra, J.M. : Localization of atrial
natriuretic peptide, ANP- (99-126) binding sites in the rat thymus and spleen
with quantitative autoradiography. Requl. Pept. 15: 341-346, 1986.
281
ZOl MH 00433-07 ICS
Niwa, M. , Shigematsu, K. , and Saavedra, J.M. : Changes in substance P
receptors of the intermediolateral cell column of the thoracic spinal cord in
young spontaneously hypertensive rats. In Nakaraura, K. (Ed,): Brain and
Blood Pressure Control. Elsevier Science Publishers, Amsterdam, 1986, pp.
225-230.
Plunkett, L.M. , Shigematsu, K. , Kurihara, M. , and Saavedra, J.M. :
Localization of angiotensin II receptors along the anteroventral third
ventricle area of the rat brain. Brain Res. 405: 205-212, 1987.
Saavedra, J.M. : Angiotensin II and rat atrial natriuretic peptide binding
sites: alterations in specific brain nuclei of spontaneously hypertensive
rats. In Na>camura, K. (Ed.): Brain and Blood Pressure Control. Elsevier
Science Publishers, Amsterdam, 1986, pp. 113-118.
Saavedra, J.M. : Atrial natriuretic peptide (6-33) binding sites: decreased
number and affinity in the subfornical organ of spontaneously hypertensive
rats. J. Hypertens. 4(suppl. 3): S313-S316, 1986.
Saavedra, J.M. , and Alexander, N. : Angiotensin II receptors in adrenal
gland, pituitary gland and brain of sino-aortic denervated rats. J.
Hypertens. 4(suppl. 5): S154-S157, 1986.
Saavedra, J.M. , Correa, F.M.A. , Kurihara, M. , and Shigematsu, K. : Increased
number of angiotensin II receptors in the subfornical organ of spontaneously
hypertensive rats. J. Hypertens. 4(suppl. 5) :S27-S30, 1986.
Saavedra, J.M. , Israel, A., Correa, F.M.A. , and Kurihara, M. : Increased
atrial natriuretic peptide (6-33) binding sites in the subfornical organ of
water deprived and Brattleboro rats. Proc. Soc. Exp. Biol. Med. 182:
559-563, 1986.
Saavedra, J.M. , Israel, A., and Kurihara, M. : Increased atrial natriuretic
peptide binding sites in the rat subfornical organ after water deprivation.
Endocrinology 120: 426-428, 1987.
Saavedra, J.M. , Israel, A., Plunkett, L.M. , Kurihara, M. , Shigematsu, K. , and
Correa, F.M.A. : Quantitative distribution of angiotensin II binding sites in
rat brain by autoradiography. Peptides 7: 679-687, 1986.
Saavedra, J.M. , and Krieger, E.M. : Early increase in adrenomedullary
catecholamine synthesis in sinoaortic denervated rats. J. Auton. Nerv. Syst.
18: 181-183, 1987.
Saavedra, J.M. , Plunkett, L.M. , Correa, F.M.A., Israel, A., Kurihara, M. , and
Shigematsu, K. : Quantitative autoradiography of angiotensin and atrial
natriuretic factor binding sites in brain nuclei of spontaneously
hypertensive rats. In Buckley, J. P., and Ferrario, CM. (Eds.): Brain
Peptides and Catecholamines in Cardiovascular Regulation. Raven Press, NY,
1987, pp. 245-256.
282
ZOl MH 00433-07 LCS
Shigematsu, K. , Niwa, M. , Kurihara, M. , Castren, E. , and Saavedra, J.M. :
Alterations in substance P binding in brain nuclei of spontaneously-
hypertensive rats. Am. J. Physiol. 252: H301-H306, 1987.
Shigematsu, K. , Niwa, M. , and Saavedra, J.M. : Substance P binding sites:
increased concentration in specific brainstem nuclei of spontaneously
hypertensive rats. In Nakainura, K. (Ed,): Brain and Blood Pressure Control.
Elsevier Science Publishers, Amsterdam, 1986, pp. 219-224.
Shigematsu, K. , Saavedra, J.M. , and Kurihara, M. : Specific si±)stance P
binding sites in rat thymus and spleen: in vitro autoradiographic study.
Recall. Pept. 16: 147-156, 1986.
Shigematsu, K. , Saavedra, J.M. , Plunkett, L.M. , Kurihara, M. , and Correa,
F.M.A. : Angiotensin II binding sites in the anteroventral-third ventricle
(AV3V) area and related structures of the rat brain. Neurosci. Lett. 67: 37-
41, 1986.
In Press
Castren, E. , Kurihara, M. , Gutkind, J.S., and Saavedra, J.M. : Atrial
natriuretic peptide receptors in thymus and spleen of young spontaneously
hypertensive rats. Biologically Active Atrial Peptides, ASH Symposium
Series. Vol. II.
Castren, E., Kurihara, M. , Gutkind, J.S., and Saavedra, J.M. : Specific
angiotensin II binding sites in the rat stellate and superior cervical
ganglia. Brain Res.
Castren, E., Kurihara, M. , and Saavedra, J.M. : Autoradiographic localization
and characterization of angiotensin II binding sites in the spleen of rats
and mice. Peptides .
Castren, E., and Saavedra, J.M. : Repeated immobilization stress increases
angiotensin II receptor density in rat hypothalamic paraventricular nucleus.
Proceedings of the IVth International Meeting on Catecholamines and Other
Transmitters in Stress.
Fuchs, E., Flugge, G. , Kurihara, M. , and Saavedra, J.M. : Binding sites of
atrial natriuretic peptide (ANP) in Tupaia belangeri central nervous system.
An in vitro receptor autoradiographic study. Peptides.
Fuchs, E., Flugge, G. , Shigematsu, K. , and Saavedra, J.M. : Binding sties for
atrial natriuretic peptide in tree shrew and primate adrenal glands.
Proceedings of World Congress on Biologically Active Atrial Peptides.
Gutkind, J.S., Kurihara, M. , Castren, E., Saavedra, J.M. : Atrial natriuretic
peptide receptors in rat syirpathetic ganglia: alterations in genetically
hypertensive rats. Biologically Active Atrial Peptides, ASH Symposium Series
Vol. II.
283
ZOl MH 00433-07 ITS
Israel, A., and Saavedra, J.M. : High angiotensin converting enzyme (kininase
II) ac±ivity in cerebrospinal fluid of adult spontaneously hypertensive rats.
J. Hypertens.
McKenna, D. , Mathis, C.A. , Shulgin, A.T, , Sargent III. T. , and Saavedra, J.M.
Autoradiographic localization of binding sites for -^^^I-DOI, a new
psychotomimetic radioligand, in the rat brain. Eur. J. Pharmacol.
Saavedra, J.M. : B-phenylethylamine, phenylethanolamine, tyramine and
octopamine. In Weiner, N. , and Trendelenburg, U. (Eds.): Handbook of
Pharmacology : Catecholamines II . Spr inger-Verlag .
Saavedra, J.M. : Brain Epinephrine in Hypertension and Stress. Proceedings
of Brain Epinephrine Symposium.
Saavedra, J.M. : Regulation of atrial natriuretic peptide receptors in the
rat brain. Cell. Mol. Neurobiol.
Saavedra, J.M. : Regulation of brain atrial natriuretic peptide receptors.
Proceedings IVth International Meeting on Catecholamines and Other
Transmitters in Stress.
Saavedra, J.M. , Israel, A., Correa, F.M.A. , and Kurihara, M. : Increased
atrial natriuretic peptide (99-126) binding sites in the subfornical organ of
water deprived and Brattleboro rats. Proceedings of World Concrress on
Biologically Active Atrial Peptides.
Saavedra, J.M. , Israel, A., Kurihara, M. , and Correa, F.M.A. : Decreased
number and affinity of rat atrial natriuretic peptide (99-126) binding sites
in the subfornical organ of spontaneously hypertensive rats. Proceedings of
World Congress on Biologically Active Atrial Peptides.
Saavedra, J.M. , Kurihara, M. , and Israel, A.: Alterations in angiotensin and
atrial natriuretic peptide receptors in brain nuclei of spontaneously
hypertensive rats. J. Hypertens. (suppl.).
284
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00447-18 LCS
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 charactars or less. Tltla must fit on one line tMtween the borders.)
Amine neurotransmitters and metabolites in mental illness
PRINCIPAL INVESTIGATOR (List other prolessionel personnel below the Principal Investigator.) (Name, title, laboratory, and institute altillalion)
William Z. Potter, M.D., Ph.D., Chief, Section on Clinical Pharmacology,
Laboratory of Clinical Science, NIMH
COOPERATING UNITS (M any)
Clinical Psychobiology Branch; Neuroscience Branch;
Child Psychiatry Branch, NIMH; and Laboratory of Clinical
Studies, NIAAA
Laboratory of Clinical Science
Section on Clinical Pharmacology
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3.45
PROFESSIONAL:
2.55
OTHER:
.9
CHECK APPROPRIATE eOX(ES)
S (a) Human subjects
D (a1) Minors
Q (a2) Intervievi^s
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Alterations of amine neurotransmitter systems (norepinephrine {f€),
serotonin {5HT) and dopamine (DA)) have been indirectly implicated in the
pathophysiology of the major mental illnesses, depression and schizophrenia.
We have applied new techniques to study the interpretation of neurotransmitter
measures fran cerebrospinal fluid (CSF), plasma and uri ne from drug-free
patients with affective illness and schizophrenia. Hew findings include the
f 0 1 1 owi ng :
1. The interrelationships between neurotransmitters as evidenced by
correlations of metabolites of NE , 5HT and DA in CSF discriminate between
responders and non-responders to antidepressant treatment. Moreover, the
same measures appear to discriminate drug free schizophrenics from normal
controls.
2. The important question of whether variations in at least one type of
neurotransmitter receptor on cells obtainable from blood can be explained
primarily on the basis of circulating agonist has been answered. Lymphocyte
beta receptor parameters have been clearly shown to vary independently of
plasma norepinephrine and epinephrine.
3. Longitudinal studies of patients with affective illness have included
repeat biochemical measures in drug-free states during which mood was
normal. Surprisingly, the exaggerated plasma NE response to going from a
supine to standing position which is consistently noted in the depressed
state persisted during the "well" state. Thus, investigation of the NE
system may reveal a trait abnomiality in persons susceptible to affective
disorder and provide a means of identifying persons at risk.
285
PHS 6040 (Rev. 1/84)
CPO BI4.«1t
other Professional Personnel:
Matthew Rudorfer Senio
John Hsiao Medic
Emile Risby Guest
Ivan Mefford Speci
Laura Fochtmann PRAT
Markku Linnoila Chief
Denni s Murphy Chief
Thomas A. Wehr Chief
David Sack Senio
David Jimerson Chief
David Pickar Chief
Judith Rapoport Chief
Trey Sunderland Senio
Project Description:
r Staff Fellow
al Staff Fellow
Researcher (NRSA)
al Expert
Fel low
r Staff Fellow
, Section on Biomedical Psych,
, Section on Clinical Studies
, Child Psychiatry Branch
r Staff Fellow
ZOl MH 00447-13 LCS
LCS/NIMH
LCS/NIMH
LCS/NIMH
LCS/NIMH
LCS/NIMH
LCS/NIAAA
LCS/NIMH
CP/NIMH
CP/NIMH
LCS/NIMH
NSB/NIMH
CHP/NIMH
LCS/NIMH
The characterization of the functional state of three amine neuro-
transmitter (NT) systems, norepinephrine (NE), serotonin (5HT) and dopamine
(DA), in depression and other major psychiatric illnesses such as schizo-
phrenia continues to be a major ongoing project. We have added to that
the exploration of ways to characterize the epinephrine (EPI) system. On-
going iriethod development and clinical studies reveal sources of variance
which we can only partly control such as the inherent "stress" responses
of NT systems to invasive procedures.
It has become increasingly clear that measures of NT systems covary
to a significant degree and may reflect some degree of functional inter-
dependence in the central nervous system. This possibility has led to
the reevaluation of NT measures in subgroups of patients and controls in
terms of their balance. In the absence of validated models for exploring
any "balance" betwen NT systems non-model dependent techniques such as
ratios of two measures or correlation matrices of three or more are employed.
Use of this approach can be applied to the cumulative data base of
measures obtained in controls and patients with depression, mania, schizo-
phrenia, eating disorders, attention deficit disorders and Alzheimer's
disease from values obtained after 1981 (the data from which new standardized
assays were made operational). Both group differences in the untreated
state and prediction of response to treatment are explored.
Methods:
Biochemical techniques are described in a separate project summary
pertaining to the central laboratory (ZOl MH 01855-03).
Selection of subjects, paying particular attention to such issues as
age of onset, frequency of recurrence of episodes, and family history is
given great emphasis. Whenever feasible, extended (over 1 month) drug-
free periods are required before biological samples are obtained--a 3-
286
ZOl MH 00447-18 LCS
week period is our current minimum although our data show that even this
is inadequate in some patients who have been on tricyclic antidepressants
and is definitely inadequate in any patient who has received chronic
neuroleptics or monoamine oxidase inhibitors. Patients are also charac-
terized according to length on a low monoamine diet as well as number of
days in hospital. This latter parameter is of particular interest since
many depressed patients are studied after brief (sometimes only overnight)
hospitalization and then transferred to outpatient status. With the
expansion of outpatient studies, some procedures are performed in some
studies without hospitalization.
"Control" subjects must be drawn fran both hospitalized and "outpatient"
age- and sex-matched individuals who are asked to be on diet. It appears
that for comparisons of urine and CSF hospitalization can be a critical vari-
able. Therefore, a comparison of "controls" under different conditions has
become an essential component of our design.
Major Findings:
1. Certain interrelationships between neurotransmitters and/or their
metabolites continue to emerge as potentially more physiologically relevant
than the absolute concentration of substances by themselves. The most
robust and consistently observed relationship remains the high correlation
between 5HIAA and HVA in the CSF although significant correlations are
also observed between MHPG and 5HIAA or HVA in comparison of several hun-
dred pairs of values. For instance, non-responders to drug treatment do
not show the usual patterns of correlation between the three NT metabolites
in CSF found in controls and responders to treatment. Moreover, in a pre-
liminary analysis of data provided by the 4E schizophrenia research unit,
schizophrenics as a group showed a lower degree of correlation than normal
controls.
2. In a separate but related series of analyses a previously noted dis-
parity of absolute concentrations of 5HIAA and HVA between control groups
has been explored. We find that the ratio of HVA to 5HIAA and the ratio
of HVA to MHPG are quite stable across control groups even when the mean
values for any single metabolite differ dramatically. And at least in
the case of the HVA/5HIAA ratio, it was substantially lower in two separate
groups of depressed patients compared to controls and intermediately
lower in schizophrenics. In contrast, initial analyses of borderline
personality disorder patients reveal a nonnal HVA/5HIAA ratio. Thus, the
ratio provides a new means of identifying biochemical abnormalities in
certain patient groups.
3. Attanpts to characterize epinephrine in CSF and venous blood using
the most sensitive assay available raise questions about the validity of
previous claims since resting concentrations are often below the level of
detection. In stress paradigms ("learned helplessness," orthostatic chal-
lenge and cold exposure of upper extremity) consistent elevations of venous
EPI are not observed although other groups have shown robust increases of
arterial EPI under comparable circumstances. Thus, alternate approaches
to studying EPI function in man are necessary.
287
01 MH 00447-18 LCS
4. Sufficient studies have been completed comparing plasma measures of
NE and EPI to establish that variations on density or function of lymphocyte
beta receptors can not be explained on the basis of circulating catecholamines.
5. Interpretation of plasma concentrations of a metabolite of at least
one NT, i.e. HVA from DA, must be reassessed in light of the demonstration
that variation in the renal clearance of HVA might account for variations
in plasma concentration rather than the rate of production of HVA. Moreover,
we have just found that the total excretion of HVA in urine is correlated
at the 0.8 or above level with that of both major NE metabolites, VMA and
MHPG. Thus, in the periphery it seems unlikely that the bulk of HVA
concentration is directly related to DA function in the brain.
6. Expansion of baseline studies from cross-sectional to longitudinal
investigations of the course of affective illness has enabled the address-
ing of state-versus-trait issues. Using plasma and CSF basal measures,
bipolar depression cannot be distinguished from drug-free normal mood in
the same individuals. Hypomania in these patients is associated with
relative increases in resting plasma norepinephrine concentration and
in CSF MHPG and HVA concentrations. The most striking finding to emerge
is the persistence into euthymia of depression-associated exaggerated
reactivity of plasma norepinephrine to an orthostatic challenge. Should
this result hold up in further subjects to be studied during the coming
year, investigations of noradrenergic reactivity in relatives of depressed
patients or other high-risk individuals will be undertaken to assess the
potential of this measure as a marker of a depression diathesis.
Significance to Biomedical Research and to the Program of the Institute :
The major theories about the biological causes of the most prevalent
severe psychiatric disorders, depression and schizophrenia, center on
monoamine neurotransmitter systems. This project applies sophisticated
laboratory assays directly to human studies of monoamine metabolism.
Results expand our understanding of the role of norepinephrine and other
neurotransmitters, mainly in depression. The personal and social costs of
this illness are great. Insofar as careful clinical research, drawing on
basic biochemical techniques, can identify biological factors in these
disorders, specific phannacologic treatments can be developed and tested
i n therapeutic tri als.
Proposed Course:
We will apply current and develop alternate methods of looking at
relationships between neurotransmitter systems to the combined populations
of controls and patients made possible by centralization of assays (see
separate project No. ZOl MH 01855-02). We believe that this approach
provides the best chance of breakthroughs in our ability to use neuro-
transmitter and their metabolite concentrations as tools in diagnosis,
prediction of treatment and understanding pathophysiology. Conversely,
we are impressed by the essentially "nonnal" range of values observed for
any single parameter in the resting, drug-free state in a variety of neuro-
psychiatric disorders.
288
ZOl MH 00447-18 LCS
A. In light of the emerginy group differences in degree of covariance
and/or ratios of neurotransmitter metabolites we will focus more on studies
of determinants of these composite parameters in control as well as
patient populations. Since the first findings related to possible inter-
active measures have emerged from studies of CSF we will consider metabolite
ratios and covariance as a function of other substances (e.g. peptide
hormones) which have previously been reported to correlate with one or
another.
B. Alternate approaches to investigating EPI function in man will be
pursued in collaboration with groups who are using either insulin or
deoxyglucose challenges in patients and volunteers since these metabolic
manipulations can produce a centrally-mediated robust release of adrenal
EPI as shown in preclinical studies. Urinary measures of EPI and its
metabolite metanephrine will also be investigated with the focus on
whether they provide information independent of plasma EPI. This will
necessarily include measures of the renal clearance of EPI.
C. Our most far-reaching plan is to assess whether the summing of both
DA and NE metabolites in urine can provide an index of "whole-body"
hydroxylase activity. To this end, free vs total DOPAC must first be
considered along with the possible efects of diet on this other major DA
metabolite. Pharmacologic manipulations in both humans and animals can
then be used to see if decreasing tyrosine hydroxylase activity produces
consistent and predictable decreases of the sum of its products in
urine. Ultimately this may allow us to establish individual tyrosine
hydroxylase "phenotypes" in anticipation of applying molecular genetic
studies on this enzyme.
Publications
Agren, H., Koulu, M., Saavedra, J.M., Potter, W.Z., and Linnoila, M.:
Circadian covariation of norepinephrine and serotonin in locus coeruleus
and dorsal raphe nucleus in the rat. Brain Res. 397: 353-358, 1986.
Agren, H., Mefford, I.N., and Potter, W.Z.: Does serotonin turnover
regulate dopamine turnover New Biochemical evidence in man. In Shagass,
C, Josiassen, R.C., Bridger, W., Weiss, K., Stoff, D., and Simpson, G.M.
(Eds.): Biological Psychiatry 1985: Proceedings of the IVth World Con-
gress of Biological Psychiatry. Elsevier Science Publishing Company,
Inc. , New York, 1986, pp.
Agren, H. , Mefford, I.N., Rudorfer, M.V., Linnoila, M. , and Potter, W.Z.:
Interacting neurotransmitter systems. A non-experimental approach to the
5HIAA-HVA correlation in human CSF. J. Psychiat. Res. 20: 175-193, 1986.
Agren, H., and Potter, W.Z.: Effects of drug wash-out on CSF monoamines
and psychoendocri ne variables. Psychopharmacol . Bull. 22: 937-941, 1986.
289
ZOl MH 00447-18 LCS
Potter, W.Z., Rudorfer, M.V., and Goodwin, F.K.: Bioloijical findings in
bipolar disorders. In Hales, R.F., and Frances, A.J. (Eds.): American
Psychiatric Association Annual Review: Volume Six. American Psychiatric
Press, Inc., Washington, O.C, 1987, pp. 32-60.
In Press
Buckholtz, N.S., Davies, A.O. , Rudorfer, M.V., Golden, R.N. , and Potter,
W.Z.: Lymphocyte beta-adrenergic receptor function in depression. Biol .
Psychiat. , (in press).
Linnoila, M. , Roy, A., Lane, E., Virkkunen, M., Rudorfer, M. , and Potter,
W.Z.: Characterization of noradrenergic state from norepinephrine and
its metabolites in patients with alcoholism, depression and disorders of
impulse control. In Dahlstrom, A. et al . (Eds.): Proceedings of the 6th
International Catecholamine Symposium. Alan R. Li ss. New York, N.Y. , (i n
press) .
290
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00428-08 LCS
PERIOD COVERED
October 1, 1986 throuc^ September 30, 1987
TITLE OF PROJECT (80 characters or lass. THh must tit on one line between fhe tnrders.)
Protein Cacboxyl Methylation; A Post Translational Modifier of Protein Function
PRINCIPAL INVESTIGATOR (List ottter prolassional personnel botow (he Principal Investigator.) (Name, title, laboratory, and institute atliliation)
PI: J.M. Saavedra, Chief, Unit on Preclinical Neuropharmacology/LCS/NIMH
COOPERATING UNITS (If any)
LAB/BRANCH
Laboratory of Clinical Science
Section on Clinical Fharmacology
INSTITUTE AND LOCATION
NIMH. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed ttte space provided.)
This project has been combined with ZOl MH 00433-07 LCS.
291
PHS 6040 (Rey. 1/84)
CPO •14-CII
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
'roject number
ZOl MH 01850-10 LCS
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (BO Characters or less. TJtle must tit on one line tMtween the txjrders.)
Clinical pharmacology of antidepressants
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliatlon)
William Z. Potter, M.D., Ph.D., Chief, Section on Clinical Pharmacology,
Laboratory of Clinical Science, NIMH
COOPERATING UNITS (If any)
Clinical Psychobiology Branch; Clinical Neuroscience Branch; and Laboratory of
Clinical Studies, NIAAA
Laboratory of Clinical Science
Section on Clinical Pharmacology
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
5.2
PROFESSIONAL:
3.6
1.6
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
D (a1) Minors
D (a2) Interviews
0 (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The therapeutic mechanism of action of antidepressant medications in
humans remains unknown. Comparison of effects on specific neurotransmitters
and their metabol ites in cerebrospinal fluid (CSF), plasma and urine in
the same patients continues. Findings of special interest during the last
year include the following:
1. Unique effects of electroconvulsive therapy (ECT) in humans continue
to emerge: unlike all antidepressant drugs studied it does not reduce
whole body norepinephrine (NE) turnover as measured in urine or NE and
serotonin turnover as measured by MHPG and 5HIAA, respectively, in CSF.
In fact ECT increases both 5HIAA and the dopamine (DA) metabolite, HVA in
CSF.
Z. Based on these clinical findings we carried out experiments on ECT in
rats and found a selective increase in the D^ subtype of DA receptor in
substantia nigra and caudate. Since ECT has been reported to have thera-
peutic effects in mania, psychosis and Parkinsonism as well as depression
these DA effects likely have clinical and mechanistic implications.
3. In keeping with the theme that more than one neurotransmitter change
is involved in antidepressant action, we have found that drugs with
specific classes of initial biochemical effects have unique in vivo
profiles of effects in humans only when changes of MHPG, 5HIAA and HVA
are simultaneously taken into account. We have employed a 3-dimensional
graph to obtain clear discrimination between five classes of drugs using
these amine metabolites.
4. Alprazolam, a potent anti-anxiety agent with possible antidepressant
properties, produces unusually robust decreases of ACTH and Cortisol
following intravenous administration. This finding may provide for a new
test for the responsivity of the HPA axis; i.e. is it more difficult to
suppress in certain psychiatric illnesses? 293
PHS 6040 (Rev. W84)
ZOl MH 01850-10 LCS
Other Professional Personnel
Matthew Rudorfer
John Hsiao
Emile Risby
Ivan Mefford
Laura Fochtmann
Markku Linnoila
Dennis Murphy
Thomas A. Wehr
David Sack
David Jimerson
David Pickar
Judith Rapoport
Trey Sunderland
Project Description:
Senior Staff Fellow LCS/ NIMH
Medical Staff Fellow LCS/NIMH
Guest Researcher (NRSA) LCS/NIMH
Special Expert LCS/NIMH
PRAT Fellow LCS/NIMH
Chief LCS/NIAAA
Chief LCS/NIMH
Chief CP/NIMH
Senior Staff Fellow CP/NIMH
Chief, Section on Biomedical Psych. LCS/NIMH
Chief, Section on Clinical Studies NSB/NIMH
Chief, Child Psychiatry Branch CHP/NIMH
Senior Staff Fellow LCS/NIMH
Our central aim is to understand the effects of major somatic anti-
depressant treatments on the monoamine neurotransmitter systems in man.
Systematic studies of drug action in normal volunteer controls and de-
pressed patients control li ng for pharmacokinetic and pre-drug physiologic
variance has permitted demonstration of both predicted and unexpected bio-
chemical alterations following treatment with drugs or convulsive therapy
having widely differing acute primary effects.
Comparison of biochemical effects in CSF, plasma and urine in the
same patients is continuing with new, efficient high performance liquid
chromatography assays, and, when coupled with physiologic, behavioral and
neuroendocri ne measures, allows for clearer systems interpretations of
changes. State-of-the-art measures of norepinephrine (NE), serotonin
(5HT), dopamine (DA) and their metabolites are made under controlled
conditions both cross sectional ly in time and longitudinally in order to
identify interrelationships, to test assumptions about the regulation of
these neurotransmitter systems, and therefore to definitively describe
effects of antidepressants as they relate to these neurotransmitter systems.
Methods:
The neurotransmitter systems of patients with either unipolar or
bipolar major affective disorder are characterized after at least a 3-week
drug-free period and then between the 3rd and 5th week following antide-
pressant treatment. Certain parameters, such as urinary transmitter and
metabolite concentrations, are studied repeatedly following the beginning
of each treatment. Patients admitted at steady-state of an antidepressant
drug are also studied serially during the withdrawal phase. Parallel studies
are performed in healthy volunteers and animal models when feasible as
described below.
Treatments are ideally administered so as to produce maximal effects
on the presumed target biochemical system such as inhibition of NE uptake
after desipramine (DMI), of 5HT uptake after clomipramine (CMI), and of
294
ZOl MH 01850-10 LCS
MAO-Type A after clorgyline using control of pharmacokinetic variance
(blood levels of DMI , CMI and desmethyl CMI) or biochemical indices (MHPG
decrease after clorgyline). In the case of lithium and ECT, standard
regimens are followed.
Novel putative antidepressants with no clear biochemical specificity
such as alprazolam are also studied. Biochemical effects are studied
after acute and chronic dosing, and, in the case of ECT, serially (generally
weekly) throughout the course of treatment.
Studies in college age volunteers housed on the unit are of shorter
duration (up to two weeks of active drug) and include DMI, lithium, and,
in single intravenous doses, alprazolam.
Specialized pharmacokinetic and baseline biochemical studies are per-
formed in volunteers age- and sex -matched to our accumulated patient popu-
lation. These volunteers come to the clinic on the day of the study or
are admitted for an overnight accommodation to the research unit.
Analysis of NE, 5HT, DA and their metabolites is carried out as
described in a separate report, ZOl MH 01855-03 LCS. Using radiolabelled
iodocyanopindolol as an antagonist lymphocyte beta receptor parameters
are determined with ligand analysis of complex agonist displacement curves
both before and after selected treatments.
To elucidate the mechanisms of action of ECT, we are administering
ECS to rats (80 mAmp x .5 sec via earclip electrodes e'^ery other day for a
total of 8 treatments) and assessing its effects from a number of vantage
points. In particular, we have applied conventional membrane binding
assays and quantitative autoradiographic techniques to examine ECS induced
changes in a variety of receptor types. We are also obtaining micropunch
specimens from brain and collecting 24 hour urines for assessing regional
and total body catecholamine concentrations and turnover. Using the
technique of tail artery cannulation which permits serial sampling of
blood in unstressed freely moving rats, we have been able to conduct
neuroendocrine challenge tests (paralleling those done on the clinical
unit) in ECS treated rats. In addition, the cannulated rat model is ideal
for assessing the acute effects of ECS on the neuroendocrine system.
Findings to Date:
1. Our ongoing treatment protocol with electroconvulsive therapy (ECT),
which has now yielded some paired data in 15 patients, demonstrates
important differences between the actions of convulsive therapy and those
of antidepressant drugs. In contrast to every other effective antidepres-
sant phannacological treatment which we have studied, ECT does not reduce
whole-body norepinephrine (NE) turnover. ECT does consistently lower
basal plasma NE concentration by an average of 30%, but plasma NE measured
after an orthostatic challenge also falls by an equivalent amount after
the course of treatment, leaving the initial relative noradrenergic hyper-
activity unchanged, despite clinical improvement in all but one patient.
295
ZOl MH 01850-10 LCS
Thus, ECT is unique in its spectrum of effects on the ^E system raising
the possiblilty that effects on other neurotransmitters may play a greater
role than hitherto supposed (see below).
2. CSF monoamine metabolite concentrations also show a unique pattern of
alteration after ECT, with little average change in MHPG but substantial
rises in 5HIAA (not observed after any chronic antidepressant drug treat-
ment) and even moreso in HVA (by 24% and 35%, respectively). However,
preliminary investigations into the responsivity of the serotonergic
system, using the prolactin stimulation by an intravenous challenge of
clomipramine as a phanmacological role before and after a course of ECT,
are negative. These findings suggest that dopaminergic effects of ECT
may be important, a possibility which is suggested by preclinical findings
summarized below.
3. Using conventional membrane binding techniques we have been able to
confirm previous reports of decreased 03 and g binding after ECS. Using
[3H]-ketanseri n we have found no significant differences in 5HT2 receptor
numbers or in 5HT1 receptors or uptake sites. Despite previous negative
results from other laboratories which utilized conventional binding tech-
niques, by employing quantitative autoradiography we have found increases
in Dl receptors in substantia nigra pars reticulata, caudate and accumbens
and corresponding changes in [3H]-forskoli n binding which suggests parallel
changes in adenylate cyclase. Autoradiographic studies of receptor binding
in organs other than brain have revealed a decrease of B receptors in the
heart ventricles, the first such- demonstration of which we are aware after
any antidepressant treatment. With our cannulated rat model we have been
able to verify the expected increases in PRL levels with ECS itself. Con-
sistent with the lack of change we find in serotonin receptors after ECS,
there is no difference in the PRL response to citalopram (a serotonin up-
take inhibitor) in ECS vs. sham treated rats. Coupled with our clinical
investigations these animal studies point to the possibility of some im-
portant interaction of norepinephrine and dopamine systems in the mode of
action of ECT.
4. Cerebrospinal fluid measures continue to prove useful in characterizing
drug effects. In addition to individual monoamine metabolite concentrations,
their relationships to one another are under study as we build upon our
earlier investigations of drug effects on the noradrenergic system. The
ratio of CSF HVA/5HIAA undergoes predictable changes with biochemically
different antidepressants. In collaboration with Dr. flans Agren, a fonner
Visiting Fellow now in the Department of Psychiatry of Uppsala University
in Sweden, we have examined results from several separate Swedish and
American studies on three serotonin uptake inhibitors (zimelidine, clo-
mipramine and citalopram), two norepinephrine uptake inhibitors (nor-
triptyline and desi prami ne) , a dopamine uptake inhibitor (bupropion) and
a dopamine agonist (bromocriptine) as well as two MAO inhibitors (clorgyline
and deprenyl). In brief, serotonin uptake inhibitors all consistently
increase the HVA/5HIAA ratio, norepinephrine and dopamine uptake inhibitors
do not affect it whereas MAOI inhibitors and the dopamine agonist reduce
the ratio. Furthermore, if drug effect is analyzed in tenns of effects
296
ZOl MH 01850-10 LCS
on all three neurotransmitter metabolites (HVA, 5HIAA and MHPG) which can
be displayed as a three dimensional plot, the values cluster according to
type of treatment. Thus, we have established two methods of classifying
biochemical drug effects in man that clearly discriminate classes which
were not able to be discriminated using changes in amine metabolites
taken singly.
5. We have continued our investigation of the serotonin reuptake inhibitor,
intravenous clomipramine (CMI), as a pharmacologic probe of the serotonin sys-
tem in depressed patients and healthy volunteers before and after ECT and
lithium, respectively. Using low (10-12.5 mg) doses of CMI which produce
no desmethyl CMI and minimal nausea in subjects, this challenge is specific
to the serotonergic system, resulting in stimulation of plasma prolactin
ACTH and Cortisol concentrations with no effect on growth hormone. As
noted above, preliminary studies of depressed patients reveal no
effect of ECT on CMI responses. Furthermore, lithium treatment in
volunteers does not alter the hormonal responses to CMI. These findings
suggest that if there are ECT or lithium induced alterations in serotonin
function they cannot be demonstrated with this serotonergic challenge.
6. Chronic lithium (14 days at 0.6-0.8 meq/1 ) in volunteers had no sig-
nificant effect on plasma MHPG, HVA or the NE response to an orthostatic
challenge on whole body NE turnover. Preliminary results reveal no evi-
dence of effects on lymphocyte and platelet adrenergic receptor parameters
using membrane preparations. This does not conflict with the findings of
reduced isoproternol -stimulated cAMP in whole lymphocytes reported by
other groups. Thus, lithium-induced alterations in models of post-synaptic
signal transduction are not accompanied by any evidence of changes in
pre-synaptic function or receptor regulation (i.e. NE turnover or release).
7. Our initial study of the effects of intravenous alprazolam (APZ) in
normal volunteers has been canpleted. The most striking findings were
that APZ is surprisingly potent in reducing plasma ACTH and Cortisol
while increasing growth hormone. In light of studies under way in Dr.
Gold's group, the effects on the HPA axis may reflect a direct inhibition
of CRF. If so, APZ could be developed into an alternate challenge of the
HPA axis in patients.
Significance to Biomedical Research and to the Program of the Institute:
Understanding of the mechanism(s) of action of antidepressant treat-
ments produces improved therapeutics, new drugs, tools for studying and
investigating the underlying pathophysiology of depression and therefore,
ultimately, provides the basis for prevention.
From a therapeutic point of view phannacoki netic studies have been
crUical to removing problems related to inappropriate dosing. Moreover,
the systematic study of biochemically selective (clorgyline) and novel,
presumably less toxic agents (alprazolam), as well as ECT -- a 50-year-old
but still poorly understood intervention -- provide treatments which are
effective in many patients who do not respond to standard antidepressants.
297
ZOl MH 01350-10 LCS
Of ultimate importance is the continued finding that changes of the
noradrenergic system are always involved in the action of somatic antide-
pressant treatments. Although simple deficit or excess catecholamine hy-
potheses of depression do not explain drug action, it seems clear that to
understand the mechanism we must understand the role of NE , individually
and as it interacts with other transmitter systems. This aspect of inter-
actions between systems may provide a means of predicting antidepressant
response which, if feasible, would have a major impact in clinical care
of a large population of patients.
Proposed Course:
A. To further develop our new understanding of effects of ECS in discrete
regions in rats we will expand our work in quantitative autoradiography
to other receptor types (D2, Substance P, Neuropeptide Y). We will also
use these techniques to examine ECS effects on ion channels (calcium
and sodium), enzymes (ACE) and second messenger systems (forskolin). In
parallel studies we will compare ECS-induced receptor changes with those
associated with lithium, insulin coma and other antidepressant treatments.
B. As a bridge from animal to clinical studies we will explore effects
of various antagonists of specific neurotransmitter systems on the neuro-
endocrine response to ECS itself. In a complementary approach central
ECS induced changes will be correlated with peripheral measures (urinary
catecholamines, lymphocyte receptors, receptors in other organs) since
these latter measures may be obtained directly or indirectly in hormones.
C. A new putative antidepressant and indirect facilitator of noradrenergic
function will be studied in patients and volunteers. This drug, idozoxan,
is a selective alpha 2 antagonist which is also of interest as a possible
means of enhancing learning during periods of stress. It will be of
particular interest to understand the adaptive biochemical changes in
humans which occur after chronic administration.
D. In light of our findings reported in ZOl MH 00447-18 that renal clear-
ance may be an important determinant of plasma concentrations of at least
one neurotransmitter metabolite, HVA, we will investigate drug effects on
renal clearance of those substances of greatest interest for neuropsychiatric
research. Lithium will be the first drug investigated in this manner
since it is known to affect some components of kidney function.
Pub! ications
Rudorfer, M.V., Karoum, P., Ross, R.J., Potter, W.Z., and Linnoila, M.:
Differences in lithium effects in depressed and healthy subjects. Clin.
Pharmacol . Ther. 37: 66-71, 1985.
Weingartner, H. , Rudorfer, M.V., and Linnoila, M.: Cognitive effects of
lithium treatment in normal volunteers. Psycho Pharmacol ogy. 86: 472-474,
1985.
298
ZOl MH 01850-10 LCS
Rudorfer, M.V., and Potter, W.Z.: The tricyclic antidepressants. In
Cutler, N.R., and Narang, P,K. (Eds.): Drug Studies in the Elderly.
Plenum Press, New York, 1986, pp. 167-187.
Rudorfer, M.V., and Linnoila, M.: Electroconvulsive therapy. In Johnson,
F.N. (Ed,): Lithium Therapy Monographs, Vol 1: Lithium Combination
Treatment. Karger, Basel, Switzerland, 1987, pp. 164-178.
Rudorfer, M.V., Linnoila, M. , and Potter, W.Z.: Accidental antidepressants:
Search for specific action. In Dahl , S.G., Gram, L.F., Paul, S".M. and
Potter, W.Z. (Eds.): Clinical Pharmacology in Psychiatry. IV. Selectivity
in Psychotropic Drug Action - Promises or Problems. Springer-Verlag ,
Heidelberg, 1987, pp. 157-166.
In Press
Agren, H., Nordin, C, and Potter, W.Z.: Antidepressant drug action and
CSF monoamine metabolites: New evidence for selective profiles on mono-
ami nergic interactions. In Proceedings of the 6th International Catecholamine
Symposium. Dahlstroin, A. et al . (Eds.), Alan R. Liss, Inc., New York, N.Y.
(in press) .
Agren, H. , and Potter, W.Z.: Effects of drug wash-out on CSF monoamines
and psychoendocrine variables. Psychopharmacol . Bui 1 . (in press).
Golden, R.N. , DeVane, C.L., Laizure, S.C, Rudorfer, M.V., Sherer, M.A.,
and Potter, W.Z.: Bupropion: The role of metabolites in clinical outcome.
Arch. Gen. Psychiatry (in press).
Golden, R.N. , Markey, S.P., Risby, E.D., Cowdry, R.W., and Potter, W.Z.:
Antidepressants reduce whole-body norepinephrine turnover while maintaining
6-hydroxymelatoni n output. Arch. Gen. Psych, (in press).
Golden, R.N., Rudorfer, M.V., Sherer, M.A. , Linnoila, M. , and Potter,
W.Z.: Bupropion: Biochemical effects and clinical response in depressed
patients. Arch. Gen. Psychiatry (in press).
Hauger, R.L., Scheinin, M. , Siever, L., Linnoila, M. , and Potter, W.Z.:
Dissociation of presynaptic noradrenergic receptor changes from clinical
response to low dosage clorgyline treatment in depressed patients. Clin.
Pharm. Ther. (in press).
Potter, W.Z., Rudorfer, M.V., Lesieur, P., Risby, E.D., and Linnoila, M.:
Biochemical effects of selective 5HT-reuptake inhibitors in man. In
Gastpar, M. (Ed.): Selective 5-HT Reuptake Inhibitors: Novel or Common
Place Agents. S. Karger, Basel (in press).
Potter, W.Z., Rudorfer, M.V., and Linnoila, M.: Effects of antidepressants
on NE and its metabolites in cerebrospinal fluid, plasma and urine. In
Proceedings of the 6th International Catecholamine Symposium. Dahlstrom,
A. et al. (Eds.), Alan R. Liss, New York, N.Y. (in press).
299
ZOl MH 01850-10 LCS
Risby, E.O., Hsiao, J.K., Sunderland, T., Agren, H., Rudorfer, M.V., and
Potter, W.Z.: The effects of antidepressants on the HVA/5HIAA ratio. Clin.
Pharmacol . Ther. (in press).
Rudorfer, M.V., Linnoila, M. , and Potter, W.Z.: Combined lithium and
electroconvulsive therapy: Pharmacokinetic and pharmacodynamic interactions,
Convulsive Therapy (in press).
Rudorfer, M.V., and Potter, W.Z.: Phannacoki netics of antidepressants.
In Meltzer, H. et al . (Eds.): Psychopharmacology, the Third Generation of
Progress (in press).
300
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01855-03 LCS
PERIOD COVERED
October 1, 1985, through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between ttre tmrders.)
Central Neurodhemistry Service
PRINCIPAL INVESTIGATOR (List other prolesshnal personnel below the Principal Investigator.) (Name, title, leboratorf, and institute alfiliatlon)
William Z. Potter, M.D., Ph.D., Chief, Section on Clinical
Pharmacology, Laboratory of Clinical Science, NIMH
Ivan N. Mefford, M.D. Special Expert/LCS/NIMH
Sanford P. Markey, M.D. Chief/AB/LCS/NIMH
COOPERATING UNITS (It any)
Section on Analytical Biochemistry and Section on Biomedical
Psychiatry, LCS, NIH; Laboratory of Clinical Studies, NIAAA
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Clinical Pharmacology
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
4.2
PROFESSIONAL
0.7
3.5
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use startdard unreduced type. Do not exceed the space provided.)
The central neurochemistry service functions as a centralized laboratory for
analysis of neuroturansmitters and metabolites in body fluids collected within
the intramural program, routine analysis include norepinephrine in plasma
urine and cerebrospinal fluid (CSF) , epinephrine in plasma urine and CSF,
dopamine and dopamine sulfate in urine, plasma and CSF, catecholamine
metabolites, HVA, MHPG and DOPAC in CSF, plasma and urine, serotonin in
platelets, platelet poor plasma and CSF and 5-HIAA in plasma, CSF and urine.
GC-MS assays are used for total urinary norepinephrine . epinephrine.
dopamine. VMA. MHPG^ HVA. DOPAC, metanephrine and normetanephr ine . HPDC with
anperometxic detection is used for all other assays. Inplemetation of
microbore HPLC analysis using novel sufactant chromatographic approaches
allows measurement of free catecholamines as well as serotonin in CSF. This
allows exandnation of duimal rhythms and of drug effect:s on these amines in
extracellular fluid. Some 16,000 assays were performed on over 8,000 samples
vdiich were processed during the last year.
301
PHS 6040 (Rev. 1/84)
SPO SI4.*lt
ZOl MH 01855-03 I£S
Proiec± Description;
The Central Neurochemistry Service provides a centralized analytical
facility vdiose focus is the measurement of neurotransmitters and metabolites
in physiological fluids generated by the clinical intramural research effort.
Four technicians are presently provided by NIMH and one by NIAAA. These
individuals perform routine assays and participate in development of new
assays four of these use primarily HPDC vtiile one performs assays and methods
development on GC-MS in collaboration with the Section on Anailytical
Biochetnistry (Dr. Markey) .
Methods;
As noted above, the major analytical effort involves hi^ performance
liquid chromatography (HPLC) with electochemical detection. Using recently
developed reagents for separation of biogenic amines and microbore
technology, selective detection of catecholamines at the 45 pg/ml level has
been accoirplished. Novel ion-pairing reagents allow "on column" concentra-
tion of saiiples (amines) eliminating tedious derivatization and extraction
steps.
1. i^)erometric detection vAien coupled to microbore HPDC; offers significant
(-50 fold) signal enhancement v*ien compared to conventional HPLC or to
coulometric detection.
2. Using a "non-eluting matrix" approach, biogenic amines can be selec-
tively concentrated "on column" eliminating the necessity of derivatization
and extraction.
3. HVA, 5HIAA and MHPG can be determined simultaneously in a single plasma
extract. Separate assays for plasma 5HIAA and HVA were combined with the
MHPG assay, eliminating separate sample preparation steps and analyses.
4. Extraction of plasma MHPG, normally the rate limiting step in this
assay, was modified to double the sanple load able to be processed.
5. Columns can be prepared "in house" via slurry packing v4iich is a
considerable savings in time and cost. This is now offered as a service to
other NIMH laboratories.
FindincTs;
1. Using atiperometric detection, we are able to quant itate concentrations
of epinephrine and serotonin in the 100 femtamole/ml range in cerebrospinal
fluid. These methods (a combination of 1 and 2) are now routinely applied to
CSF samples. Epinephrine concentrations are routinely found to be 1-5 pg/ml
vAiile serotonin is usually found in the 10-100 pg/ml range.
2. Concentrations of NE, MHPG, HVA, DOPAC, and 5HIAA are measurable in the
picomole/ml range in CSF and/or plasma by anperometric detection. Thousands
of sairples have been analyzed and the results are described in reports from
the various clinical investigators using these assays.
302
ZOl MH 01855-03 I£S
3. Evidence has been accumulated demonstrating that only arterial blood is
a suitable source for measurement of epinephrine for stress indices.
4. Resting plasma norepinephrine is highly correlated with CSF norepine-
phrine suggesting that plasma may provide a suitable single measurement.
Significance to Biomeidal Research and to the Program of the Institute;
Neurotransmitter system function is ittplicated in major psychiatric
illness, in behavioral medicine (e.g. responses to psychological and
physiological stress) and in the mode of action of psychoactive as well as
cardiovascular drugs. Iitproved methods for studying these neurotransmitter
systems are crucial to understanding their operation in humans since adequate
animal models or in vitro systems do not exist.
Only by fully and accurately quantitating neurotransmitters and their
metabolites will it be possible to distinguish alterations of output vs those
of metabolism and to relate amount to function. Ihese techniques provide the
best current hope of biochemically identifying individuals with psychiatric
disease, at risk for such illness and/or most likely to respond to specific
treatment.
Proposed Coijrse:
With full-time professional direction of the laboratory, we plan to
achieve the following over the next year:
1. Continue to assess the usefulness of plasma MHPG vs plasma norepine-
phrine and noremetanephrine as an index of noradrenergic function.
2. Measure free amines and metabolites in plasma and urine to study the
renal clearance of these ccmpounds.
3. Study the effects of various antidepressant therapies on CSF serotonin
concentrations .
4. Assess the functional role of epinephrine formation in brain brain via
CSF measurements in psychiatric populations and following drug intervention.
303
ZOl MH 01855-03 LCS
Publ ications
Agren, H., Mefford, I.N., and Potter, W.Z.: Does serotonin turnover
regulate dopamine turnover New Biochemical evidence in man. In Shagass,
C, Josiassen, R.C., Bridger, W., Weiss, K,, Stoff, D., and Simpson, G.M.
(Eds.): Biological Psychiatry 1985: Proceedings of the IVth World Con-
gress of Biological Psychiatry. Elsevier Science Publishing Company,
Inc., New York, 1986, pp.
Agren, H., Mefford, I.N. , Rudorfer, M.V., Linnoila, M., and Potter, W.Z.:
Interacting neurotransmitter systems. A non-experimental approach to the
5HIAA-HVA correlation in human CSF. J. Psychiatr Res. 20: 175-193, 1986.
Agren, H., and Potter, W.Z.: Effects of drug wash-out on CSF monoamines
and psychoendocri ne variables. Psychopharmacol . Bui 1 . 22: 937-941, 1986.
Donnelly, M., Zametkin, A., Rapoport, J., Ismond, D., Weingartner, H.,
Lane, E., Oliver, J., Linnoila, M. , and Potter, W.Z.: The treatment of
childhood hyperactivity with desipramine: Plas.na and urinary catecholamine
levels and clinical response. Clin. Pharmacol . Ther. 39: 72-81, 1985.
Pickar, 0., Roy, A., Breier, A., Doran, A., Wolkowitz, 0., Colison, J.,
and Agren, H.: In: Psychobiology of Suicidal Behavior, Annals, N.Y.
Acad. Sci., 487, 189-196, 1986.
Roy, A., Agren, H. , Pickar, D. ,. Linnoila, M., Doran, A., Cutler, N. , and
Paul, S.M.: Reduced CSF concentrations of homovanillic acid and homovanillic
acid to 5-hydroxyi ndoleacetic acid ratios in depressed patients: Relation-
ship to suicidal behavior and dexamethasone nonsupression. Am. J. Psychiatry
143: 1539-1545, 1986.
Roy, A., Jimerson, D.C., and Pickar, D.: Plasma MHPG in depressive
disorders and relationship to the dexamethasone supression test. Am. J.
Psychiatry 143: 846-851, 1986.
Roy, A., Linnoila, M., and Pickar, D.: Relative activity of metabolic
pathways for norepinephrine in endogenous depression. Acta Psychiatr. Scand.
73: 524-623, 1986.
Roy, A., Pickar, D., Linnoila, M., Doran, A.R., and Paul, S.M.: Cerebrospinal
fluid monoamine and monoamine metabolite levels and the dexamethasone
supression test in depression. Arch. Gen. Psychiatry 43: 355-350, 1986.
Roy, A., Virkkunen, M., Guthrie, S. , and Linnoila, M.: Indices of
serotonin and glucose metabolism in violent offenders, arsonists and
alcoholics. Annals of N.Y. Acad. Sci . 487: 202-220, 1985.
Roy-Byrne, P.P., Rubinow, D.R., and Linnoila, M.: Relation between plasma
prolactin and plasma homovanillic acid in normal subjects. Neuro psycho-
biology 16: 85-87, 1986.
304
ZOl MH 01855-03 LCS
Roy-Byrne, P.P., Uhde, T.W., Sack, D.A., Linnoila, M., and Post, R.M.:
plasma HVA and anxiety in patients with panic disorders. Biol . Psychiatry
21: 849-853, 1986.
van Kammen, D.P., van Kammen, W.B., Mann, L.S., Seppala, T., and Linnoila,
M.: Dopamine metabolism in the cerebrospinal fluid of drug free
schizophrenic patients with and without cortical atrophy. Arch. Gen. Psychiatry
43: 978-983, 1986.
Flament, M., Rapoport, J., Murphy, D.L., Berg, C, and Lake, C.R.:
Biochemical changes during clomipramine treatment of childhood obsessive-
compulsive disorder. Arch. Gen. Psychiatry 44: 219-225, 1987.
Gjerris, A., Sorensen, A.S., Rafaelsen, O.J., Werdelin, L., Ailing, C,
and Linnoila, M.: 5-HT and 5-HIAA in cerebrospinal fluid in depression.
J. Affective Disord. 12: 13-22, 1987.
Marshall, T.H. , Jacobson, K.A., Kirk, K.L., and Linnoila, M. : Liquid
chromatographic assay for cerebrospinal fluid normetanephrine. Life Sci .
40: 1513-1521, 1987.
Pickar, D., Wolkowitz, 0., Doran, A.R., Labarca, R., Roy, A., Breier, A.,
and Narang, P.K.: Clinical and biochemical effects of verapramil administra-
tion to schizophrenic patients. Arch. Gen. Psychiatry 44:113-118, 1987.
Potter, W.Z., Rudorfer, M.V., and Goodwin, F.K.: Biological findings in
bipolar disorders. In Hales, R.E., and Frances, A.J. (Eds.): American
Psychiatric Association Annual Review: Volume Six. American Psychiatric
Press, Inc., Washington, D.C., 1987, pp. 32-60.
Roy, A., Guthrie, S., Pickar, D., and Linnoila, M.: Plasma norepinephrine
response to cold challenge in depressed patients and nonmal controls.
Psychiatry Res. 21: 161-168, 1987.
Roy, A., Pickar, D. , Linnoila, M., Chrousos, G.P., and Gold, P.W.: Cerebro-
spinal fluid corticotropi n-releasing hormone in depression: Relationship
to noradrenergic function. Psychiatry Res. 20:229-237, 1987.
Sunderland, T., Tariot, P.N., Cohen, R.M., Newhouse, P. A., Mellow, A.M.,
Mueller, E.A., and Murphy, D.L.: Dose-dependent effects of deprenyl on
CSF monoamine metabolites in patients with Alzheimer's disease. Psycho-
pharmacology 91: 293-296, 1987.
Virkkunen, M. , Nuutila, A., Goodwin, F.K., and Linnoila, M.: Cerebrospinal
fluid monoamine metabolite levels in male arsonists. Arch. Gen. Psychiatry
44: 241-247, 1987.
305
ZOl MH 01855-03 LCS
In Press
Agren, H., Nordin, C, and Potter, W.Z.: Antidepressant drug action and
CSF monoamine metabolites: New evidence for selective profiles on mono-
aminergic interactions. In Proceedings of the 6th International Catecholamine
Symposium. Dahlstrom, A., et al . (Eds.), Alan R. Liss, Inc., New York, N.Y.
(i n press) .
Buckholtz, N.W., Davies, A.O. , Rudorfer, M.V. , Golden, R.N. , and Potter,
W.Z.: Lymphocyte beta-adrenergic receptor function in depression. Biol .
Psychiat. , (in press).
Golden, R.N. , DeVane, C.L. , Laizure, S.C, Rudorfer, M.V., Sherer, M.A.,
and Potter, W.Z.: Bupropion: The role of metabolites in clinical outcome.
Arch. Gen. Psychiatry (in press).
Golden, R.M., Markey, S.P., Risby, E.D., Cowdry, R.W., and Potter, W.Z.:
Antidepressants reduce whole-body norepinephrine turnover while maintaining
6-hydroxymelatoni n output. Arch. Gen. Psychiatry (in press).
Golden, R.N., Rudorfer, M.V., Sherer, M.A. , Linnoila, M. , and Potter,
W.Z.: Bupropion: Biochemical effects and clinical response in depressed
patients. Arch. Gen. Psychiatry (in press).
Hauger, R.L., Scheinin, M., Siever, L. , Linnoila, M. , and Potter, W.Z.:
Dissociation of presynaptic noradrenergic receptor changes from clinical
response to low dosage clorgyline treatment in depressed patients. Clin.
Pharm. Ther. (in press).
Linnoila, M., Roy, A., Lane, E., Virkkunen, M., Rudorfer, M., and Potter,
W.Z.: Characterization of noradrenergic state from norepinephrine and
its metabolites in patients with alcoholism, depression and disorders of
impulse control. In Dahlstrom, A. et al . (Eds.): Proceedings of the 6th
International Catecholamine Symposium. Alan R. Liss, New York, N.Y. , (in
press) .
Potter, W.Z., Rudorfer, M.V., Lesieur, P., Risby, E.D., and Linnoila, M.:
Biochemical effects of selective 5HT-reuptake inhibitors in man. In
Gastpar, M. (Ed.): Selective 5-HT Reuptake Inhibitors: Novel or Common
Place Agents. S. Karger, Basel (in press).
Potter, W.Z., Rudorfer, M.V., and Linnoila, M.: Effects of antidepressants
on NE and its metabolites in cerebrospinal fluid, plasma and urine. In
Proceedings of the 6th International Catecholamine Symposium. Dahlstrom, A.
et al. (Eds.), Alan R. Liss, New York, N.Y. (in press).
Risby, E.D., Hsiao, J.K., Sunderland, T. , Agren, H. , Rudorfer, M.V., and
Potter, W.Z.: The effects of antidepressants on the H\/A/5HIAA ratio. Clin.
Pharmacol . Ther. (in press).
306
ZOl MH 01855-03 LCS
Zametkin, A. J.: The effect of methyl pheni date upon urinary catecholamine
excretion in hyperactivity: A partial replication. Biol. Psychiatry, (in
press) .
307
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01860-01 LCS
PERIOD COVERED
October 1, 1986, through September 30, 1987
TITLE OF PROJECT (SO characters or lass. Title must tit on one line between the txrders.)
The Role of Epinephrine in Brain
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attillatlon)
Ivan N. Mefford, Ih.D.
COOPERATING UNITS (if any)
Section on Analytical Biochemistry and Section on Biomedical
Psychiatry, LCS, NIH; Laboratory of Clinical Studies, NIAAA
Laboratory of Clinical Science
SECTION
Section on Clinical Pharmacology
INSTITUTE AND LOCATION
NIH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
0.3
PROFESSIONAL:
0.3
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Epinephrine is the least prevalent catecholamine in mammalian brain,
localized in the most primitive and medial aspects. Traditional approaches
to studying epinephrine in brain have treated it primarily as a neurotrans-
mitter. Over the past two years our view has changed, to consider epine-
phrine primarily as an extraneuronal metabolite of norepinephrine, thus
functioning as hormone in forebrain. This has pronpted the study of the role
of epinephrine in sedation and intoxication and the inplementation of
equilibrium dialysis as a method for monitoring extracellular fluid (ECF)
epinephrine content. Inhibition of the epinephrine forming enzyme,
phenylethanolamine-N-methyl transferase (FNMT) produced a potent and long-
lasting antagonism of both barbiturate and ethanol intoxication in rats. ECF
epinephrine exceeds norepinephrine at baseline throughout the hypothalamus in
the anethestized rat, suggesting that epinephrine may act as the primary
agonist as extxa junctional alpha2 receptors in this part of the brain.
Monitoring CSF epinephrine in rhesus monkeys demonstrates marked increases in
epinephrine content during barbituratate infusion, consistent with this
hypothesis .
309
PHS 6040 (Rev. 1/84)
cpo •K.eit
ZOl MH 01860-01 LCS
Project Description:
The goal of this work is to understand and describe the metabolism and
physiological of endogenously formed epinephrine in the mammalian brain.
This project is being studied using several approaches.
Metabolism of epinephrine. Accumulated pharmacological data suggests
that enzymatic formation in the hypothalamus is dissociated from the storage
sites of epinephrine. Evidence suggests that this synthesis may occur in
non-neuronal elements. I am examining the possibility that this synthesis
occurs in glial cells, specifically astrocytes. This is being studied in
both astrocyte cultures and astrocytes isolated from adult rat brain. Future
studies will examine the properities of uptake of norepinephrine into these
cells.
Pharmacological manipulation of epinephrine synthesis . Considering
epinephrine as an extraneuronal metabolite of norepinephrine suggests that
the physiologically relevant pool of epinephrine is found in extracellular
space. This pool is actively modified by the release of norepinephrine from
selected neuronal populations, particularly the projections of the A]^ and A2
cell body groups. Epinephrine synthesis via FNMT should provide slow eleva-
tion of this pool, but a rather short time course for clearance following
enzyme inhibition. Numerous pharmacological manipulations of noradrenergic
release and reuptake and metabolism can be studied including MAO inhibitors,
uptake inhibitors, alpha2 receptor agonists and antagonists and inhibitors of
epinephrine synthesis. The effects of these manipulations can be measured in
extracellular fluid using equilibrium dialysis.
Methods:
Ecpjilibrium dialysis. Dialysis probes are prepared to provide optimum
recovery and regional selectivity. Dialysis tubing, 250 um in diameter is
used to prepare probes as described by (Zetterstrom, Sharp) and Ungerstedt.
Collected dialysates are analyzed for amines using microbore HPLC with
anperometric detection. Further selectivity is obtained by using IGEPON T-77
as a chromatographic modifier.
All other tissue, plasma and/or CSF analyses are accomplished using
published HPDC techniques.
Findings:
1. Extracellular fluid epinephrine exceeds norepinephrine in anesthetized
rat.
2. CSF epinephrine increased markedly in response to barbiturate
administration in awake, unanesthetized monkey.
310
ZOl MH 01860-01 LCS
3. Astrocytes cultured from foetal rat hypothalamus have epinephrine -
synthesizing capacity.
4. FiMI inhibition provides marked prophylaxis against ethanol or
barbiturate intoxication, but not anesthesia.
Significance;
Understanding the metabolism and functional significance of epinephrine
in mammalian brain may provide a great deal of insight into the mechanism of
action of several classes of drugs. If, as this research proposes, epine-
phrine is an extraneioronal metabolite of norepinephrine, any drug affecting
norepinephrine release, reuptake storage and metabolism would affect the
hormonal pool of epinephrine. It is proposed that one of the functions of
epinephrine in brain is tonic regulation of the level of arousal and reac-
tivity to sensory stimuli. Some evidence suggests that epinephrine synthesis
is iitportant in reward mechansims. Consequently, epinephrine synthesis may
be inportant in antidepressant efficacy. Our work, already coitpleted,
suggests an irtporatant role for epinephrine in intoxication and tolerance to
sedative hypnotics.
Proposed Course;
Test the hypothesis that hypothalamic epinephrine is an extraneuronal
metabolite of norepinephrine, primarily from A-^ and A2 projections, by;
A) Assessing the actions of intoxicants, anesthetics and sedative
hypnothics in awake unanesthesized animals on extracellular epinephrine,
norepinephrine, dopamine and serotonin.
B) Assessing effects of classical adrenergic drugs, anphetamine, cocaine,
tricyclies, neuroleptics and MAO inhibitors on extracellular epinephrine,
C) Studying synthesis of epinephrine and uptake of norepinephrine in non-
neuronal brain cells.
311
ZOl MH 01860-01 LCS
Publications
Mefford, I.N.: Distribution of epinephrine in mammalian brain. Clin.
Neurophannacol. 9(4): 177-179, 1986.
In Press
Canpbell, B.G., Bobker, D.H. , Mefford, I.N., and Weber, E. : Both the sigma-
receptor specific ligand (+)3-PPP and the PCP receptor-specific ligand TCP
act in mouse vas deferens via augmentation of electrically evoked
norepinephrine release. Eur. J. Riarnacol. (in press).
Mefford, I.N. : Are there epinephrine neurons in rat brain? Brain Res.
Reviews (in press) .
Mefford, I.N.: Ethanol and brain epinephrine. In Linnoila, M. (Ed.),
Moderator, Alcohol Intoxication and Withdrawal. Annals of Internal Medicine
(in press) .
Mefford, I.N. : Distribution of epinephrine in brain. Procr. in
Neuropsychopharmacol . and Biolocrical Psychiatry. 1987, (in press) .
312
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00787-08 LCS
PERIOD COVERED
October 1. 1986 through September 30. 1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line between the txjrders.)
Brain Mechanisms of Isolation Call 1n Squirrel Monkey (Saimiri sciureus!
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: P. D. Maclean Intramural Research Scientist LCS, NIMH
Other: J. D. Newman Research Physiologist LCE, NICHD
COOPERATING UNITS (If any)
Laboratory of Comparative Ethology, NICHn
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Comparative Studies of Brain and Behavior
INSTITUTE AND LOCATION
NIMH, NIH, PoolesvUle, Maryland 20837
TOTAL MAN- YEARS:
0.7
PROFESSIONAL:
0.4
OTHER:
0.3
CHECK APPROPRIATE BOX{ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues Q (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
There are indications that in the therapsid-mammal ian transition changes
in the auditory and vocal apparatus afforded audiovocal communication as a
means of maintaining maternal -of fspri ng contact and contact of members of a
group. The just completed present project is part of an investigation concerned
with identifying the cerebral representation of the separation cal 1 , a basic
mammalian vocalization that serves the above noted functions. For this purpose,
squirrel monkeys are tested for their ability to produce spontaneous calls in
isolation before and after ablations of different parts of the brain. The
present study has focused on the midline frontolimbic cortex, one of two cortical
areas where stimulation elicits vocalization in monkeys. Evidence derived by
the process of elimination indicates that the spontaneous calls depend on the
concerted action of a continuous band of rostral limbic cortex comprising parts
of areas 24, 2b, and j^. Ablation of the midii ne frontal neocortex peripheral
to this limbic zone is compatible with criterion performance in the production
of the call. The present report also includes ancillary observations in regard
to separation calls emitted while the subject is awakening from sodium pento-
barbital (Nembutal) anesthesia, as well as following the intraventricular
administration of oxytocin .
313
PHS 6040 (Rev 1/84)
CPO Bt 4-B1
ZUl MH 00787-08 LCS
Project Description:
Objectives: Since the mammal-like reptiles (the antecedents of mammals)
were probably egg laying, and since their auditory apparatus resembled that
of lizards, these and other considerations suggest that they may not have en-
gaged in parental care or audiovocal communication. In the therapsid-mammali an
transition, two small bones of the jaw joint of the mammal-like reptiles be-
came transformed into the malleus and incus of the highly tuned mammalian ear.
Present evidence indicates that the separation cry is universal among mammals,
serving initially to maintain maternal-offspring contact and then, later,
contact of members of a group. Hence the separation cry perhaps ranks as the
earliest and most basic mammalian vocalization. The present investigation is
concerned with identifying the cerebral representation of the separation cry,
using squirrel monkeys as subjects. The present phase of the work has focused
on the rostral midline frontolimbic cortex, one of two cortical areas in
monkeys where stimulation elicits vocalization.
Methods Employed: Subjects are squirrel monkeys two or more years of
aye and of either sex, belonging to the two main species characterized by the
ocular patch as "gothic" and "roman" and having distinctive separation calls.
The subjects are tested for their ability to produce spontaneous separation
calls before and after bilateral ablation of respective parts of the frontal
lobe. Since the monkeys are tested while isolated in a sound reducing chamber,
such experimentally induced vocalizations are referred to as isolation calls.
Criterion performance is the production of 20 or more calls during a period
of 15 min. The presence or absence of alterations in the pattern of the call
is demonstrated by spectrographic analysis.
Major Findings: The present phase of the study has been concluded with
the submission of a full length report for publication. The results may be
briefly summarized by reference to the accompanying Figs. 1 and 2. Figure lA
shows Rosabal's cytoarchitectural areal parcel lation. For facilitating com-
parison of lesions, the other figures show numbers of Rosabal's areas inserted
into millimeter squares conforming to the planes of the brain atlas. The cor-
tical area critical for the spontaneous call may be inferred by the process
of the elimination. Pregenual lobotomy or lobectomy (vertical line in Fig. IB,
subjects SC-8 and X-5), but not prefrontal lobectomy (SC-7), resulted in fail-
ure to produce spontaneous isolation calls. The results of the pregenual
lesions do not answer the question as to whether or not severance of connections
with the midline or lateral cortex rostral to the lesion or the midline cortex
caudal to the lesion accounts for the deficit. The findings in a fourth sub-
ject (shaded area in Fig. IC, subject R-5) indicated that the cortex essential
for the call is located on the medial frontal surface. A successive narrowing
down of the lesion indicated that the spontaneous call depends on the concerted
action of a continuous band of rostral midline limbic cortex comprising parts
of areas 24, 25, and 12 (Fig. 2B). Elimination of all the midline neocortex
peripheral to this zone (Fig. 2A) or lesions of parts of the limbic zone itself
(e.g. Fig. 2D) had no enduring effect on the production of the call.
314
ZOl MH 00787-08 LCS
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ZOl MH 00787-08 LCS
Ancillary Findings. Incidental to the present experiments, it has been
noted that monkeys emit separation calls upon awakening from sodium pentobar-
bital (Nembutal) anesthesia. In the case of rodent pups, it has been reported
that their ultrasonic separation calls may be induced by a decline in body
temperature upon their removal from the nest (Okon, 1972). Recordings have
been made on nine monkeys while awakening from pentobarbital anesthesia (P-084;
Z-5; SC-7; SC-11; 1979; 1976; 368-P; 1991; 1989). Seven of nine subjects pro-
duced typical separation calls while awakening that persisted for periods
ranging from 9 to 108 min. Peak calling ranged from 3.5 to 13 per min. For
these pilot observations, conditions did not permit the central of ambient
temperature. The findings in this small number of cases did not indicate
that calling was initiated by a decline in body temperature. For example,
one subject did not begin to call until its rectal temperature increased from
98° to 99°. It was observed in one case that one subject (1991) that emitted
no isolation calls under usual conditions achieved a peak rate of nine calls
per minute upon awakening from anesthesia. One subject (Z-5) in which mid-
line frontolimbic ablation virtually eliminated the isolation call, produced
calls at a rate of 3.5/min upon awakening from anesthesia. This finding sug-
gests that calling during recovery from pentobarbital anesthesia depends on
subcortical mechanisms, possibly at the midbrain level. Acetylcholine elicits
vocalization when applied to the central gray matter, and it is known that
acetylcholine is released during light stages of barbiturate anesthesia.
The adjuvant role of oxytocin in parturition and in milk secretion has long
been recognized. Apropos of the nursing situation and the maintenance of
maternal -of fspri ng contact, it is of special interest that in a recent experi-
ment performed with Gessa, the injection of 0.2 ug of oxytocin into the third
ventricle of a squirrel monkey resulted in the production of separation calls
that compared in rate and duration to what was observed in this same subject
while awakening from pentobarbital anesthesia. Parenthetically, this monkey
showed the manifestations of yawning, stretching, and penile erection that
Gessa and co-workers (1987) observed upon injecting oxytocin into the paraven-
tricular nucleus of the rat.
Significance to Biomedical Research and the Program of the Institute:
Neurologists have frequently commented upon the persisting ignorance of
specific cerebral mechanisms underlying laughing and crying. This lack of
information is of major significance because in an ethnographic sense, the
manifestations of crying and laughter would rank along with language as re-
flecting the evolution and status of the human condition. In preceding re-
ports on this project it has been pointed out how the experimental findings,
together with various clinical data, suggest that the evolution of the thala-
mocingulate division of the limbic system has partly involved the provision
of a neural substrate for laughing and crying, as well as a reciprocal inner-
vation of these two conditions. At the same time, attention has been called
to the relevance of the research on the separation cry to such mental health
problems as childhood separation anxiety, the "failure-to thrive" syndrome,
grief reactions, depression (including premenstrual and post-partum) , and
various forms of addiction, particularly the addiction possibly determined by
the high concentration of opiate receptors in the cingulate cortex. Functional
anatomical aspects of the study are considered in the accompanying related
project ZOl MH 00796-02 LCS.
316
ZOl MH 00787-08 LCS
Proposed Course: In view of the additional anatomical findings described
in accompanying project ZOl MH 00796-02 LCS, it will be important to test not
only the effects lesions of the various nuclei within the bounds of the inter-
nal medullary lamina, but also of the ventral anterior nuclei. The pilot
observations in regard to separation calls evoked by the administration of
pentobarbital deserve further investigation.
Publications:
Hotton, III, N., Maclean, P.O., Roth, J .J . , and Roth, E.C.:
The Ecology and Biology of Mammal -Like Reptiles. Washington,
Smithsonian Institution Press, 1986, 326 pp.
MacLean, P.O.: Brain evolution relating to family affiliations.
Social Science Information (Sur Les Sciences Sociales) (in press).
MacLean, P.O.: The triune brain. In Adelman, G. (Ed.):
Encyclopedia of Neuroscience. Cambridge, Birkhauser Boston,
Inc. , (in press) .
MacLean, P.D.: A rei nterpretation of memorative functions of the
limbic system. In Goldberg, E. (Ed.): Festschrift for Aleksandr
Romanovich Luria. New York, The IRBN Press, (in press).
MacLean, P.O.: Anokhin's operational architectonics with respect
to memory. In Sudakov, K. (Ed): Systems Research in Physiology,
New York, Gordon and Breach, (in press).
317
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT MUMBER
Z01 MH 00796-02 LCS
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Vtle must lit on one line between the borders.)
Cytochemical Tracing of Thalamic Connections with Midline Frontal Cortex
PRINCIPAL INVESTIGATOR (List other pmfessionel personnel below the Principal Investigator) (Nante, title, latxratory, and institute attiliation)
PI: P. D, HacLean Intramural Research Scientist LCS, NIMH
Other:
COOPERATING UNITS (it any)
UB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Comparative Studies of Brain and Behavior
INSTITUTE AND LOCATION
NIMH, NIH, Poolesville, Maryland 20837
TOTAL MAN- YEARS:
0.6
PROFESSIONAL:
0.3
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space
In a parent project on the cerebral
(see ZOl MH 00787-08 LCS), it was found t
cortex caudal to the polar area in squi rr
in the case of rhesus monkeys) result in
provided.)
representati
hat ablation
el monkeys d
di scernible
the th^alamus. The present project is employing cytoc
r
for obtaining clarification of this matter. Last yea
description of dive'rse thalamic nuclei, as well as ex
shown to be connected with different midline frontal
focuses on additional f inklings suggesting an orderly
parts of the ventral anterior nucleus to different mi
the light of assorted experimental and clinical evide
ulate division of the limbic system and the striopall
tions are implicated in crying and laughter, the anat
link-up of mechanisms implicated in both the affect a
manifestations.
on of the separation cal 1
of the midl ine frontal
id not (as was also known
retrograde degeneration in
hemi cal traci ng technique-s
r's report included a
trathalamic structures,
areas. The present report
projection of respective
dline frontal areas. In
nee that the thai amoci ng-
idonigral thai amic connec-
omical findings suggest a
nd expression of these
319
PHS 6040 (Rev. W84)
SPO 91 4-SII
ZOl MH 00796-02 LCS
Project Description:
Objecti ves : In a recently completed study on the role of the midline
frontolimbic cortex (see accompanyiny report ZOl MH 00787-08 LCS) it was found
in the squirrel monkeys used as subjects that except for the medial polar cor-
tex, midline frontal ablations resulted in no clearly discernible retrograde
degeneration in the medial dorsal nucleus or elsewhere in the thalamus. This
finding was in agreement with long known observations that such lesions failed
to produce such degeneration in rhesus monkeys. For example, in 1964 Akert
wrote, "The existence in rhesus monkey of athal ami c frontal areas is already
suggested by the work of Walker (1938, Fig. 39).... [F]rontal granular cortex
consists of two principal regions: one ( lateral -ventral ) which receives cen-
tral projections from the medial dorsal nucleus, and another (dorsal-medial)
which receives no essential projections from the thalamus and at most may be
supplied by sustaining ones." In the present project, cytochemical tracing
techniques are being employed to obtain clarification of this matter. Although
the findings described in last year's report are in general agreement with
what other workers have reported with respect to midline frontal connections
with diverse thalamic nuclei, there are certain gaps and inconsistencies,
particularly in regard to the ventral anterior nuclei, that, as will be ex-
plained, are especially important to resolve from the standpoint of clarifying
mechanisms accounting for crying and laughing in human beings.
Methods Employed: Adult squirrel monkeys representative of the two main
species (the so-called "gothic" and "roman" types) are used for these studies.
The findings to date have been obtained by employing a modification of a tech-
nique utilizing wheat germ agglutinin conjugated to horseradish peroxidase
(WGA-HRP). The findings are now to be supplemented by other cytochemical
tracing techniques that will help to resolve questions in regard to collateral
innervation of midline frontal areas. The results of exploratory first trials
with florescent dyes (fluorogold, rhodamine, fast blue) and silver protein
are not yet available.
Major Findings: This report focuses on additional findings relating to
the ventral anterior nuclei which include a lateral principal part (VApp) and
a medial magnocel lular part (VAmc). Generally speaking, the parayenual part
of the rostral cingulate cortex and of the subcallosal cortex is identified
with retrograde labeling of cells of VAmc that border upon the lateral part
of the mammil 1 othalamic tract and envelop the lower part of the tract, sug-
gesting the appearance of a bed nucleus. In the frontal plane (c. AP 9.5-AP
9) where the two tracts appear above the level of the third ventricle, label-
ing of cells extends into the medial part of the ventral lateral nucleus (VLm).
It is to be noted at this point that both VAmc and VLm receive afferents from
the substantia nigra, whereas the princpal part of VA receives projections
from the internal segment of the globus pallidus. With injections of WGA-HRP
further and further towards the frontal pole and then backwards in the neocor-
tex to the rostral supplementary area, the labeling in the VA complex appears
to extend further and further laterally as though, in a pictorial sense, one
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ZOl MH 00796-02 LCS
were opening up a fan. With injections involving midline parts of areas 8
and 6 overlying the supragenual cingulate cortex, the greater part of VApp is
labeled. WGA-HRP applied to these areas also results in labeling within the
oral part of the ventral lateral nucleus (VLo). This nucleus, it should be
noted, receives projections from the internal segment of the pallidum. The
anterograde labeling seen with WGA-HRP method suggests that the frontal areas
project back to the same nuclei from which they receive afferents.
Significance to Biomedical Research and the Program of the Institute:
As pointed out in last year's report, there is clinical evidence that the
striopal li do-thal ami c circuits are involved in crying and laughter. There
are also clinical indications that the thai amoci ngulate division of the
limbic system is implicated in these same manifestations. Since the frontal
lobes are known to play an important role in emotions and mood, the anatomical
findings afforded by recent techniques reveal connections by which there may
be a link-up between the stri opal 1 idoni gro-thal amic complex that would serve
as a substrate for both the affect and expression of crying and laughter. To
be sure, the only direct overlap of the thalamic connections of the two systems
in question are provided by VAmc and VLm. The nigra projects to both of
these nuclei, while the the pallidum has some projections to VLm. However,
there is evidence (Carmel ,1970) that VApp and VAmc are not only connected
with each other, but also with intralaminar and other thalamic nuclei.
The question arises as to how the cerebellum would participate in
mechanisms of crying and laughter, including the alternating waves of these
manifestations. The cerebellar projections to the thalamus are described as
having no overlap with the parts of VA and VL under consideration, nor is
there clear evidence of intrathal ami c connections (Asanuma et al . , 1983).
This is a question deserving further investigation, particularly in the light
of cerebellar symptoms that may develop in patients with lesions of the
premotor parts of the frontal lobes.
The anatomical findings in regard to VA help to clarify the observations
by Starzl and Magoun and by Hanberry and Jasper some 35 years ago that this
nucleus appeared to be central to a short-latency, diffuse projecting system
affecting predominantly the frontal cortex, but also accounting for widespread
cortical responses elsewhere. Hence the work of the present project is
relevant not only to mechanisms of crying and laughter, but also to global
functions of the frontal lobe, including functions depending on an integration
of past memory, memory of ongoing experience, and a "memory of the future."
Proposed Course: To be continued with addition of techniques for
demonstrating collateral innervation.
Publications:
Maclean, P.O.: The midline frontolimbic cortex in the evolution
of crying and laughter. In Perecman, E. (Ed.): The Frontal Lobes
Revisited. New York, The IRBN Press, 1987, pp. 121-140.
321
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Zni MH 00797-02 LCS
PERIOD COVERED
TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.)
Neurobiology of Attachment
PRINCIPAL INVESTIGATOR (Ust Other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: T. R. Insel Staff Physician LCS NIMH
Others
L. P. Miller
Guest Worker
LCS mi
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Clinical Science, NIMH
SECTION
Section on Comparative Studies of Brain and Behavior
INSTITUTE AND LOCATION
NIMH. NIH. Poolesville. Maryland 20837
TOTAL MAN-YEARS:
1.3
PROFESSIONAL:
OTHER:
Q^
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
n (a2) Interviews
D (b) Human tissues Exl (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This is the second year of this project which investigates attachment
and separation in infants and parents. Studies in rat pups have focused
on the ultrasonic isolation call as a behavioral measure of separation
distress. Following up on our earlier results demonstrating potent effects
of the benzodiazepines on ultrasonic isolation calls during this year, we
demonstrated increased occupancy of benzodiazepine receptors with in vivo
receptor autoradiography during separation. These new results suggest a
physiologic role for this receptor system in the separation response. We
are currently testing this hypothesis further using intraventricular
injecti ons of endogenous benzodiazepine ligands into 1 -week-old rat pups
to investiate effects on the rate of isolation calls.
Our continuing studies of parental behavior have demonstrated that
the brain is a target organ for the neuropeptide oxytocin and that oxytocin
receptors increase in selected limbic regions during the postpartum period.
As lesions in these regions slightly faci 1 i tate maternal behavior, oxytocin
may inhibit rather than increase neural activity at these sites.
323
PHS 6040 (Rev. 1/&4)
SPO SI4>>II
ZOl MH 00797-02 LCS
Project Description:
Qbjecti ves : We began this year with three objectives:
(1) To investigate a physiologic role for the benzodiazepine receptor
in the rat pup separation response.
(2) To determine the significance of the oxytocin receptor increase
in the postpartum period.
(3) To extend our oxytoci n-parental behavior studies to nonhuman
primates.
Methods Employed: The infant response to brief separations has been
investigated in rat pups from 1 to 14 days of age. Rat pups, when separated
at these ages, emit ultrasonic calls which can be detected, quantified, and
characterized using a computer-based sound spectrum analyzer. Our studies have
investigated the pharmacologic modification of these calls by testing pups
isolated for 2 minutes prior to subcutaneous drug administration, replacing the
pup in its litter for 30 minutes, and retesting during a second 2-minute isola-
tion period. In non-pharmacol ogic studies we investigated the influence of
several other factors such as temperature, age, and presence of littermates on
production of ultrasonic isolation calls.
In vi vo 1 abel i ng of benzodiazepine receptors involves injecting
3h-R0-15 1788 (3 Ci/pup) subcutaneously. Following injections, pups remain
with their littermates or are separated for various periods of time. At
20 minutes posti njections, pups are sacrificed, and brains immediately removed
and frozen. Frozen 20 sections are exposed to ^h sensitive film for 8 weeks.
The autoradiographic images are analyzed using a computer-based densitometric
system. Nonspecific binding can be determined by pretreatiny a subset of
pups with diazepam (b my/kg).
To determine the significance of the oxytocin increase in the postpartum,
we embarked on a series of lesion experiments in 15-day pregnant females.
Attempts at making cytotoxic lesions were not successfiil , probably due to the
proximity of the target to the ventricle. Electrolytic lesions were more
successful, using a radi of requency lesion maker with an electrode placed
stereotaxically into the bed nucleus of the stria terminalis. Maternal
behavior was scored on the day of delivery and for 3 days postpartum by
adapting a quantitative technique first published by Pedersen et al . (Science
215:648,1982).
Parental behavior in pygmy marmosets was monitored and scored as noted
in our previous annual report. Due to a decrease in the percentage of young
raised successfully by our breeding pairs, this project was postponed until
a more reliable baseline of parental behavior could be obtained.
324
Z01 m 00797-02 LCS
Major Findings:
(1) Rat pups separated for 25 minutes from their littermates show
a significant decrease in ^h-rQ-IS 1788 binding in frontal and cingulate
cortex but not in several other cortical and subcortical regions examined.
This decrease in binding probably results from release of an endogenous
ligand which displaces ^h-RQ-IB 1788 from the receptor.
(2) Lesions of the bed nucleus of the stria terminalis do not decrease
maternal behaviors and may, slightly increase some of the components of the
maternal response. Taken together with our behavioral evidence that anosmia
interacts with oxytocin's promaternal effects, these lesion data demonstrate
that oxytocin's role may be to inhibit cannibalism or aggression towards the
young as one part of a physiologic system of checks and balances.
(3) Pygmy marmosets remain a valuable research resource because of
their pattern of maternal and paternal behavior; however, our current
breeding colony will need considerable rehabilitation before invasive studies
can be initiated.
Significance to Biomedical Research and the Program of the Institute:
Although the past decade has seen an explosion of research in the neurobiology
of cognition, locanotion, and feeding, there has been a conspicuous absence of
research into the neural substrates of such primary social behaviors as mother-
infant attachment, pair-bonding, and affiliative behavior. This absence seems
particularly noticeable in mental health research where the inability "to love
and work" have long been recognized as a common feature of diverse forms of
psychopathology and early experiences of loss or isolation have been shown to
affect object relations in adulthood.
The demonstration that the same receptor which has been implicated in
the pharmacologic modulation of anxiety is also activated physiologically
during the infant's separation response provides the first biological evidence
for Freud's dictum that "...anxiety proves to be a product of the psychic
helplessness of the infant..." (1939).
Finally, this project has provided the first evidence for a morphologic
change in brain with parturition--the significance of this change to postpartum
mood and behavior remain to be assessed.
Proposed Course: The role of the benzodiazepine receptor in separation
distress is currently being investigated further using direct intracerebro-
ventricular injections of endogenous ligands into awake pups. In addition,
other aspects of the separation response such as corticosterone and
cardiovascular changes will need exploration in the coming months.
Maternal behavior continues to be an area of research excitement: the
role of oxytocin receptors in males, the duration of the postpartum increase
in brain oxytocin receptors, and the change in oxytocin content all need
investigation. We will be extending our lesion studies to oxytocin cell
325
ZOl m 00797-02 LCS
bodies and extending preliminary oxytocin immunohistochemical studies to an
oligo-deoxyribonuclease probe for oxytocin mRNA using in situ hybridization.
We hope to return to the pygmy marmosets for studies involving ICV
administration of oxytocin to determine if this peptide affects paternal
as well as maternal behavior. This research awaits a more stable period
of nonnative parental behavior in our colony.
Publ i cations:
Insel , T.R.: Postpartum increases in brain oxytocin binding.
Neuroendocrinology 44: 515-518, 1986.
Insel, T.R., Hill, J.L., Mayor, R.B.: Rat pup ultrasonic isolation
calls: Possible mediation by the benzodiazepine receptor complex.
Pharmacol. Biochem. Behav. 24: 1263-1267, 1986.
Insel, T.R. , and Hill, J.L.: Infant separation distress in genetically
fearful rats. Biol. Psychiatry 22:705-707, 1987.
Wamboldt, M.Z., and Insel, T.R.: The ability of oxytocin to induce
short latency maternal behavior is dependent on peripheral anosmia.
Behav. Neurosci. 101:439-441.
Insel, T.R.: The biology of parenthood. /\mer. Health (in press).
Insel, T.R., Miller, L.P., Gelhard, R.E., and Hill, J.L.: The neural
basis of the rat pup ultrasonic isolation calls. In Newman, .1.0. (Ed.):
The Physiologic Control of Mammalian Vocalization. New York, Plenum
Press, i n press.
Wamboldt, M.Z., Gelhard, R., Insel, T.R.: Gender differences in caring
for infant Cebuella Pygmaea, role of infant age and relatedness.
Dev. Psychobiol. (in press).
326
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
February 1. 1987 to September 30. 19R7
PROJECT NUMBER
ZOl MH 00798-01 LCS
TITLE OF PROJECT (80 characters or less. Title musf fit on one line between ttie txirders.)
Studies on the Development of the Cerebral Cortex
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfilietlon)
PI: B. B. Stanfield Special Expert LCS NIMH
COOPERATING UNITS (If any)
LAB/BRANCH
Laboratory of Clinir.al Sdenr.e, NIMH
SECTION
Section on Comparative Stiiriies nf Rrain anrl Rehavior
INSTITUTE AND LOCATION
NIMH, NIHAC PonlRc^vilip Maryland 9n».97
TOTAL MAN-YEARS
ns7
PROFESSIONAL
n.4n
n.i7
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues KIl (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project comprises ongoing studies previously supported extramurally which are being
continued in the LCS as well as studies initiated since the PI joined the LCS on February 1 ,
1987. This work on the development of the cerebral cortex which relies heavily on
neuroanatomical techniques focuses on the role of eliminatory events which occur during
normal brain development. Much of our effort has concentrated on the transient occipital
cortical component of the pyramidal tract which we previously identified. We have recently
found that in animals in which the occipital cortex receives an induced aberrant
somatosensory input through the lateral geniculate nucleus as a result of neonatal enucleation
and rostral cortical lesion, some occipital neurons will maintain a spinal projection. In
addition, we have recently identified axonal eliminatory phenomena which occur during the
development of the fornix, of the projections of the locus coeruieus. and of the major
ascending thalamic afferent systems. Finally, we have continued our studies of the
projections extended and maintained by heterotopic cortical transplants made during
development. All of our findings in these experiments point to cortical locale as a decisive
factor in determining which of the initially extended projections a cortical neuron will
maintain.
327
PHS 6040 (Rev. 1/84)
CPO B1 4-oie
Z01 MH 00798-01 LCS
Other Collaborative Professional Personnel Engaged on the Project:
D.D.M. O'Leary Assistant Professor Washington Univ. Sch. of Med., St. Louis, MO
Project Description:
Objectives: The overall goal of this project is to gain a better understanding of
the development of the cerebral cortex. We have concentrated our studies on the role
certain regressive or eliminatory phenomena play during normal cortical development.
During the past ten years or so it has become increasingly clear that these are critical in
shaping the projection patterns which are found in the adult cortex.
Methods Employed: The experiments completed or in progress can be grouped as
five separate studies. These will be described separately:
1.) In an effort to determine what normally brings about the removal of the
occipital pyramidal tract collaterals, we initiated a series of experiments involving
neonatal lesions. We first removed the normal definitive targets of the occipital
pyramidal tract neurons (which we had previously identified as the superior colliculus
and the basilar pontine nuclei) to test whether the occipital pyramidal tract neurons
would maintain their pyramidal tract collateral in the absence of their normal targets.
The superior colliculus can easily be removed in young pups by aspiration, but this is
not possible with the basilar pontine nuclei, not only because of their inaccessible
position at the base of the brain, but also because of their proximity to the pyramidal
tract itself which must be left intact in this experiment. Thus, the basilar pontine
nuclei were removed indirectly. This could be accomplished by lesioning the cerebellum
since this leads to a rapid and massive retrograde degeneration of the pontine nuclei.
After the animals had grown beyond the stage at which the transient occipital pyramidal
tract projection is normally eliminated, the retrogradely transported fluorescent
marker. Fast Blue was injected into the pyramidal decussation and the occipital cortex
was examined for the presence of any residual pyramidal tract neurons.
In a separate series of experiments we tested for the maintenance of occipital
pyramidal tract axons in animals which at birth had received bilateral enucleations as
well as lesions of the rostral one-half to two-thirds of the cerebral cortex. The
rationale for this paradigm stems from recent observations we had made in eyeless mice
(both congenitally eyeless and neonatally enucleated) with similar neonatal cortical
lesions. We had found that in these mice the lateral geniculate nucleus comes to be
innervated by medial lemniscal axons. We wondered if this would happen in these
enucleated and lesioned rats and, if so, whether this somatosensory input to visual cortex
could prevent the elimination of occipital pyramidal tract axons. Thus, when these
neonatally lesioned and enucleated rats matured, we injected either Fast Blue into the
pyramidal decussation or the anterograde tracer, WGA-HRP, into the dorsal column
nuclei, and in some cases we did both injections.
2.) The results of the work described above emphasized the importance of the
input relayed through the thalamus during development in determining the final
projection patterns of the cortex. Yet outside of the visual system, little is known
regarding the development of thalamic afferents. Thus, we have examined the
development of the major ascending afferents to the thalamus in fetal and postnatal rats
using TMB histochemistry following WGA-HRP injections into either the dorsal column
328
Z01 MH 00798-01 LCS
nuclei, the inferior coiliculus, or the deep cerebellar nuclei, to label fibers of the
medial lemniscus, the brachium of the inferior coiliculus or the brachium
conjunctivum, respectively.
3.) We have used both anterograde and retrograde tracing techniques to study the
development of the fornix in rats. We undertook this study since incidental observations
in material from an earlier study suggested that this primary efferent pathway of the
hippocampal formation may exhibit a major transient component during development.
4.) In order to explore whether the phenomenon of collateral elimination occurs
during the development of a brainstem nucleus, we injected Fast Blue into the spino-
medullary junction of rats at various ages and examined the distribution coeruleospinal
cells within the locus coeruleus.
5.) Our observation that the distribution of pyramidal tract neurons is
widespread during the first postnatal week and includes the occipital cortex, whereas no
pyramidal tract neurons are found in the adult occipital cortex, led us to the suggestion
that the differences seen in the projections of the various regions of the adult cortex are
not intrinsic to the neurons found in these regions. Consistent with this idea is our
subsequent finding that neurons within pieces of fetal occipital cortex which
transplanted to the rostral cortex of a newborn host are able to extend pyramidal tract
axons and maintain these beyond the age at which occipital pyramidal tract axons are
normally eliminated. In order to explore further the projections extended and
maintained by cortical neurons transplanted to a new cortical locale, we carried out
additional experiments, transplanting rostral cortex to an occipital locale as well as
occipital cortex to a rostral locale, and utilizing ^H-thymidine autoradiography to
identify the transplants and Fast Blue to examine the projections at different survival
times and to additional targets.
Major Findings: The major findings of the studies described above can be
summarized as follows:
1 .) We have found that the early removal of the definitive targets of the
transient occipital pyramidal tract neurons does not prevent the loss of these cells'
pyramidal tract axons. However, many occipital cortical neurons can maintain
pyramidal tract axons following neonatal enucleation and rostral cortical ablation.
Further, this procedure induces an aberrant innervation of the dorsal lateral geniculate
nucleus by medial lemniscal axons and the distribution and number of occipital neurons
which maintain a pyramidal tract axon seem related to the location and magnitude of the
induced lemniscal innervation of the lateral geniculate nucleus. These results not only
demonstrate that this normally transient projection can be maintained, but underline
the importance of the kind of thalamic input the cortex receives in influencing the
projections which that cortex will maintain.
2.) We have found that by the day of birth, each population of axons of the major
ascending afferents to the thalamus has already entered into and arborized within their
appropriate thalamic relay nucleus. The overall distribution of each ascending afferent
system, however, differs dramatically between young and mature rats. In neonatal rats,
a substantial proportion of axons extend beyond the thalamus and often enter the internal
capsule (many of these appear to bypass the thalamus altogether). In addition, axons
which enter into and arborize within their appropriate terminal fields in the thalamus,
frequently overshoot their targets and extend into adjoining thalamic nuclei. These early
329
Z01 MH 00798-01 LCS
overgrowths are all subsequently eliminated and the restricted adult distribution of each
afferent system is evident by P30. Taken together with similar observations on the
development of retinal fibers these results indicate that developmental overgrowths may
be a general feature of the development of the major thalamic afferent pathways.
3.) Our study of the fornix indicates that these axons reach the caudal
hypothalamus a day or two before birth. Before any fibers of the fornix can be identified
entering into their principal target, the mamillary nuclei, a prominent contingent of
fibers course past the mamillary complex. This postmamillary component continues to
grow into the midbrain and pontine tegmentum during the first postnatal week as the
projection into the mamillary nuclei is elaborated. During the second and third
postnatal weeks, the postmamillary component of the fornix becomes progressively
smaller until it is completely eliminated. The cells of origin of this transient
postmamillary component of the fornix are found within the subicular complex of the
hippocampal region. Most, if not all, of the cells of origin of the postmamillary
component of the fornix survive the period during which this projection is eliminated.
And recently, using a delayed double dye injection paradigm, we have shown that at least
some of the subicular cells which transiently extend axons beyond the mamillary bodies
maintain a projection to the mamillary bodies. Interestingly, the axons of the fornix
which enter and eventually arborize within the mamillary nuclei and are maintained in
the adult seem to arise during development as interstitial collaterals from parent fibers,
the distal portions of which are subsequently eliminated. Further, the fact that although
a postmamillary component of the fornix is not present in adult rats, such a pathway has
been described in other species, such as cats, suggests that interspecific variations in
projection pattern can result from the differential elaboration or elimination of an
initially quite similar pattern of connections.
4.) Our observations on the development of the locus coeruleus indicate that
coeruleospinal cells are present throughout the locus coeruleus just after birth, but are
confined to its ventral portion by the end of the fourth postnatal week. We have shown
that this change is not brought about by cell death, since neurons retrogradely labeled
through their spinal axon following a neonatal injection of tracer are still present in the
dorsal locus coeruleus even if the animal is not killed until the fourth postnatal week.
Thus, the dorsal coeruleospinal neurons in newborn rats do not die but rather lose their
spinal collateral. These results demonstrate that collateral elimination which we and
others have repeatedly shown to occur during cortical development, may be a more
generally occurring phenomenon than has previously been appreciated. Interestingly, in
the locus coeruleus, as in the cortex, collateral elimination may be largely responsible
for the spatial segregation of projection neuron populations which emerges during
development as the adult pattern.
5.) Our observations on heterotopic cortical transplants made during
development indicate that the projections which the cells in such transplants maintain
are appropriate for the locale of the transplant rather than for the transplant site of
cortical origin. This is true even though the transplanted tissue could be shown to
initially extend transient projections, like those of the adjacent host cortex, to sites
appropriate for its region of origin, but these were subsequently eliminated, while those
projections to sites appropriate for the new cortical locale were maintained. These
results are consistent with the notion that, at least as far as the connections they are
able to maintain are concerned, the various regions of the neocortex may not be as
distinct during development as might be thought. Rather, the distribution of cortical
projection neuron populations seen in the adult does not seem to be "preprogrammed"
330
Z01 MH 00798-01 LCS
but results through a process of collateral elimination which restricts initially
widespread distributions of projection neuron populations. Further, this restriction
can be influenced by factors extrinsic to the cortical neurons themselves, such as their
position within the tangential plane of the cortex.
Significance to Biomedical Research and to the Program of the Institute: Our
studies on the eliminatory events that occur during brain development have helped to
establish that these events constitute a major and widely present feature of the normal
development of the central nervous system. In addition, these studies have helped to
elucidate how such frankly regressive events may play critical roles in ensuring that
development results in the establishment of appropriate neuronal connections.
Our work on the transient occipital pyramidal tract projection and our studies
utilizing heterotopical cortical transplants suggest that the restriction of the initially
widespread distributions of cortical projection neuron populations through collateral
elimination allows the acquisition of regionally specific patterns of cortical projections
without the necessity of these being prespecified to individual neurons. That is,
individual neurons during development may be of a particular cortical projection neuron
phenotype, but need not be intrinsically specified for the specific target appropriate for
their position within the tangential plane of the cortex. In additon to limiting the amount
of cellular prespecification necessary for normal cortical development, such stratagems
introduce the potential for pliability and plasticity into the cortex during development
and possibly during phylogeny as well.
Proposed Course: During the following year our work will proceed along the
following lines:
We will continue and complete our studies of the projections extended and those
maintained by heterotopic cortical transplants made during development.
We will continue and complete our analysis of the maintenance of the occipital
pyramidal tract neurons and the medial lemniscal innervation of the lateral geniculate
nucleus in neonatally lesioned and enucleated rats.
We will continue our delayed double dye study of the maintained targets of
subicular neurons with transiently extend axons through the postmamillary fornix.
In order to explore what factors may be involved in the elimination of the
postmamillary component of the fornix in rats, we will initiate a study to examine the
fate of this projection in animals in which the mamillary bodies have undergone
transneuronal degeneration following an early lesion to the cingulate cortex.
We will initiate a study to examine the distribution of locus coeruleus neurons
with spinal projections and those with rostrally directed axons in tottering (tg/tg)
mutant mice in which a hyperinnervation of some locus coeruleus targets in the absence
of any increase in the number of locus coeruleus neurons has been reported.
331
Z01 MH 00798-01 LCS
Publications:
Chen, K.S., and Stanfield, B.B.: Evidence that selective collateral elimination
during postnatal development results in a restriction in the distribution of locus
coeruleus neurons which project to the spinal cord in rats. Brain Res. 410:
154-158, 1987.
Stanfield, B.B., Nahin, B.R., and O'Leary, D.D.M.: A transient postmamillary
component of the rat fornix during development: Implications for interspecific
differences in mature axonal projections. J. Neurosci. (in press).
332
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
February 1. 1987 to September 30. 1987
PROJECT NUMBER
ZOl MH 00799-01 LCS
TITLE OF PROJECT (80 characters or lass. Title must fit on one line between the borders.)
Studies on Postnatal Neuronoqenesis
PRINCIPAL INVESTIGATOR (List other protessional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and institute affiliaticn)
PI: B.B. Stanfield Special Expert LCS NIMH
Otiiers: T.R. Insel Staff Physician LCS NIMH
COOPERATING UNITS (if any)
Laboratory of Clinical Science. NIMH
SECTION
Section on Comparative Studies of Brain and Behavior
INSTITUTE AND LOCATION
NIMH. NIHAC. Poolesville. Maryland 20837
TOTAL MAN- YEARS:
Q.37
PROFESSIONAL:
0-27
0.10
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues U (c) Neither
n (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. !3o not exceed the space provided.)
This project comprises work previously supported extramurally which is being continued in
the LCS as well as studies initiated since the PI joined the LCS on February 1, 1987. These
studies have utilized ^H-thymidine autoradiography and neuroanatomical tract tracing
techniques to study aspects of the continuing neuronogenesis in the adult dentate gyrus. We have
found that dentate granule cells generated in the adult rat can extend axonal projections for
appreciable distances, and we have initiated a study in pygmy marmosets to see if new granule
cells are generated in an adult primate.
333
PHS 6040 (Rev. 1/84) OPO sw-Bit
Z01 MH 00799-01 LCS
Project Description:
Objectives: We iiave studied the continuing neuronogenesis which occurs in adult
rats in a limited segment of the cerebral cortex, the dentate gyrus. Until recently it was
believed that neuronogenesis in mammals is completed before, or immediately after,
birth, however, it is now clear that while this is true for the vast majority of neurons,
in the rat dentate gyrus the production of granule cells continues at a slow rate well into
adulthood and that cells generated in the adult may, in an older adult rat, account for
almost half of the neurons present in the dentate gyrus. We are interested in learning
more about this phenomenon with the eventual goal of understanding the mechanisms
which control this slow accretion of neurons.
Methods Employed: Previous ^H-thymidine autoradiographic studies in rodents
have shown that while the full complement of neurons in most brain regions is produced
prenatally, in a few sites, such as the dentate gyrus, the cerebellum and the olfactory
bulb, the bulk of neuronogenesis occurs in the immediate postnatal period. I^ore recent
evidence indicates that after this perinatal surge in neuron production, dentate granule
cells continue to be produced at a slow yet identifiable rate throughout most, if not all,
of a rat's life. In order to determine whether the cells which incorporate the
^H-thymidine in the adult rat dentate gyrus are in fact neurons which extend axonal
projections, we injected a series of animals with ^H-thymidine on postnatal day 100.
Four weeks later we injected the retrograde tracer. Fast Blue, into the mossy fiber
layer of the hippocampus, which contains the axons of the granule cells. After
processing the sections from these brains for autoradiography, we examined them under
bright- and dark-field illumination and fluorescence epi-illumination.
In order to determine if new granule cells are added to the dentate gyrus in adult
primates, we have recently initiated a study utilizing cell counts as well as
^H-thymidine autoradiography in the pygmy marmoset (Cebuella pygmaea). The pygmy
marmosets were chosen due to the small brain and body size (an average adult weighs
only about 150 g) and the relatively short period of time between birth and adulthood in
these animals.
Major Findings: When we examined the sections from the animals injected with
3H-thymidine on postnatal day 100, as expected we found an appreciable number of
^H-thymidine labeled cells in the granule cell layer of the dentate gyrus. Further,
many of these ^H-thymidine labeled cells were labeled with the retrograde tracer as
well. Thus, these ^H-thymidine labeled cells are neurons which were generated on
postnatal day 100 and which over the next thirty days extended an axon for a
considerable distance. This not only demonstrates the remarkable ability of axons to
grow and presumably to establish contacts within the normal adult neuropil, but also
indicates that within the dentate gyrus this is a normally occurring ongoing process
which results in the continuing addition of new neuronal elements to the hippocampal
circuitry.
Significance to Biomedical Research and to the Program of the Institute: These
studies on the continuing neuronogenesis in the adult dentate gyrus have helped to
establish that, in rodents at least, new neurons are generated in the adult, that these new
neuronal elements do not simply replace neurons which are lost and that they are able to
extend axonal processes and become integrated into the pre-existing circuitry of the
334
Z01 MH 00799-01 LCS
hippocampus. Further studies will hopefully help us to understand the role of this
continuing neuronogenesis in the function of the dentate gyrus and why, if new neurons
can be continuously added here, does this not occur throughout the brain.
Proposed Course: During the following year our worl< will proceed along the
following lines:
We will continue our study of axonal extension by adult generated dentate granule
cell and in addition use various time periods between the ^H-thymidine injection and the
tracer injection to establish a time course for this axonal extension. This will provide us
with information on the differentiation of these adult generated neurons which can then
be compared with the differentiation of neurons generated during the early development
of the dentate gyrus.
We will continue our study of the dentate gyrus of pygmy marmosets to determine
if ^H-thymidine will be incorporated by dentate granule neurons in adults of this
species and if the total number of dentate granule neurons changes significantly during
the lifetime of this primate.
Publications: None
335
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1,19^
PROJECT MUMBER
ZOl MH 02219-04 LCS
through September 30, 1987
TITLE OF PROJECT (80 characters or less. We must tit on one tine between the borders.)
Animal Models of Anxiety
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute ettiliatlon)
PI: T. R. Insel Staff Physician LCS, NIMH
COOPERATING UNITS (if any)
Laboratory of Comparative Ethology, NICHD; Addiction Research Center, NIDA,
NIDA, Baltimore, MD
LAB/BRANCH
Laboratory of Clinical Science, NIMH
SECTION
Section on Comparative Studies of Brain and Behavior
INSTITUTE AND LOCATION
IMH, NIH, Poolesville. Maryland 20837
TOTAL MAN- YEARS;
1.3
PROFESSIONAL;
0.5
OTHER;
0.8
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
□ (b) Hunnan tissues [3 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our approach to the neurobiology of anxiety continues to be developmental
with a focus on the role of early experience on the development of neurotrans-
mitter and neuropeptide systems. We tested a specific hypothesis: that early
stress activates brain corticotropin releasing factor (CRF) pathways and has
long-term effects on brain CRF receptors with mixed results. As part of this
project the development of CRF neurons, receptors, and second messenger was
determined in fetal and postnatal rat brain. Receptors were found to be
dramatically increased over adult levels soon after birth. Treating infants
with CRF had short-term effects on development, long-term effects on behavior,
but no lasting effects on brain CRF receptors. In related studies, neither
noradrenergic lesions nor the behavi oral paradigm of learned helplessness was
found to alter brain CRF receptors.
337
PHS 6040 (Rev 1/84)
ZOl MH 02219-04 LCS
Other Collaborative Professional Personnel Engaged on the Project:
E. DeSouza Section Chief ARC NIDA, Baltimore, MD
T. Minor Research Psychologist UCLA Brain Research Inst.
Project Description:
Objecti ves : This year was dedicated to testing a developmental hypothe-
sis for the dysregulation of the hypothal amic-pi tuitary-adrenal (HPA) axis
observed clinically with affective or certain anxiety disorders. It appears
that during development there are critical periods (probably coinciding with
the differentiation of neural elements) when ligands have organizational or
"positive programming" effects on their receptor fields. For instance,
postnatal administration of morphine to rat pups has been reported to induce
an i ncrease (not a decrease) in brain opiate receptors and to be associated
with analgesia in adulthood. As loss of a parent has been associated with
both affective and HPA axis dysregulation in adulthood, we reasoned that (a)
early loss would increase endogenous CRF and (b) increases in CRF at a critical
period in development would have long term effects on both behavior (response
to separation in adulthood) and endocrine regulation (hyper-secretion of CRF
with separation). The second, part of this hypothesis was tested this year.
Methods Employed: Our first task was to define a critical period in
development during which to intervene with exogenous CRF. We reasoned that the
period of maximal expression of CRF receptors would provide such a period, as
treatment during the normal overshoot of axons, dendrites, and cell bodies
might protect against the elimination of postsynaptic elements. We studied
CRF receptors by homogenate binding and in vitro receptor autoradiography in
brains from rat fetuses 15, 17, 19, and 21 days old and from postnatal days 2,
8, 14, 21, and 28. Using a 32P-cAMP assay, we also investigated the linkage
of the CRF receptor to its second messenger, adenylate cyclase. And, with a
synthetic ol igo-ribonuclease probe, we have used in situ hybridization to
investigate the anatomy of brain CRF mRNA.
Based on results from these ontogeny studies, rat pups were injected
with CRF (1 ug or 10 ug/day) from postnatal days 1 through 8. A subgroup of
pups were tested with single peripheral or central (ICV) injections of CRF at
day 8 to investigate acute behavioral effects. Outcome measures for adults
injected as pups were open field tests, corticosterone response to CRF (3 ug
or 10 ug/kg, IM), corticosterone response to separation (60 minutes), and
brain and pituitary CRF receptor number.
Finally to examine whether CRF penetrated the blood-brain barrier in rat
pups, the Oldendorf method was employed in 21 day old pups and in adults.
Major Findings:
1) CRF receptors appear in rat brain by fetal day 17, increase to more
than 300% over the adult level by postnatal day 8, and reach adult
levels by postnatal day 14. The cyclase generating complex itself
338
ZOl MH 02219-04 LCS
is relatively slow to mature - GTP , NaF, and forskolin (probes of
different components within the adenylate cyclase complex) were not
at their adult levels of effectiveness until postnatal day 14. CRF
message was expressed as early as fetal day 17, with surprisingly
high levels in the cortex.
2) After acute peripheral CRF injections in 8 day pups, a weak but sig-
nificant increase in corticosterone was observed but no change in
isolation calls was noted. Acute central injections were associated
with a significant, dose-dependent decrease in ultrasonic isolation
calls, with only a slight increase in corticosterone.
3) Chronic neonatal treatment with CRF was associated with early eye
opening, increased exploratory behavior in the open field, no altera-
tion in corticosterone response to CRF or separation, and an increase
in pituitary but not brain CRF receptors.
4) CRF administered peripherally does penetrate the blood brain barrier
of a 21 day old pup, but not in adult rats.
Significance to Biomedical Research and the Program of the Institute:
These studies provide a model with which to study how early experience might
reult in long term morphologic and behavioral effects. A careful chronologic
characterization of the anatomic and functional ontogeny will be important for
any such study to elucidate the rules by which ligands might influence the
survival of their receptors during the period of postnatal sculpting of brain
connections. In addition, the choice of neural system (CRF, benzodiazepine,
excitatory amino acid) for study, will require an understanding of which sys-
tems are activated during those events in development which have long term
consequences on behavior (see adjoining report on the Neural Basis of Separa-
tion and Attachment). Although our hypothesis regarding postnatal CRF, and
adult depression can now be rejected, our results provide evidence that (1)
early experience can alter behaviors relevant to exploration and (2) CRF
treatment early in life may induce CRF receptors in the pituitary.
Proposed Course: During the coming year we plan to follow several leads
from these studies. The ontogeny of second messengers in brain can now be
investigated using labelled forskolin and phorbol ester (for adenylate cyclase
and phosphoinositol proteins respectively). The role of excitatory amino acids
in development will also be investigated in the coming months - first by des-
cribing the ontogeny of NMDA, kainic acid, and quisqualate receptors in brain,
and then by treatments with excitatory amino acid antagonists (e.g. MK-801).
Publications :
Tamborska, E., Insel, T.R., and Marangos, P.: "Peripheral" and "central"
type benzodiazepine receptors in Maudsley rats. Eur. J. Pharmacol . 126:
281-287, 1985.
339
ZOl MH 0221^-04 LCS
Insel , T.R.: The neurobiol oyy of anxiety: A tale of two systems.
In Shaw, B.F., Segal, Z.R., Vallis, T.M., and Cashman, F.E. (Eds.):
Anxiety Disorders. New York, Plenum Press, 1987, pp. 35-51, 1987.
Insel, T.R., Lane, E.A., Sheinin, M., and Linnoila, M.: Acute and
chronic effects of desipramine administration to rhesus monkeys.
Eur. J. Pharmacol. 136: 63-68, 1987.
Insel, T.R.: The development of CRF, CRF receptors, and receptor linkage
to cyclase. In DeSouza, E. , and Nemeroff, C. (Eds.): Corticotropin
Releasing Factor. CRC Press (in press).
Marangos, P.J., Insel, T.R., Montgomery, P., and Tamborska, E.: Brain
adenosine receptors in Maudsley reactive and non-reactive rats. Brain
Res, (in press).
Wamboldt, M.Z., and Insel, T.R.: Pharmacologic models of anxiety. In
Handbook of Anxiety. New York, Pergamon Press (In press).
■i/(f)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00382-13 LCS
PERIOD COVERED
nrtnhpr 1. 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title rrtust fit on one line t>etween the txirders.)
Loca1i7fltion and nharacterization of Brain Neurochemicals
PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator) (Name, title, latioratory, and institute attiliation)
David M. Jacobowitz Chief, Histopharmacology Section
Jaime Kapitulnik Visiting Associate
LCS, NIMH
LMC, NCI
COOPERATING UNITS (it any)
Laboratory of Molecular Carcinogenesis, National Cancer Institute
Laboratory of Clinical Science
Histopharmacology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAL:
1.3
CHECK APPROPRIATE BOX{ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
(1) The presence of cytochrome P-450 in rat brain was studied by immunohisto-
chemistry. Immunoreactive nerves were observed only in brain sections incubated
with antiserum to 3-methylcholanthrene-induced cytochrome P-450. The most abundant
concentration of nerve fibers with cytochrome P-450 immunoreactivity was observed
in the globus pallidus. Immunoreactive fibers were also observed in the caudate-
putamen, amygdala, septum, ventromedial nucleus of the hypothalamus, medTal
forebrain bundle, ansa lenticularis, and ventromedial portion of the internal
capsule and crus cerebri. Cell bodies with cytochrome P-450 immunoreactivity were
observed in the caudate-putamen and in the perifornical area of the hypothalamus.
These results indicate that rat brain contains a form of cytochrome P-450 with
antigenic relatedness to the hepatic 3-methylcholanthrene-inducible cytochrome
P-450C. This cytochrome P-450 isozyme was detected in brain areas which metabolize
morphine and convert estradiol and estrone into catechol estrogens, which suggest
an important role for this enzyme in the metabolism of both exogenous and
endogenous compounds in brain.
(2) A detailed immunocytochemical study of the localization of neuropeptides,
enzymes and other neuroregulators in the dorsal tegmental region of the rat
brainstem was carried out. Of the neurochemicals screened, atrial natriuretic
factor (ANF), choline acetyl transferase (ChAT), cholecystokinin (CCK), calcitonin
gene-related peptide (CGRP), dynorphin B (Dyn B), galanin (GAL), somatostatin
(bOM), substance P (SP), neurotensin (NT), neuropeptide Y (NPY), vasopressin (VP),
vasoactive intestinal peptide (VIP), serotonin (5HT), gTutamic acid decarboxylase
(GAD), and tyrosine~fTydroxylase (TH) were studied. The multiplicity of neuro-
chemicals within this area suggests a possible influence on a variety of functions
modulated by the lateral dorsal tegmental nucleus and other closely associated
tegmental nuclei.
PHS 6040 (Rev. 1/84)
SPO SI4-9I*
ZOl MH 00382-13 LCS
Project Description
Objectives: 1) To study the presence of cytochrome P-450 in the rat
brain by immunohistochemistry,
2) To identify and localize a variety of neurochemicals within perikarya
and fibers of the laterodorsal tegmental nucleus (ntdl) which is a cluster of
cells located just medial to the locus coeruleus in the pontine brainstem.
Methods Employed: 1) Immunocytochemistry of cytochrome P-450. 2) Immuno-
cytochemistry of ANF, ChAT, CCK, CGRP, Dyn B, GAL, SOM, SP, NT, NPY, VP, VIP,
5HT, GAD, TH.
Major Findings:
1) The most abundant concentration of nerve fibers with cytochrome P-450
immunoreactivity was observed in the globus pallidus of both normal and
colchicine-treated rats. The caudate-putamen contained many extremely fine
varicose fibers. Fibers were also observed in the amygdala, septum,
ventromedial nucleus of the hypothalamus, medial forebrain bundle, ansa
lenticularis, and ventromedial portion of the internal capsule and crus
cerebri. Cell bodies with cytochrome P-450 immunoreactivity were observed in
the caudate-putamen and in the perifornical area of the hypothalamus.
2) We have described a detailed study of the localization of neuropep-
tides, enzymes and other neuroregulators in the dorsal tegmental region of
the rat brainstem.
Significance to Biomedical Research and the Program of the Institute:
1) The cytochrome P-450 isozyme was detected in brain areas which
metabolize morphine and convert estradiol and estrone into catecholestrogens,
which suggest an important role for this enzyme in the metabolism of both
exogenous and endogenous compounds in the brain.
2) The multiplicity of neurochemicals within this area suggest a
possible influence on a variety of functions modulated by the lateral dorsal
tegmental nucleus and other closely associated tegmental nuclei.
Proposed Course of the Project: 1) The possibility that cytochrome P-450
is involved in the MPTP toxicity in monkeys which results in the Parkinson
syndrome will be pursued. 2) Lesions of the lateral dorsal tegmental nucleus
will be performed. The influence of these lesions on "boxing behavior"
following prefrontal cortex injection of carbachol will be studied.
Publications:
Hamill, G.S., Skofitsch, G. and Jacobowitz, D.M.: Immunocytochemical local-
ization of atrial natriuretic factor, galanin and calcitonin gene-related
peptide within the rat interpeduncular nucleus. Brain Res. Bull. 17: 83-93,
1986.
342
ZOl MH 00382-13 LCS
Jacobowitz, D.M. and Skofitsch, G.: Calcitonin gene related peptide in the
central nervous system: Neuronal and receptor localization, biochemical
characterization and functional studies. In: Moody T. (ed.). Neural and
Endocrine Peptides and Receptors. Plenum Publishing Co., 1986, pp. 247-288.
Kapitulnik, J., Gelboin, H.V., Guengerich, P.P. and Jacobowitz, D.:
Immunohistochemical localization of cytochrome P-450 in rat brain.
Neuroscience 20: 829-834, 1987.
Millan, M.A., Jacobowitz, D.M., Hauger, R.L., Catt, K.J. and Aguilera, G.:
Distribution of corticotropin releasing factor (CRF) receptors in primate
brain. Proc. Natl. Acad. Sci_. USA 83: 1921-1925, 1986.
Zamir, N., Skofitsch, G. and Jacobowitz, D.M.: Distribution of immunoreactive
melanin-concentrating hormone in the central nervous system of the rat. Brain
Res. 373: 240-245, 1986.
343
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00388-11 LCS
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders.)
Coexistence of Peptides and Neurotransmitters
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
David M. Jacobowitz Chief, Histopharmacology Section LCS, NIMH
Toshio Ohhashi Guest Worker LCS, NIMH
COOPERATING UNITS (if any)
Laboratory of Clinical Science
Histopharmacology
INSTITUTE AND LOCATION
NIMH. ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
1.6
PROFESSIONAL:
1.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Immunohistochemical studies have revealed that calcitonin gene-related peptide
(CGRP) coexists with acetylcholine in motor cells of the spinal cord. Therefore
this study was undertaken to investigate a possible interaction between the
cholinergic nerve neurotransmitter and CGRP on neuromuscular transmission in the
isolated rat diaphragm. Electrical stimulation of the isolated phrenic nerve
resulted in twitch contractions which were dose-degendently potentiated by CGRP in
concentrations ranging from 1.2 X 10"^ M to 3 X 10 -^ M. The potentiating action of
CGRP (3 X lO"'' M) returned to control level in about 25 min and was rarely
tachyphylactic. The action of CGRP was dependent upon the stimulation pulse width
ranging from 0.2 to 1.0 msec. Rat calcitonin (4.5 X 10"'^ M) caused a minimal change
in the amplitude of twitch contractions. CGRP had no effect on the quiescent
striated muscle. Twitch responses to direct electrical stimulation was also
enhanced by CGRP (6 X lO"^ M - 6 X 10"^ M) in the absence and presence of 10"^ M
d-tubocurarine. These results suggest that CGRP modulates the action of
acetylcholine at the motor-end plates of striated muscle. The possibility that an
alteration in the normal peptide content in nerve endings (motor-end plates) of the
body may lead to a variety of muscle malfunctions is of great clinical signifi-
cance and should be studied in humans.
PH', f/j/lO <U>:, MM)
OPO B1 4-BI*
ZOl MH 00388-11 LCS
Project Description:
Objectives: To investigate a possible interaction between the cholinergic
nerve neurotransmitter acetylcholine and calcitonin gene-related peptide (CGRP)
on neuromuscular transmission in the isolated rat diaphragm.
Methods Employed: Electrical stimulation of the isolated phrenic nerve-
diaphragm preparation.
Major Findings:
(1) Electrical stimulation of the isolated phrenic nerve resulted in
twitch contractions which were dose-dependently potentiated by CGRP in
concentrations ranging from 1.2 X 10"^ M to 3 X 10"^ M. The potentiating action
of CGRP (3 X 10"^ M) returned to control level in about 25 minutes and was
rarely tachyphylactic.
(2) The action of CGRP was dependent upon the stimulation pulse width
ranging from 0.2 to 1.0 msec.
(3) CGRP had no effect on the quiescent striated muscle. Twitch responses
to direct electrical stimulation was also_enhanced by CGRP (6 X 10"^ M - 6 X
10''' M) in the absence and presence of 10"^ M d-tubocurarine.
Significance to Biomedical Research and the Program of the Institute: The
mo 1 0 r end plate in the diaphragm muscle is yet another example of the ever
increasing reports of neuronal sites containing classical neurotransmitters
coexisting with peptides. It would seem that acetylcholine and CGRP coreleased
could serve to interact cooperatively to result in a potentiation of the muscle
contraction. The demonstration that an increase in the pulse width results in a
progressive increase in the contractile response following phrenic nerve
stimulation suggests that the CGRP potentiating action may come into play when
there is a need for greater muscle contraction. In this way the modulatory
action of CGRP may serve to increase the capacity for the muscle contractile
response to acetylcholine. The present results suggest that CGRP modulates the
action of acetylcholine at the myoneural junction in striated muscle. This is
of great physiological and clinical significance.
Proposed Course: Immunocytochemical work on the possible coexistence of
CGRP and acetylcholinesterase (AChE), an enzyme present in motor-end plates
will be studied in a variety of striated muscles and species including the
human musculature.
Publications:
Crawley, J.N., Stivers, J. A. and Jacobowitz, D.M.: Neuropeptides modulate
carbachol-stimulated "boxing" behavior in the rat medial frontal cortex. In
Moody, T. (ed.). Neural and Endocrine Peptides and Receptors. Plenum Press,
1986", pp. 321-332.
Hamill, G.S., Skofitsch, G. and Jacobowitz, D.M.: Immunocytochemical local-
ization of atrial natriuretic factor, galanin and calcitonin gene-related
peptide within the rat interpeduncular nucleus. Brain Res. Bull . 17: 83-93,
1986.
346
ZOl MH 00388-11 LCS
Jacobowitz, D,M. and Skofitsch, G.: Calcitonin gene related peptide in the
central nervous system: Neuronal and receptor localization, biochemical
characterization and functional studies. In Moody, T. (ed.). Neural and
Endocrine Peptides and Receptors. Plenum Press, 1986, pp. 247-288.
Sills, M.A. and Jacobowitz, D.M.: Chronic administration of either nialamide or
desipramine decreases wet-dog shakes in rats produced by the TRH-analog
MK-771. Brain Res. 401: 195-199, 1987.
Skofitsch, G. and Jacobowitz, D.M.: Quantitative distribution of galanin-like
immunoreactivity in the rat central nervous system. Peptides 7: 609-613, 1986.
Skofitsch G., Sills, M.A. and Jacobowitz, D.M.: Autoradiographic distribution
of ^25j_ga-|api-p binding sites in the rat central nervous system. Peptides 7:
1029-1042, 1986.
Zamir, N., Skofitsch, G. and Jacobowitz, D.M.: Distribution of immunoreactive
melanin-concentrating hormone in the central nervous system of the rat. Brain
Res. 373: 240-245, 1986.
347
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
PROJECT NUMBER
ZOl MH 00396-09 LCS
October 1. 1986 t.n .Spptpmhpr ^n, iq«7
TITLE OF PROJECT (BO characters or less. Title must tit on one line between the borders )
A Study of Proteins Within t.hp r.N.S hy Twn-nimpn<;innal r:pi Fiprtrnphnrpcis
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
David M. Jacobowitz Chief, Histopharmacology Section LCS, NIMH
William E. Heydorn Pharmacologist FDA
Anne-Marie O'Carroll Visiting Fellow LCS, NIMH
Takemi Fukuda Guest Researcher LCS, NIMH
Lois Winsky Guest Researcher LCS, NIMH
Jitendra Patel Visting Associate BPB, NIMH
COOPERATING UNITS (if any)
Division of Neuropharmacological Drug Products, Food and Drug Administration
Laboratory of Clinical Science
Histopharmacology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20205
TOTAL MAN- YEARS:
3.4
PROFESSIONAL:
2.4
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects □ (b) Human tissues E (c) Neither
D (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The identification of interesting proteins within the CNS utilizing
2-dimensiorial gel electrophoresis (2DE) continues. 1) One of the largest proteins
on our brain gels, "protein 36" has been isolated and purified from homogenates
of rat brain. Protein 36 is a homodimer with a molecular weight of 64 kD and a
monomeric weight of 37 kD with a pi of 6.5. Immunocytochemical studies using a
rabbit antibody raised to this protein revealed that protein 36 is localized in
large pyramidal cells, dendrites and axons of layer V of the cerebral cortex. 2)
A second protein ("protein 94") has been isolated from the bovine brain and
purified. This is a cAMP-stimulated phosphoprotein (M.W. 94 kD) which was
originally identified in the rat neostriatum. Purification was monitored by
autoradiography of ^^P-phosphorylated samples separated by SDS-PAGE. Immunocyto-
chemical studies revealed tjji^t protein 94 was localized in astrocytes. 3|^We
have detected at least 3 Ca -binding proteins in the rat cortex by ""^Ca
autoradiography. Two proteins were identifie^^as calmodulin and the B subunit of
"calcineurin. In addition, an unidentified Ca -binding profein of molecular
weight about 18 kD and pi 5.4 was revealed. 4) Seven high affinity calcium
binding proteins have been detected following ammonium sulfate precipitation.
Three of the proteins have been identified (calmodulin, B subunit of calcineurin
and vitamin D-dependent calcium binding protein). The identity of the other 4
proteins are unknown. 5) We have studied phosphoproteins present in seven
discrete microdissected brain areas. Marked regional differences in the pattern
of phosphorylation among the brain areas studied under basal conditions and
fol lowing activation of cyclic AMP-dependent protein kinase and calcium/
calmodul in-dependent protein kinase and pr"otein kinase C were noted. 6) A series
ot studies examining proteins within auditory nuclei are under progress. Results
indicate several differences in proteins visible on two-dimensional gels across
auditory nuclei or more generally between sensory nuclei and other brain areas.
PHS 6040 (Rev. 1/84) GPO si 4-9IB
ZOl MH 00396-09 LCS
Project Description:
Objectives: 1) To isolate and purify a protein ("protein 36") that exists
in high concentration on our brain gels. 2) To isolate and purify the 94 kD
ph^iphoprotein and raise antibodies to th^^protein. 3) Identification of
Ca -binding proteins using 2-DE and "^^Ca -autoradiography. 4) To study
phosphoproteins in discrete regions of the brain using two-dimensional gel
electrophoresis. 5) To study and compare proteins within different nuclei of
the auditory system of rabbits and to determine whether Pavlovian conditioning
training or daily exposure to auditory and pupillary stimulation produce any
change in either the content or phosphorylation of proteins within auditory or
other relevant nuclei in rabbit brain.
Methods Employed: 1) Two-dimensional gel electrophoresis; 2) Silver
staining of proteins on polyacrylamide gels; 3) Electrophoretic transfer of
proteins to nitrocellulose paper and subsequ^tjit identification of proteins by
use ^1 specific antisera; 4) Detection of Ca -binding proteins by
"^^Ca -autoradiography; 5) Autofluorography of radiolabelled proteins. 6)
NH4)2S04 fractionation; 7) Biochemical separation techniques - ammonium sulfate
precipitation; ion-exchange and gel filtration chromatography; 8) Immunization
of rabbits for the production of antiserum; 9) Immunocytochemical methods; 10)
Radiolabel phosphate (from ATP) incorporation into proteins from microdissected
brain tissue; 11) Microdissection of discrete regions of the rat and rabbit
brain; 12) Classical conditioning of the nicitating membrane response of
rabbits.
Major Findings:
(A) We have isolated and purified a soluble protein designated "protein 36"
which is found in the rat brain as one of the larger protein spots appearing on
the 2DE gels. Protein 36 is a homodimer with a molecular weight of 64 kD and a
monomeric weight of 37 kD with a pi of 6,5, Immunocytochemical studies using a
rabbit antibody raised to this protein revealed that protein 36 is localized in
large pyramidal cells, dendrites and axons of layer V of the cerebral cortex.
The hippocampus contained cells in the stratum radiata and processes in the
stratum pyramidalis. A variety of cell types were also observed in the globus
pallidus, thalamus and hypothalamus,
(B) Subcellular fractionation studies have shown the 94 kD protein to be
localized predominantly to the cytosolic fraction. The pl value of the
phosphorylated form of the denatured form of the protein was found to be
approximately 4.7 while that of the unphosphorylated form of the protein was
found to be approximately 3.8 by isoelectric focusing column chromatography.
Production of a rabbit antiserum to the protein allowed immunohistochemical
localization studies to be carried out. In the rat and monkey cortex grey and
white matter astrocyte-1 ike cells were observed. In the rat cerebellum,
Bergmann fibers and cell bodies were observed in the molecular layer. In
addition, processes which enveloped the purkinje cells were also seen.
(C) At least three Ca -binding proteins were detected in rat cortex by "^^Ca -
autoradiography of two-dimensional electrophoretograms. The identities of two
of these Ca -binding proteins were determined to be calmodulin and the B
subunit of calcineurin. The identification was based upon the following
350
ZOl MH 00396-09 LCS
criteria: 1) comigration of polyacryl amide gels with the appropriate purified
proteins, 2) staining of nitrocellulose blots with sg^cific antisera for
calmodulin and calcineurin and 3) ability to bind Ca . This information is
useful in that it identifies two major brain proteins visible on silver-stained
two-dimensional polyacrylamide^^els. In addition, this data reveals the
location of an unidentified Ca -binding protein of molecular weight 'v^ 18,000
daltons and pi 5.4 on these gels.
(D) A total of seven high-affinity calcium-binding proteins have been detected
in rat brain. Of these seven proteins, three are detectable in a crude tissue
punch of rat cortex while four are seen only after protein enrichment with
ammonium sulfate. Three of the seven proteins detected in this study have been
identified: calmodulin, the B subunit of calcineurin and the type II intestinal
vitamin D-dependent calcium-binding protein. A fourth protein, soluble in a
saturated solution of ammonium sulfate, is probably one fo the subunits of the
S-100 protein knowiji^to bind calcium. The identities of the other four proteins
visualized by '+5Ca -autoradiography in this study are unknown. These results
demonstrate that rat brain contains a number of high-affinity calcium-binding
proteins. However, to consistently detect a number of these proteins, enrich-
ment using differential protein solubility in ammonium sulfate is necessary.
(E) Phosphoproteins present in seven discrete microdissected brain areas have
been studied using a combination of the micropunch technique, two-dimensional
gel electrophoresis and autoradiography. Under basal conditions (no exogenous
protein kinase activating factors added), about 40 discrete phosphoproteins
were visualized over a pH rang of 4.8-7.1 and a molecular weight range of
100,000-10,000 daltons. Approximately twelve of these labeled proteins
correspond to silver-stained proteins visible on two-dimensional electrophoreto-
grams of brain tissue. Three distinct regional differences in the basal pattern
of protein phosphorylation were noted among the seven brain areas studied.
Addition of calcium plus calmodulin to the incubation mixture markedly
increased the phosphorylation of three acidic proteins in all seven brain areas
studied. Activation of cyclic AMP-dependent protein kinase caused a qualitative
increase in the degree of phosphorylation in all seven brain areas, with over
fifty total proteins being labeled. Among the most prominent proteins visible
after activation of cyclic AMP-dependent protein kinase were two poorly
focusing proteins of molecular weight 94,000 and 83,000 daltons. Based upon
physicochemical characteristics, a number of phosphoproteins labeled in this
study have been identified. These results demonstrate that there are marked
regional differences in the pattern of protein phosphorylation among micro-
dissected areas of the rat brain. In addition, these data identify which
silver-stained proteins visualized on two-dimensional electrophoretograms of
brain tissue are phosphoproteins.
(F) A protein which appears to be specifically localized within several
auditory and some other sensory nuclei was not seen in hippocampus, cortex,
cerebellum or facial motor nucleus but was present in large amounts in the
cochlear nucleus, inferior colliculus and trigeminal sensory nuclei. Lesions of
the auditory nerve may decrease the content of this protein in cochlear nucleus.
Another protein was detected which appeared to be specifically localized within
the lateral superior olive. In vitro studies indicated marked variations in the
degree to which phosphorylation was stimulated by the addition of 8-bromo
351
ZOl MH 00396-09 LCS
cyclic AMP or calcium plus calmodulin. For example, in the cochlear nucleus,
only decreases in the phosphorylation of a few proteins were seen with the
addition of the cyclic AMP analog. In contrast, increased radiolabel phosphate
incorporation into protein was seen under this condition in the inferior
colliculus.
Significance to Biomedical Research and the Program of the Institute: While
many proteins remain to be identified certain important strides have been made
in this direction. The information contained in this report will provide other
workers in the field with a basis for reference and comparison. This is likely
to accelerate the process of protein identification on 2DE gels and to enhance
the value of this technique in the study of CNS proteins.
A major purpose of our investigation of proteins has been to establish
immunochemically defined markers for neural cells and other cell structures.
Underlying this approach has been the basic premise that antibodies can be
valuable tools for studying a wide variety of biological systems including the
nervous system. Measurement in blood and cerebrospinal fluid may provide new
tools for diagnosis and monitoring of neurological diseases in addition to
further immunohistochemical studies of pathological nervous tissue.
Astrocytes (glial) cells constitute a large fraction of the volume of the
mammalian brain cortex. Much work needs to be done to unravel the multiple
roles of astrocytes in brain function. The development of an antibody is
significant in advancing our knowledge of astrocyte morphology and function in
the brain.
Relatively little is known regarding the biochemistry of pharmacology of
the auditory system. The results described in this report represent preliminary
findings of a comprehensive study of auditory proteins and their phosphorylation.
The identification of sensory specific proteins indicate the occurrence of
unique biochemical events which may be functionally related to sensory process-
ing. In addition, the examination of brain tissue from animals receiving
auditory stimulation and/or Pavlovian conditioning may provide insights as to
how a stimulus is coded within the brain and the degree to which this coding is
dependent on its relevance to other environmental events.
Proposed Course of the Project: Future efforts will be directed at both identi-
fying and learning more about the novel calcium-binding proteins and phospho-
proteins. In addition, isolation and purification of unknown proteins will
continue. We will set up cell cultures of astrocytes and study various types
(fibrous, protoplasmic, oligodendrocytes) that react specifically with our
astrocyte specific antibody.
Publications:
Fukuda, T. and Jacobowitz, D.M.: Purification and immunocytochemical detection
of a protein that reveals layer V pyramidal cells in the rat cortex.. Brain
Res. , in press.
Heydorn, W.E., Creed, G.J., Creveling, C.R. and Jacobowitz, D.M.: Studies on
catechol -0-methyl transferase in rat brain using two-dimensional gel electro-
phoresis. Neurochemistry International 8: 581-586, 1986.
352
ZOl MH 00396-09 LCS
Heydorn, W.E., Creed, G.J., Nguyen, K.Q. and Jacobowitz, D.M.: Effect of
5,7-dihydroxytryptamine on the concentration of individual proteins in
different areas of the rat brain. Brain Res. , 368: 193-196, 1986.
Heydorn, W.E., Creed, G.J., Patel , J. and Jacobowitz, D.M.: Distribution of
proteins in different subcellular fractions of rat brain studied by two-
dimensional gel electrophoresis. Neurochemistry International 9: 357-370, 1986,
Heydorn, W.E., Gierschik, P., Creed, G.J., Milligan, G., Spiegel, A. and
Jacobowitz, D.M.: The B subunit of the guanine nucleotide regulatory proteins:
Identification on two-dimension gels of brain tissue, existence of multiple
charge forms and its regional and subcellular distribution in brain. J.
Neurosci. Res. 16: 541-552, 1986.
Heydorn, W.E., Creed, G.J. and Jacobowitz, D.M.: Observations and implications
on the migration of calmodulin in a 2 dimensional gel system. Electrophoresis
8: 251-252, 1987.
Narayan, R.K., Heydorn, W.E., Creed, G.J, and Jacobowitz, D.M,: Protein
patterns in various malignant human tumors by two-dimensional gel electro-
phoresis. Cancer Res. 46: 4685-4694, 1986.
Narayan, R.K., Heydorn, W.E., Creed, G.J., Kornblith, P.L. and Jacobowitz, D.
M.: Two-dimensional gel electrophoretic protein patterns in high grade human
astrocytomas. In Walker, M.D. and Thomas, D.G.T. (eds.) Biology of Brain
Tumour. The Netherlands, Martinus Nijhoff Publishers, 1986, pp. 7-14.
Rodriguez-Sierra, J. P., Heydorn, W.E., Creed, G.J. and Jacobowitz, D.M.:
Isolation of specific proteins affected by estradiol in the arcuate-median
eminence of prepuberal female rats. Brain Res. 399: 379-382, 1986.
Rodriguez-Sierra, J.F., Jacobowitz, D.M, and Blake, C.A.: Effects of neuropep-
tide Y on LH, FSH and TSH release in male rats. Peptides 8: 539-542, 1987.
Rodriguez-Sierra, J.F., Heydorn, W.E., Creed, G.J. and Jacobowitz, D.M.:
Incorporattion of amino acids into proteins of the hypothalamus of prepuberal
female rats after estradiol treatment. Neuroendocrinology, in press.
Youdim, M.B.H., Sills, M.A., Heydorn, W.E., Creed, G.J. and Jacobowitz, D.M.:
Iron-deficiency alters discrete proteins in rat caudate nucleus and nucleus
accumbens. J. Neurochem. 47: 794-799, 1986.
353
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00397-09 LCS
PERIOD COVERED
October 1, 1986 to September 30. 1987
TITLE OF PROJECT (80 charactors or less. Title must lit on one line tMtween the borders.)
Autoimmune Aspects of Disease
PRINCIPAL INVESTIGATOR (List other professional personnel tielow the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
James S. Frazier Staff Fellow LCS, NIMH
Hiroyasu Nakata Visiting Scientist LCS, NIMH
David M. Jacobowitz Chief, Histopharmacology Section LCS, NIMH
COOPERATING'UNITS (if any)
Laboratory of Clinical Science
Histopharmacology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.3
PROFESSIONAL:
1.1
CHECK APPROPRIATE BOX{ES)
D (a) Human subjects E (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Autoimmunity has been implicated in an increasing number of diseases
including psychiatric disease. Using 2D gel electrophoresis and Western immuno-
blotting techniques, the study of CNS proteins continue as we look for specific
proteins which might be target antigens for autoantibodies of pathologic origin.
First, we have found that there exists a number of autoantibodies in the sera of
noraml animals and humans that react with brain cortical proteins. These include
antibodies to: the cytoskeletal proteins actin, tubulin and GFAP; the enzymes
sGOT and neuron specific enolase; and the B-subunit of the signal transducing G
protein. Second, we have found there to be no obvious differences between the
autoantibodies seen in normals and those seen in schizophrenics. Third, we have
documented the presence in a small percentage of Type I diabetics of an antibody
to a 25,000 MW pancreatic specific protein. Fourth, we have purified and
partially characterized this protein. And fifth, have raised a rabbit antibody
that was shown by immunocytochemistry to specifically bind to islet cells of the
monkey pancreas.
355
PHS 6040 (Rev. 1/84)
QPO SM-ait
ZOl MH 00397-09 LCS
Project Description
Objectives: 1) To study the autoimmune aspects of diseases which might
directly or indirectly affect the brain. 2) To characterize the "normal"
immune state such that comparisons between normal processes and disease
processes might be made. 3) To screen sera from schizophrenic to other
psychiatric patients for disease specific autoantibodies. 4) Once target
antigens for potentially pathogenic antibodies have been identified, to
purify, charaterize and study this protein in detail.
Methods Employed:
1) Two-dimensional gel electrophoresis. 2) Silver staining of proteins
on gels. 3) Electrophoretic transfer of proteins to nitrocellulose paper and
subsequent immunoblotting with sera or CSF. 4) Ul tracentrifugation, ammonium
sulfate fractionation, cation exchange chromatography. 5) Antibody formation
via rabbit immunization. 6) Fluorescence microscopy.
Major Findings:
1. There exists in normal sera a number of autoantibodies with the capacity
to react with CNS proteins. One to fifty dilutions of sera incubated
with 2D Western immunoblots of cortical proteins revealed that 100% of 8
sera tested had an antibody to Actin, 75% to Tubulin, 75% to GFAP, 88%
to sGOT, 100% to NSE and 63% to the e subunit of G proteins. Antibodies
remained reactive out to 1 to 500 dilutions, there existed a number of
immunoreactive proteins yet to be identified.
2. Schizophrenic sera (n=21) contained antibodies with reactivity no
different from control, when sera samples were tested against cortical
proteins at a 1:50 dilution.
3. Two out of 30 Type I diabetic sera contained an antibody to a 25,000
dalton human pancreatic specific membrane-bound tetraisomeric protein
with pi values of approximately 7.0-8.0. No controls (n=12) contained
this antibody. Positive diabetic sera showed reactivity out to a
dilution of 1:2500, while controls were negative at 1:10 dilution.
Positive sera was negative when tested against human liver, adrenal,
thyroid, salivary, kidney and brain. Positive sera remained positive
when reacted against human pancreas from six donors of differing age and
sex. Positive sera was positive against immunoblots of cultured human
islet cells, but negative against rat insulinoma cells.
4. When separated by ammonium sulfate fractionation and cation exchange
chromatography, the purified pancreatic protein was injected into a
rabbit. The resultant antibody was found to have immunochemical
reactivity at a 1:3000 dilution with the islets of Langerhans of the
monkey pancreas while pre-immune sera was negative. Interestingly, the
sera showed immunoreactivity for the juxta glomerular apparatus of the
rat kidney.
356
ZOl MH 00397-09 LCS
Significance to Biomedical Research and the Program of the Institute:
The finding that there are sera of normal control autoantibodies that bind
proteins, including neuron-specific proteins suggests that alterations of the
blood-brain barrier may make possible the entrance of the antibodies into the
brain and thereby possibly potentiate, if not initiate various psychiatric and
neurologic sequela. The inability to find schizophrenic specific antibodies may
mean that autoimmunity is not a component of schizophrenia, or it may mean that
non-cortical areas of the brain need to be examined. Also, sera and CSF samples
taken during varying phases of altered cognitive states might need to be
examined.
The finding of a Type 1 diabetic specific antibody and the character-
ization of it's target protein may provide valuable insights into the
pathogenesis of this disease. More importantly, finding this antibody lends
credibility to the use of 2D gel electrophoresis and Western immunoblotting
techniques for the identification of potentially pathologic antibodies.
Further refinement of these techniques for studies of CNS immunopathology may
help define more clearly the role of autoimmunity in psychiatric disease.
Proposed Course of the Project: Further work will focus on attempts to
obtain a sequence of the diabetic protein antigen in order to learn whether
or not we are dealing with a known protein. Further work with the antiserum
will be undertaken to do immunocytochemical studies on human pancreas and
other organs (e.g., kidney).
Publications:
None
357
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
PROJECT NUMBER
ZOIMH 02377-01 LCS
nctnhpr 1. 1986 tn SpptPtnhpr '^n^ 1Q«7
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
A Stlidy of Arlpnn<;inp Rprpntnr-;- TsolfltJ
soiation and Characterization
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Hiroyasu Nakata Visiting Scientist LCS, NIMH
David M. Jacobowitz Chief, Histopharmacology Section LCS, NIMH
COOPERATING UNITS (If any)
Laboratory of Clinical Science
Histopharmacology
INSTITUTE AND LOCATION
NIMH. ARflMHA. Bethesda.
Maryland 2*0892
^
TOTAL MAN-YEARS;
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Isolation of A2 adenosine receptors from rat brain membranes have been
performed. The purification methods included solubilization by digitonin and
column chromatographies using Q-Sepharose, hydroxylapatite, Green A-agarose and
Mono Q. Approximately 1000-fold purification was achieved at the last stage of
purification and the specific binding activity of the final preparation was '^
100 pmol/mg protein assayed using [^HlN-ethylcarboxamideadenosine (NECA) as a
ligand. The apparent molecular weight of the partially purified A2 adenosine
receptors was estimated to be approximately 80,000 by HPLC (TSK 4000PW-5000PW)
experiments.
It was also found that A2 adenosine receptors could be separated from Aj
adenosine receptors or other unknown adenosine binding sites by hydroxylapatite
chromatography.
Mouse mastocytoma P815 cell membranes were demonstrated to have A2
adenosine binding sites in a relatively high concentration. These adenosine
binding sites were also solubilized by detergents without any significant
changes of ligand binding properties.
359
GPo si«-sia
ZOl MH 02377-01 LCS
Project Description:
Objectives: 1) To isolate and characterize the biochemical properties of
adenosine receptors from rat brain membranes. 2) To identify and classify
adenosine receptors of mouse mastocytoma P815 cell membranes.
Methods Employed:
1) Ultracentrifugations for subfractionation of rat brain membranes and
for isolation of solubilized receptor fractions. 2) Column chromatographies of
ion-exchange resins, hydroxyl apatite, or dye-coupled agarose gel. 3) FPLC and
HPLC. 4) Radiolabeled ligand binding assay using cell harvester. 5) One- and
two-dimensional polyacrylamide gel electrophoresis. 6) Intraperitoneal
injection of mastocytoma P815 cells to DBA/2 mice to grow the cells and
collect the ascitic fluid to harvest the grown cells.
Major Findings:
(A) Approximately 1000-fold purification of adenosine receptors which
showed A2-type pharmacology was obtained after several purification steps,
i.e., solubilization with digitonin. Fast Q- Sepharose chromatrography,
hydroxyl apatite chromatography and Mono Q chromatography. The specific binding
activity of the final receptor preparation assayed using [^HjNECA (20 nM) as a
ligand was approximately 100 pmol/mg of protein. The apparent molecular weight
of this partially purified A2 adenosine receptor was calculated to be approxi-
mately 80,000 by HPLC (TSK 5000PW-4000PW, tandem-linked columns) experiments.
(B) Ai and A2 adenosine receptors could be separated from each other by
hydroxylapatite chromatography. When solubilized preparations from rat brain
which contained both Aj and A2 adenosine receptors was applied to a
hydroxylapatite column and the column was eluted with a gradient of potassium
phosphate, three major peaks which had adenosine binding activity were found
(designated Peak A, Peak B and Peak C by the order of elution). Peak C was A2
adenosine receptors and Peak A was A^ adenosine receptors judging from their
ligand binding pharmacology. Peak B was an unknown adenosine binding protein
which should be examined further.
(C) Adenosine binding sites which showed A2-type pharmacology were
identified in membranes of mouse mastocytoma P815 cells. The dissociation
constant (Kd) was 380 nM and the maximum specific binding was 20 pmol/mg when
assayed at 0°C using [^HjNECA as a ligand. The rank order of potency for
inhibition of [^HjNECA binding was NECA > N-cyclopropylcarboxamide adenosine >
2-chloroadenosine > isobutylmethylxanthine > phenyl isopropyladenosine, which
was a typical pharmacology for A2-adenosine receptors. These adenosine binding
sites were solubilized by either sodium cholate or digitonin. The solubilized
binding sites retained the same adenosine binding characteristics as those of
membrane-bound form. The apparent molecular weight of the adenosine binding
sites solubilized with digitonin was estimated to be approximately 300,000 by
gel filtration experiments.
360
ZOl MH 02377-01 LCS
Significance to Biomedical Research and the Program of the Institute:
Adenosine and its stable analogs have pronounced physiological effects on
various tissues including nervous tissue. These include modulation of adenylate
cyclase, inhibition of both nerve cell firing and neurotransmitter release in
vivo and in vitro and a sedative action thought to be centrally mediated. Most
of these actions are thought to be mediated via the cell surface receptor to
adenosine. Therefore, it is important to characterize the biochemical
properties of the adenosine receptor to understand the function of adenosine
which has significant pharmacological effects. These adenosine receptors are
usually classified as Aj and A2. A^ adenosine receptors are linked to
inhibition of adenylate cyclase whereas A2 adenosine receptors are linked to
activation of adenylate cyclase. Although it is important to isolate these
receptors and study their biochemical functions, very little biochemical work,
especially on A2 adenosine receptors, has been done so far. As an initial step
toward complete understanding of biochemical properties of adenosine receptors,
A2-adenosine receptors were solubilized and partially purified up to 1000-fold
using rat brain membranes as starting materials. During the purification steps,
it was also found that Ai, A2 and the other unknown adenosine binding proteins
could be separated from each other on hydroxylapatite chromatography. This
finding will be useful as a convenient method for separation of Ai and A2
adenosine receptors from crude mixtures.
It is very important to find a good cell culture system for studying
adenosine receptor functions in living cells. For that purpose, several
cultured cells were screened. Mouse mastocytoma P815 cells which can be easily
grown in both cell culture systems and in ascities fluid of DBA/2 mice have
been known to secrete histamine and 5-hydroxytryptophan and such secretions are
likely to be affected by adenosine suggesting the presence of adenosine
receptors. From the ligand binding studies, it was demonstrated that P815 cell
membranes had a high concentration of adenosine binding sites which showed A2
type pharmacology. A^ type adenosine binding sites were not identified. These
results demonstrated that the mastocytoma P815 cell will be a good cell line
for the study of functions of A2-adenosine receptors in culture systems.
Proposed Course: Isolation and purification studies will continue.
Publications:
None
361
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
PROJECT NUMBER
ZOl MH 02239-03 LCS
nrtnhPr 1. 1986 to Sppt.pmhPr 31, 1Q«7
w^ "^ — — ■ ■-* ■■ ■ --— — — r "^iiinr ^ I k'^*^-* * .^ V I
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
r.nnceptual Analysis of Complex BiobphavJoral Population Systems
jRiNnPAL INVESTIGATOR (List other orofessional oersonnel below the Pnnr.injtl Ini/nKtinatnr ) {Nama titia lahf^rotnm ,
PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Pnncipal Investigator) (Name, title, laboratory, and institute affiliation)
John B. Calhoun Chief URBS LCS NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Clinical Science
SECTION
Section on Clinical Neuropharmacology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3.0
PROFESSIONAL:
1.0
OTHER:
2.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues (3 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been terminated due to the retirement of the principal
investigator.
363
PHS 6040 (Rev. 1/84)
SPO 81 4-S1I
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 tn September
PROJECT NUMBER
ZOl MH 00163-10 CHP
30, 1987
TITLE OF PROJECT (80 characters or less- Title must fit on one line between the borders.)
Treatment of Obsessional Children and Adolescentr, with Clomipramine
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Judith L. Rapoport, M.D., Chief, CHP, NIMH
Henrietta Leonard, M.D., NRSA Fellow, CHP, NIMH
Dennis L. Murphy, M.D., Chief, LCS, NIMH
Theodore Zdhn, Ph.D., Research Psychologist, LPP, NIMH
Agnes Whitaker, M.D., Columbia University
Susan Swedo, M.D., Staff Fellow, CHP, NIMH
Martha Denckla, M.D., Chief, Autism & Behav. Dis. Sec, DNB, NINCDS
Robert Freiland, M.D., LN, NIA
COOPERATING UNITS (// any)
Laboratory of Psychology and Psychopatho] ogy, NIMH; Clinical Neuropharmacology
Branch, NIMH; National Institute of Neurological & Communicative Disorders &
Stroke; Columbia University
Child Psychiatry Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland
TOTAL MAN-YEARS:
3.8
PROFESSIONAL:
2.3
1.5
CHECK APPROPRIATE BOX(ES)
[xl (a) Human subjects
H (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Obses.'".ive Compulsive Disorder (OCD) i.s now recognized as a common mental
disorder, occurring in perhaps three million people in this country alone. In
adolescents, the prevalence maybe as high as one percent.
Follow-up studies in our Brancli show a chronic course for the disorder with
increasing severity of depression and anxiety over time. Clomipramine has a
highly specific beneficial effect on obsessions while Desmethyl imipramine (DMI)
another tricyclic antidepressant did not differ from placebo.
New studies with brain imaging (PET and CT scan), as well as a survey of
patients with Sydenham's chorea, implicate abnormalities in the basal ganglia
while spinal fluid studies show that low concentration of CSF 5-HIAA, the major
serotonin metabolite, is associated with more severe OCD.
365
PHS 6040 (Rev. 1/84)
GPO SI4-Sie
ZOl MH 00153-010 CHP
Project Description:
Objectives: To examine the biological correlates, treatment, and natural history
of Obsessive Compulsive Disorder (OCD) in childhood and adolescence.
Methods Employed: A comparison of clomipramine (CMI) and desmethyl imipramine
(DMI) treatments of adolescents with Obsessive Compulsive Disorder is ongoing.
After a baseline observation week, there are two weeks of single blind
administration of placebo followed by double blind crossover administration of
clomipramine or desmethyl imipramine, each for five weeks. To date, 25 children
and adolescents have completed this protocol. Most have agreed to a lumbar
puncture during the baseline procedure.
Spinal fluid samples are being sent for immunoglobulin, as well as CSF
monoamines and metabolites, to compare with controls and relate to clinical
measures .
A survey of adolescents who had recently had Sydenham's chorea or Rheumatic
fever alone without accompanying Sydenham's chorea, has been initiated. Subjects
were surveyed with the Leyton Obsessional Inventory-Child Version that had been
developed for epidemiological purposes by our group. Those scoring above 20 were
interviewed for possible Obsessive Compulsive Disorder.
CT scans and PET scans of obsessive compulsive patients and age/sex-matched
controls are being examined. Of particular interest are ventricle size, caudate
size on the CT scans and levels of glucose utilization in the basal ganglia,
cingulate gyrus, and frontal lobes.
Major Findings: Drug Treatment Trial: There was a dramatic improvement in
obsessions and compulsions on CMI compared with DMI. This was striking for all
measures of severity, and for global functioning as well. DMI appeared to have a
mild antidepressant effect when given first, hut when it followed CMI there was
an increase in global depression ratings probably secondary to demoralization
upon return of obsessive symptoms.
CSF Monoamines: Preliminary analysis of the first 17 CSF samples for
monoamines and metabolites were carried out in the Laboratory of Clinical Science
(Dr. William Potter, Chief). There was a highly significant correlation between
5-HIAA and HVA (r^.83) CSF 5-HIAA was negatively correlated with baseline
severity; that is , the most severely ill subjects had a lower CSF 5-HIAA (r=-.73
p ■•' .01). Unlike previous reports however, we had no relationship between
baseline CSF values and drug response for these subjects.
Sydenham's chorea: Because of spontaneous outbreaks of Rheumatic fever and
in some cases, Sydenham's chorea in 1986, we were able to investigate whether
this infectious disorder which selectively affect? the basal ganglia would be
associated with obsessive compulsive symptomatology, as clinical reports from the
1940's had suggested. To date, a total of 11 adolescents with Rheumatic
366
ZOl MH 00153-10 CHP
fever and 11 with Sydenham's chorea have completed the Leyton Obsessional
Interview. The results show that there is a significantly higher interference
score regarding Sydenham's patients than by those with the Rheumatic fever.
Every subject with a score of above 20 was interviewed clinically. There were
two such subjects in the Sydenham's group and both had clinical OCD; none in the
Rheumatic fever group had such elevated scores and so none were interviewed.
This is a startling finding as our previous epidemiological data showed an
incidence of less than one percent in the general population compared with 20
percent in our children with Sydenham's chorea.
Brain Imaging studies: CT scans: To date, 10 male obsessive compulsives
and 10 controls have been compared with volumetric analysis of basal ganglia and
other brain studies. There was a decrease in the caudate volume for the
obsessive subjects compared with controls. A total of 17 PET scans have been
completed in adult obsessive compulsive patients, but results are not yet
available.
Significance to Mental Health Research: Childhood Obsessive Compulsive Disorder
occurs in up to 1% of adolescents. There has been virtually no research on this
disabling disorder, in spite of this prevalence. These studies address the
etiology, treatment, and clinical course of the disorder which will have broad
implications for the neurobiology of anxiety and of stereotyped behaviors.
Proposed Course of Project: A follow-up study of the 20 adolescents identified
in the Columbia University epidemiological study is underway. This will show the
natural course of the disorder in a community population. The drug treatment
study, brain imaging studies, and surveys of subjects with Sydenham's chorea will
be continued to enlarge the samples. Finally, a prospective treatment study of
30 obsessive compulsive adolescents who participated in the CMI/DMI comparison,
will be initiated to see if behavioral and pharmacological treatment affect long
term (two years) outcome.
Publications
Fldment, M., Rapoport, J.L., Murphy, D., Berg, C, Lake, C: Biochemical changes
during clomipramine treatment of childhood obsessive compulsive disorder. Arch.
Gen. PsYchi-^try 44: 219-225, 1987.
Berg, C, Behar, D., Zahn, T. and Rapoport, J.L.: Obsessive Compulsive
Disorder-An Anxiety Syndrome? In Gittelman, R. (Ed.): Anxiety Disorders. New
York, Guilford Press, 1987, pp. 126-139.
Rapoport, J.L.: Treatment of Obsessive compulsive adolescents with clomipramine
and desmethylimipramine; A double-blind crossover study. Psychopharmacol . Bull.,
in press.
367
ZOl MH 00153-010 CHP
Leonard, H. and Rapoport, J.L.: Relief of obsessive compulsive symptoms by LSD
and psilocin in a 17-year old obsessive compulsive boy. Am. J. Psychiatry, in
press.
368
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00161-09 CHP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Behavioral Effects of Dietary Substances in Normal and Hyperactive Children
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atliliation)
Markus Kruesi, M.D., Senior Staff Fellow, CHP, NIMH
Martine F]ament, M.D., Guest Researcher, CHP, NIMH
Marian Yarrow, Ph.D., LDP, NIMH
Carolyn Zahn-Waxler, M.D., LDP, NIMH
Thomas Uhde, M.D., BPB, NIMH
COOPERATING UNITS (if any)
Laboratory of Developmental Psychology, NIMH
Biological Psychiatry Branch, NIMH
Child Psychiatry Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland
20892
TOTAL MAN- YEARS:
.75
PROFESSIONAL;
50
.2!
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects
H (a1) Minors
n {a2) Interviews
n (b) Hunnan tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Relationships between aggression, activity, sugar, and aspartame were
investigated in 30 preschool boys: 18 had histories of behavioral reactions to
sugar and 12 were familiar peers whose parents reported their children were not
sugar reactive. Analysis of week long diet diaries found no significant
differences in sugar or carbohydrate consumption between the two groups nor were
correlations reported previously by another group between weekly sugar
consumption and observed activity and aggression replicated. Furthermore,
aggression did not increase in response in blinded sugar challenge. A
significant statistical but negligible decrease in actometer activity was
observed following aspartame. The likelihood of behavioral reactions to
aspartame seem questionable.
369
PHS B04n (Rev 1/81)
ZOl MH 00161-09 CHP
Project Description:
Obiectives : The relationship between dieldiy substances and behavior in normal
and disturbed children were investigated in a series of studies; responses to
sugar and aspartame were examined. Pathological responses such as aggressivity
to sugar and anxiety to caffeine were tested in high risk populations.
Methods Employed: Thirty preschool boys (18 alleged sugar reactors and 12
familiar peers without such a history) received glucose (1.75 gm;kg), sucrose
(1.75 gm/kg), aspartame (30 mg/kg) and a saccharin sweetened control both at home
and in an NIMH playroom. The dependent measures for the NIMH sessions were
videotaped observations of behavior, behavioral rating scales, and actometer
measured activity. Challenges were also administered in a random order double
blind manner at home. Week-long diet diaries were kept by parent(s).
Major Findings: This was the first study to investigate an etiologic role for
sugar ingestion in aggression. A previous study by other investigators had
reported correlations between sugar intake and aggressive-destructive, as well as
hyperactive behaviors and subsequently spawned intense interest in sugar's effect
upon behavior. That set of correlations was not replicated. On none of the
dependent measures was an effect attributable to sugar seen. Although children
reported to be reactive to sugar were more hyperactive than their peers at
baseline, there was no evidence for differential reactivity to sugar.
Significance to Mental Health Research: This is the first trial to investigate
an etiologic role for sugar in aggression. In addition, this study failed to
replicate correlations between habitual sugar consumption and observed behavior
that instigated much research in this area. Questions had arisen concerning
possible adverse behavioral consequences of aspartame consumption by children
either by altering central serotonin or via some unknown mechanism. This study
utilized a dose estimated to be at the 90th percentile consumption for aspartame
and did not find any behavioral effects. These results should direct research
efforts focus upon other pathogenic influences, and reassure public concerns.
Proposed Course of Project: This project has been terminated.
Publications :
Kruesi, M.J. P. and Rapoport, J.L.: Diet diid human behavior: How much do they
affect each other? Annu. Rev. Nutr . 6: 113-130, 1986.
Kruesi, M.J. P.; Carbohydrate intake and children's behavior. Food. Technol. 40:
150-152, 1986.
370
ZOl MH 00161-09 CHP
Kruesi, M.J. P., Rapoport, J.L., Berg, C, Stables, G. and Bou, E.: 7-day
carbohydrate and other nutrient intakes of preschool boys alleged to be behavior
responsive to sugar intake and their peers. In Essman, W. (Ed.): Nutritions and
Brain Function. Basel, S. Karger, 1987, pp. 133-137.
Zahn, T., Rapoport, J.L.: Acute autonomic nervous system effects of caffeine in
boys and men. Psychopharmacology 91: 40-44, 1987.
Kruesi, M.J. P., Rapoport, J.L., Cummings, E.M., Berg, C, Israond, D.I., Flament,
M., Yarrow, M. and Zahn-Waxler, C: Sugar and aspartame: Aggression and activity.
Am. J. Psychiatry, in press.
371
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZO] Mil 00178-06 CHP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO charactars or his. Title must lit on one line between rfts tyorders.)
Brain Structure and Function in Developmental Neuropsychiatr ic Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atliliation)
Judith M. Rumsey, Ph.D., Staff Fellow, CHP, NIMK
Susan Swedo, M.D., Staff Fellow, CHP, NIMH
Connie Duncan, Ph.D., Staff Fellow, LPP, NIMH
Richard Coppola, Ph.D., LPP, NIMH
Daniel Weinberger, M.D., Chief, CEDE, NIMH
Martha Denckla, M.D., Chief, Autism & Behav. Dis. Sec, DNB, NINCDS
Michael Goldberg, M.D., LSR, NEI
COOPERATING UNITS (il any)
Laboratory of Psychology and Psychopathology, NIMH; Sec. on Autism & Behav. Dis.,
DNB, NINCDS; Clinical Brain Disorders Branch, NIMH; Lab. of Sensorimotor
Research, NEI
LAB/BRANCH
Child Psychiatry Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892-
TOTAL MAN-YEARS:
3.1
PROFESSIONAL
1.80
CHECK APPROPRIATE BOX(ES)
m (a) Human subjects
S (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Regional cerebral blood flow data, using a xenon inhalation technique, shows
an increased hemispheric asymmetry and reduced antero-poster ior difference in
severe and persistent dyslexia. Analysis of EEC spectra, event-related
potentials^ and neuropsychological testing is in progress. Preliminary eye
movement data has been collected on adults with poor reading and adults with
attention deficit disorder, residual type (ADD-RT) to see if saccadic intrusions
and related variables differentiate these groups. Tasks were designed for use in
PET studies of dyslexic, ADD-RT, and autistic subjects. These include a
phonological and a syntactic task designed to activate left perisvlvian cortex,
environmental sounds, timbre and tonal memory tasks designed to activate right
temporal regions, attentional tasks designed to activate frontal cortex, and
affective and attentional-shif t tasks designed to tap into deficits associated
with autism.
The sample of autistic men previously studied with PET and the
fluorodeoxyglucose technique was increased, and a correlational pattern analysis
°f glucose metabolic data completed. Main findings were fewer large positive
correlations between frontal and parietal regions and lower correlations of
thalamus, caudate, lenticular nucleus, and insula with frontal and parietal
regions in autistic patients, relative to controls.
373
PHS 6040 (R«v. 1/84)
CPO tl4-*ll
ZOl MH 00178-06 CHP
Project Description:
Objectives : These studies have as their primary goal the identification of
neuroanatornical, neurophysiological, and neuropsychological abnormalities which
characterize the developmental disorders of dyslexia and infantile autism. The
role of attentional dysfunction in these disorders and the relationship between
dyslexia and attention deficit disorder, which show a high incidence of clinicai
overlap, are also of interest. A secondary goal is the determination of the
sensitivity and specificity of various imaging techniques, with particular
reference to developmental disorders.
Methods Employed: Methods include PET using f luorodeoxyglucose to study energy
metabolism and using ( 0-15) -labelled water to study regional cerebral blood flow
with neuropsychological activations, infrared oculography for recording eye
movements, xenon inhalation techniques for measuring cortical blood flow,
electrophysiology, neuropsychological testing, and behavioral questionnaires.
Major Findings: Regional cerebral blood flow studies of severely dyslexic men
using xenon inhalation techniques with various activation tasks have demonstrated
increased hemispheric asymmetries (including increased flow to the left
hemisphere) and reduced antero-poster ior gradients in these men, relative to
controls. The former suggests that there may be less efficient information
processing or inadequate bihemispher ic integration, rather than the decreased
ability tc activate left language cortex that has been hypothesized to be
involved in dyslexia. The reduced antero-poster ior difference may reflect a
deficit in the ability of frontal systems to respond adequately to cognitive
demands. This finding parallels the reduced antero-poster ior differences in
glucose metabolism seen in adults with ADD-RT reported by Zametkin, et al (1986).
Taken together, the two studies suggest the possibility of some common substrate
for these two frequently overlapping disorders.
Neuropsychological studies indicate that deficits in verbal learning
associated with severe dyslexia involve not only immediate memory span, but also
deficits in application of semantic strategies, which normally facilitate recall.
Correlational analyses of PET-FDG data on 14 autistic men and their controls
show fewer large positive correlations between frontal and parietal regions and
lower correlations of the thalamus, caudate, lenticular nucleus, and insula with
fronta] and parietal regions in autistic patients, with many of the latter
correlations negative in the autistic group that are positive in controls. These
results may indicate functionally impaired interactions between frontal/parietal
regions and neostriatum and thalamus, regions which subserve directed attention.
374
ZOl MH 00178-06 CHP
Eye movement data has been collected using infrared oculography in small
numbers of subjective poor readers (i.e. individuals who complain of slow
reading, but who do well on standardized tests), and adults with attention
deficit disorder, residual type and in 15 normal controls. Additional data
collection and quantification of existing data is underway to determine the
incidence and nature of saccadic intrusions in these groups. Regressions while
reading appear to distinguish subjective poor readers from the other groups.
Thus, despite their good performance on standardized reading measures, eye
movement data document the legitimacy of their reading complaints.
Significance to Mental Health Research: Application of these techniques holds
promise for understanding developmental disorders, the role attentional
dysfunction plays in them, and their relationship to other child psychiatric
disorders, such as attention deficit disorder. This research may prove useful
for understanding brain dysfunction in disorders where macroscopic brain anatomy
appears normal. Such dysfunction may involve widely distributed neural systems,
rather than mimicking the more circumscribed localization seen in acquired
disorders involving focal lesions. These studies may also aid in the development
of a more meaningful and biologically-based nosology. The eye movement data may
highlight a large and previously understudied group of "subjective poor readers".
This work will differentiate them from dyslexic subjects and possibly suggest new
treatment strategies.
Proposed Course of Project: A protocol for further study of regional cerebral
blood flow using neuropsychological activations in dyslexia, as well as in ADD-RT
and infantile autism, residual state, will be activated with PET. Nine tasks
have been developed for pretesting and are being audiotaped. These tasks will be
pretested behaviorally in normal controls and in small samples of the various
patient groups for their sensitivity to processing deficits and will be pretested
phsiologically with PET for the ability to differentially activate brain regions
of interest in these disorders. In dyslexia and ADD-RT, we are interested in
activating left anterior and posterior language cortex and prefrontal cortex with
attentional, phonological and/or syntactic tasks. Tasks involving environmental
sounds, tonal memory, and timbre will be used to activate right temporal cortex
as a contrast region. These two disorders will be deconfounded in this study.
Because autistic patients frequently show expressionless faces and flat
intonation, tasks involving the perception and expression of affective intonation
are also being pretested. A task involving the shifting of attention will also
be pretested in this group.
Once task selection is completed, we will proceed with our PET study of
dyslexia and attention deficit disorder, while completing additional behavioral
studies of autism in adults with relatively high Verbal and Performance IQs. The
latter will provide important additional information for task selection for PET
studies of autistic men.
375
ZOl MH 00178-06 CHP
A correlational pattern analysis is being applied to our cerebral blood flow
data on severe dyslexia. Of particular interest, given our findings of unusually
large hemispheric asymmetries and reduced antero-posterior differences, are
correlations between left-right homologous cortical regions and frontal-parietal
association cortex. Group differences in correlational patterns involving these
areas would suggest unusual functional associations between these sets of
regions.
Analysis of preliminary eye movement data and of electrophysiological data
is planned for this year. Results will determine our decisions concerning future
application of these methods.
We are also pursuing comparative neuropsychological studies of men with mild
and with "compensated" versus severe dyslexia and of dyslexic men with and
without attention deficit disorder. Such studies will provide a meaningful
context within which to evaluate the generality of our physiological findings.
Publications
Rumsey, J., Dorwart, R., Vermess, M., Denckla, M.B., Kruesi, M.J. and Rapoport,
J.L.: Brief report: Magnetic resonance imaging of brain anatomy in severe
developmental dyslexia. Arch. Neurol. 43(10): 1045-1046, 1986.
Rumsey, J., Andreasen, N. and Rapoport, J.L.: Thought, language, communication,
and affective flattening in autistic adults. Arch. Gen. Psychiatry 43: 771-777,
1986.
Creasey, H., Rumsey, J., Schwartz, M., Duara, R., Rapoport, J.L. and Rapoport,
S.: Brain morphometry, as measured by quantitative CT scanning, in autistic men.
Arch. Neurol. 43(7): 669-672, 1986.
Zahn, T., Rumsey, J. and Van Kammen, D.P.: Autonomic nervous system activity in
autistic, schizophrenic, and normal men: Effects of stimulus significance. J.
Abnorm. Psvchol. 96(2): 135-144, 1987.
Rumsey, J. and Denckla, M. : Neurobiological research priorities in autism. In
Schopler, E. and Mesibov, G.(Eds.): Current Issues in Autism. New York, Plenum
Publishing Corp., 1987, pp. 43-61,
Rumsey, J., Creasey, H., Stepanek, J., Dorwart, R., Patronas, N., Hamburger, S.
and Duara, R.: Hemispheric asymmetries, fourth ventricular size, and cerebellar
morphology in autism. J. Autism Dev. Disord. , in press.
Rumsey, J., Berman, K., Denckla, M., Hamburger, S., Kruesi, M. and Weinberger,
D.: Regional cerebral blood flow in severe developmental dyslexia. Arch. Neurol.,
in press.
376
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00301-05 CHP
PERIOD COVERED
October 1, 1986 to September 1987
TITLE OF PROJECT (80 characters or less. Title must fit <
Diagnosis in Child Psychiatry
I one line betweerj the borders.)
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Judith L. Rapoport, M.D., Chief, CHP, NIMH
Brack Borcherding, M.D., Clinical Associate, CHP, NIMH
Alan J. Zametkin, M.D., Staff Psychiatrist, CHP, NIMH
Eric Taylor, M.D., Sr. Registrar, Maudsley Hospital, London, Eng.
James Swanson, Ph.D., Prof, of Psychology, Univ .of Cal if ., Irvine, CA
Michael Rutter, M.D., Prof, of Child Psychiatry, Maudsley Hospital, London, Eng.
COOPERATING UNITS (it any)
Department of Psychiatry, Maudsley Hospital, London, Eng.
Department of Pediatrics, University of California, Irvine, CA
LAB/BRANCH
Child Psychiatry Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
.30
PROFESSIONAL;
10
.10
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
H (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
To investigate the basis for widely separate rates in the diagnosis of
childhood hyperactivity between the U.S. and U.K., research teams and clinician
panels in both countries completed diagnostic ratings of standardized case
histories using D3M III aiid ICD 9 diagnostic schemes. The results showed effects
of diagnostic schemes on rates for the diagnosis of hyperactivity.
However, there was additional effect of nationality of case, with U.S. cases
more likely to receive the diagnosis of Attention Deficit Disorder. To pursue
this, a new study is started comparing 24 hour motor activity in hyperactive
children and their parents in three settings: Bethesda, Maryland; Irvine,
California; and London, England.
377
ZOl MH 00301-05 CHP
Project Description:
Objectives : To examine the widely discrepant rates of diagnosis of childhood
hyperactivity between the U.S. and most European countries, hyperactive boys are
being studied with 74-hour actometer recordings of naturalistic motor activity in
three different clinical settings. The purpose of the study is to see whether,
at a given score on teacher and parent rating scales, children actually exhibit
comparable motor activity across these settings.
Methods Employed: Approximately 15 boys, ages six to 11 years, will be studied
at each setting - the Child Psychiatry Branch at NIMH, the Department of
Pediatrics at the University of California at Irvine, and the Division of Child
and Adolescent Psychiatry at the Maudsley Hospital in London, England. Boys
chosen will meet DSM III-R criteria for Attention Def icit/Hyperact ivity Disorder
and have comparable and appropriately high parent and teacher ratings of
disruptive behavior.
These children will wear activity monitors for seven consecutive days of 24-
hour recording of activity levels. The results will be compared across U.S. and
European settings. Specific comparisons of activity during different time
periods, such as sleep, play, and school subject, will also be made.
Major Findings: None to date from the actometer study. A major effect of
clinician training and diagnostic scheme has been found.
Significance to Mental Health Research: Childhood hyperkinesis has been
considered an American phenomenon and widely ascribed to dietary, cultural and/or
environmental as has been supposed and that most of the differences is accounted
for by clinician rating scheme and possibly referral differences in the two
countr ies .
Data from this project may confirm that actual activity levels are
comparable in both the English and American boys, when a similar diagnostic
i^cheme is used.
Proposed Course of Project: The above data will be collected throughout the
school year and provide a basis for further study of combinations of ratings,
activity level, and cognitive testing to compare diagnosis across different
settings .
Publications :
Rapoport, J.L., Donnelly, M. and Zametkin, A.: Situational hyperactivity in a
United States clinical setting. J. Child Psychol. Psychiatry 27: 639-646, 1986.
378
ZOl MH 00301-05 CHP
Taylor, E. and Rapoport, J.L.: Diagnosis of Hyperactivity - U.S. S UK
Differences. In Sargeant, J. and Bloomingdale, L. (Eds.): Research Diagnostir
Criteria for Attention Deficit Disorder. New York, Spectrum Publishers, in press,
Rapoport, J.L.: DSM III-R and Child Diagnosis. In Last, C. and Hersen, M.
Issues in Diagnostic Research. New York, Academic Press, in press.
[Eds.
379
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02240-03 CHP
PERIOD COVERED
October 1, 1986 to September 30, 1937
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Neurobiology of Attention Deficit Disorder
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Judith L. Rapoport; M.D., Chief, CHP, NIMH
Alan J. Zanietkin, M.D., Staff Psychiatrist, CHP, NIMH
Markus J. P. Kruesi, M.D., Staff Psychiatrist, CHP, NIMH
William Z. Potter, M.D., Ph.D., Chief, Section on Clinical Pharmacology, LCS,
NIMH
Markku Linnoila, M.D., Ph.D., Chief, LCS, NIAAA
Robert M. Cohen, M.D., Ph.D., Chief, Section on Clinical Brain Imaging, LCM, NIMH
COOPERATING UNITS (it any)
Section on Clinical Pharmacology, LCS, NIMH
National Institute on Alcohol Abuse and Alcoholism
Section on Clinical Brain Imaging, LCM, NIMH
Child Psychiatry Branch
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.75
PROFESSIONAL;
2.1
.75
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
H (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK
To loca
cerebral blc
TypPf parent
deoxyf luorog
To date
have been do
rnechanisni of
Disorder wit
methyl phen id
chronic effe
of dopamine,
Thr- rel
aggression,
the spinal f
disorder, is
concentratio
■>'lu:ts. Per
along with p
hdve been st
'3 0(1 ten age-
lower CSF T-
(Use standard unreduced type. Do not exceed the space provided.)
lize possible abnormal patterns of cerebral glucose metabolism and
od flow in adults with Attention Deficit Disorder (ADD) Residual
s of diagnosed hyperactive children are being studied using the 2-
lucose method of Positron Emission Tomography (PET).
, 30 PET scans have been performed without medication and 16 scans
ne during treatment with dextroamphetamine. To understand the
action of stimulant medication in the treatment of Attention Deficit
h Hyperactivity (ADDH), a comparison of pemoline (Cylert),
ate, and dextroamphetamine was initiated to examine the acute and
cts of these medications in the same subjects on peripheral measures
norepinephrine, prolactin, and growth hormone.
ationships between the behavioral dimensions of impulsivity.
and concentrations of monoamine metabolites, particularly 5-HIAA, in
luid of children with conduct disorder and/or attention deficit
b'.' i ng examined. Assoc iat ; c.ris between decreased C.TF .'"^•HIAA
ns and impulsive aggressive behavior have been reported in impulsive
ipheral measures of serotonin and catecholamines are being obtained
ersonality, impulsivity and aggression measures. Seventeen subjects
udied to date. A preliminary comparison between ten Conduct Disorder
matched Obsessive Cominil slve children, sliows a trend (p = .09) for
HIAA coni-'-nl ration in conduct disorder. Within the conduct di.sorder
r values are assorlc^tpd w;th more destructive behavior.
381
ZOl MH 02240-03 CHP
Project Description:
Object ives : To study the pathophysiology of ADDH and Conduct Disorder. Specific
monoamine and neuroanatomical hypotheses are being tested using pharmacological
probes, examination of spinal fluid, blood and urinary excretion of monoamines
and metabolites, and with Positron Emissions Tomography (PET). Understanding
biological contributors to these disorders will aid in development of more
effective treatment.
Methods Employed: In earlier studies we have demonstrated that methylphenidate,
dextroamphetamine, and pemoline have different effects on peripheral measures of
catecholamine function. Current work examines whether in the same sample of
children, all three drugs act differently by studying children during a multiple
stimulant drug crossover study. Biochemical correlates of response will be
examined .
Spina] fluid monoamine metabolites are being obtained from children with
conduct disorder, attention deficit disorder, children with obsessive compulsive
disorder as well as a pediatric contrast group. Platelet serotonin, as well as
plasma catecholamines, are being collected from the psychiatric subjects.
Measures include ratings of impulsivity, aggression, anxiety, depression, and
obsessional ity .
Major Findinqf^: Pemoline appears as clinically effective as methylphenidate and
dextroamphetamine. When children are examined on the day hospital setting, there
are very few non-responders to any of the three stimulant medications. Isolated
cases of selective response and of tolerance to stimulant drug response are of
considerable theoretical interest .ind are being r-ludied in greater depth.
To document the presence in children of a concentration gradient for
dopamine and serotonin metabolites in the CSF, (which is we"!] ert.Hbl ished in
adults), sequential CSF aliquots were obtained from eight prepubertal subjects
and 18 subjects who were Tanner stage II to V. Three of eight prepubertal
subjects had slopes for 5-HIAA concentration/ml collected that were not
statistically different from zero. In contrast, across tiie whole, group slopes
were similar to ttiose reported in adults. Comparison of ten age-matched conduct
disorder and obsessive compulsive disorder children showed a trend (p - .09) for
lower concentrations of 5-HIAA in the conduct disordered group. Within the
conduct disorder group (N-13) there is a significant negative correlation
(r=-.63) between CSF 5-HIAA and Brown-Goodwin Scale ratings of aggressive acts.
The PET studies are not yet analysed.
Significance to Mental Health Reseaxcli: Attention Deficit Disorder is a
relatively common childhood disability that persists into adulthood for about 30%
of the cases. Examination of pathophysiology of childhood ADDH and Conduct
Disorder will permit more accurate diagnosis -.uid treatment of children and adults
with d spectrum of impulse disorders.
382
ZOl MH 02240-03
Conduct disorder is the most frequent cause for referral of childi'tT; dni5.
adolescents for psychiatric services. The preliminary trend for lower 5-HIAA
concentration in the CSF of conduct disorder subjects is consistent with studies
in adults relating impulsive and aggressive behavior with decreased 5-HIAA
concentrations. Identification in children of a concentration gradient for the
monoamine metabolites, 5-HIAA and HVA, establishes the need for methodological
control over the aliquot collected - a point not addressed in previously in
pediatric behavioral studies.
Proposed Course of Project: The sample si7.e of the pemoline study will be
increased to approximately 20 and the data will then analyzed. PET scans will
continue to enlarge the sample size of drug treated subjects both on and off
stimulant medication.
The data on CSF concentration gradients and the contrasts between pre and
post pubertal children is being analyzed, as are preliminary examination of
dimensions of personality variables and aggression histories across subjects in
relation to CSF biochemistry.
A follow-up study is underway of all subjects for whom CSF chemistries are
available to see if 5-HIAA and/or HVA predict follow-up status.
Publ ications:
Kruesi, M.J. P., Linnoila, M., Rapoport, J.L., Brown, G.C. and Petersen, R.:
Carbohydrate craving, conduct disorder and low CSF 5-HIAA. Psychiatry Res . 16:
83-86, 1985.
Zametkin, A.J., Linnoila, M., Karoum, F. and Salle, R.: Pemoline and the urinary
excretion of catecholamines and indolamines in children with attention deficit
disorder. Am. J. Psychiatry 143:3: 359-362, 1986.
Donnelly, M., Zametkin, A.J., Rapoport, J.L., Ismond, D., Weingarten, H., Lane,
E., Oliver, J., Linnoila, M. and Potter, W.Z.: Treatment of childhood
hyperactivity with desipramine: Plasma drug concentration, cardiovascular
effects, plasma and urinary catecholamines and clinical response. Clin.
Pharmacol. Ther. 39:1: 72-81, 1986.
Rapoport, J.L., Donnelly, M., Zametkin, A.J. and Carrougher, J.: Situational
hyperactivity in a U.S. clinical setting. J. Child Psychol. Psychiatry 27:5:
639-646, 1986.
Zametkin, A.J. and Rapoport, J.L.: The Pathophysiology of Attention Deficit
Disorder. A Critical Review, In Lahey, B. and Kasdin, A. (Eds.): Advances in
Clinical Child Psychology. New York, Plenum, 1986, pp. 177-216.
383
ZOl MH 02240-03
Zametkin, A.J., Rapoport, J.L., Potter, W.Z. and Karoum, F.: Treatment of
hyperactive children with d-phenylalanine . Am. J. Psychiatry 144: 792-793, !9B7.
Zametkiii, A. .7. and Rapoport, J.L.: The Noradrcner'^ic Hypothesis of Attention
Deficit Disorder: A Critique. In Meltzer, H. (Ed.): Ppvchopharmacoloqy : Third
Generation of Progress. New York, Raven Press, 1987.
Zametkin, A.J. and Rapoport, J.: The neurobiology of attention deficit disorder
with hyperactivity. Where have we come in 50 years? J. Am. Aca'. Child Adolesc.
Psychiatry, in press.
Zametkin, A. J., Potter, W.Z. and Rtjpoport, J.L.: The effect of methylphenidate
upon urinary catecholamines and behavior in hyperactive children: A replication.
Biol. Psychiatry, in press.
Prendergast, M., Taylor, F:., Rapoport, J . [■ . , Zametkin, A.J., Donnelly, M., Aist,
M.B. and Bartko, J.: a cross-national study of DSK-IIJ and ICD-9 psychiatric
diagnosis of children with disorders of conduct and/or alteiition. J. Child
Psychol. Psychiatry, in press.
384
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00084-13 CNG
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Genetic-Biologic Studies of Psychiatric Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I. E. Gershon Chief CNG, NIMH
Others: W.H. Berrettini Medical Officer CNG, NIMH
L. DeLisi Asst. Professor SUNY at Stony Brook
J.I. Nurnberger Jr. Dir. of Res., Inst, of Psych. Res., Indiana U.
J. Hamovit Research Social Worker CNG, NIMH
J. Guroff Social Science Analyst CNG, NIMH
D. Kazuba Psychologist CNG, NIMH
COOPERATING UNITS (if any)
LCS, NSB, NIMH, Chestnut Lodge, Rockville, MD
LAB/BRANCH
Clinical Neurogenetics Branch
SECTION
Section on Clinical Genetics
INSTITUTE AND LOCATION
NIMH, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
4.5
OTHER;
4.3
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
n (a1) Minors
D (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Family study of schizophrenia: 108 relatives of chronic schizophrenics, 129
relatives of chronic psychosis schizoaf fectives , and 380 relatives of
psychiatrically normal controls, were studied with systematic diagnostic
interviews, information from relatives, and review of medical records where
appropriate. Comparing relatives of schizophrenics with relatives of schizo-
af fectives, there was no tendency for schizoaffective diagnosis or actue or
chronic psychoses to aggregate separately. Increased bipolar disorder was
found in relatives of schizoaf fectives but not in relatives of schizophrenics.
Major affective disorder (bipolar and unipolar) was increased in relatives of
both types of psychotic proband. A separate family study of bulimia is
ongoing.
Cultured lymphoblastoid cell lines have been prepared from a series of 40
affected-sib-groups with schizophrenia, 16 pedigrees of bipolars, and 5
schizophrenia pedigrees. These are being used in genetic linkage and
association studies.
Mathematical analysis of the power of linkage methods has provided estimates
of the robustness, in the presence of genetic heterogeneity, of the affected-
sib-pair and pedigree lod score linkage methods (study of unaffected offspring)
for inherited quantitative traits. We have also analyzed the power of the
high-risk paradigm. These analyses allow precise estimates of the required
numbers of individuals for valid linkage and high risk studies.
385
PHS 6040 (Rev 1/84)
GPO 914-SIB
ZOl MH 00084-13 CNG
Investigators (continued)
I.D. Dauphinais NRSA Fellow CNG, NIMH
J. Gelernter Medical Staff Fellow CNG, NIMH
J. Gejman Visiting Associate CNG, NIMH
I. Family studies
A. Schizophrenia
The initial report of this study, a collaboration with the Clinical
Neuroscience Branch and Chestnut Lodge, has been accepted for publication.
237 relatives of 48 patients with chronic psychosis, diagnosed as
either schizophrenia or schizoaffective disorder, along with 380 relatives
of psychiatrical ly normal controls, were studied with systematic diagnostic
interviews, information from relatives, and review of medical records
where appropriate. A variety of nonbipolar psychotic disorders was found
in the relatives of the patients. Comparing relatives of schizophrenics
with relatives of schizoaf fectives, there was no tendency for schizoaffective
diagnosis or acute psychoses to aggregate separately from schizophrenia.
Increased bipolar disorder was found in relatives of schizoaf fectives but
not relatives of schizophrenics. Major affective disorder (bipolar and
unipolar) was increased in relatives of both types of psychotic probands
(see table next page). If we subdivide the ill probands according to whether
or not they had a history of substance abuse, relatives of probands with
substance abuse had greater frequency of affective disorder and of substance
abuse, but there were not significant differences in number of relatives
with non-bipolar psychoses.
These results have important implications for molecular genetic
linkage studies of schizophrenia. In large pedigrees, one can lose
considerable statistical power by failing to identify as "affected"
relatives who have variant forms of illness. This study supports an
inclusive definition of "affected" — schizoaffective, drug abuse with
chronic psychosis, and possibly major affective disorder, in these families.
Of course, the schizotypal personality disorders previously associated
with schizophrenia would also be included. Another implication of these
family study results is to look for common linkage markers in schizophrenia
and schizoaffective disorder.
386
ZOl MH 00084-13 CNG
Proband
Diagnosis
Schizophrenia
Chronic
(24 families)
Schizo-
Af fective
Chronic
(24 families)
Control
(67 families)
Illness Frequencies in First-Degree Relatives
_ Age-Corrected Morbid Risk, per cent
Schizo-
All Affective All Bi- Other &
Psychosis* Acute polar** Unipolar Normal
6.2
11.7
2.ll
5.0
0.0
0.32
2.2
9.6
0.83
14.7
9.3
6.7^,5
71.9
69.4
90.1
Number of
Relatives
108
129
380
* Includes schizophrenia, schizoaffective chronic and indeterminate,
unspecified functional psychosis, other psychiatric disorder with
hospitalization.
**Includes bipolar I, bipolar II, cyclothymic personality.
^ p = 0.03 vs schizophrenia, p < 0.0001 vs schizoaffective
2 p < 0.01 vs schizophrenia
3 p < 0.0001 vs schizophrenia
^ p = 0.01 vs schizophrenia
5 All affective (all bipolar and unipolar): p = 0.007 vs schizophrenia,
p < 0.001 vs schizoaffective
All probabilities by Fisher's exact test.
B.
Bulimia
In collaboration with Dr. Jimerson of Laboratory of Clinical Science,
a family study of bulimia is ongoing. 42 probands have been enrolled in
the study, and 67 relatives of patients have been examined. The controls
are the same as in the schizophrenia family studies. We propose to
determine whether affective disorders and eating disorders are co-aggregating
in families of these patients. The study should be complete within 18
months.
387
ZOl MH 00084-13 CNG
III. Population genetic studies
1. Power of linkage methods
A. Affected sib-pair method
In preliminary studies, we determined the number of affected-sib-pairs
needed to find linkage for some specific modes of Inheritance and for a small
amount of heterogeneity. We have extended these studies and examined the
required sample size for a wide range of assumptions that are likely to be
relevant for the major psychiatric disorders. We have considered the
merits of applying this methodology for screening the human genome for
linkage to psychiatric disorders as well as replicating linkages found in
specific pedigrees in a more general population sample. For example, to
find linkage to affective disorders (assuming 7% population prevalence
and autosomal dominant inheritance with reduced penetrance), as many as
160 affected sib-pairs would be needed to find linkage up to 10% recombination
if only 50% of families were linked to the marker locus. To determine if
the chromosome 11 linkage found in an Amish pedigree could be replicated in
a sample of affected-sib-pairs, only about 1/3 of this sample size would
be required because the linkage is close. About the same number would be
required to replicate this finding of chromosome 21 linkage to Alzheimer's
disease. However, if only a small proportion of families were linked (25%),
then 220 affected-sib-pairs would be needed. In general, the affected-sib-
pair method will be more powerful for "rarer" disorders like schizophrenia
than for "common" disorders like major affective disorder. In any case,
a new linkage will be difficult- to detect unless it accounts for about 50%
of families. However, this method will usually be powerful for determining
whether a linkage previously found in a limited sample for sub-population
is present in the general population, and for estimating the proportion
of families in the population that are linked.
B. Lod score method
We have examined the power of the lod score method for pedigrees of
"moderate" size (approximately 15 individuals) under conditions of hetero-
geneity. For example, a highly penetrant, dominant gene for bipolar
illness (given the parameters found to fit linkage in the Amish pedigree)
that accounted for 25% of families, about 30 families of this size would
be needed to insure a high probability to detect linkage. However, if
only 10% of families were linked, we would only have 50% chance to detect
linkage with 30 families. Thus, a substantial number of families are
required to find linkage if this gene accounts for only a small proportion
of families. If penetrance of the gene were reduced (50%), the overall
power is slightly lower. These sample size estimates are conservative
since, given the rapid progress in the development of the human gene map,
most linkage studies will examine multiple marker loci simultaneously
(whose map positions are known) which increases the power of linkage
detection over pairwise tests.
ZOl MH 00084-13 CNG
2. Power of high risk methods for biological traits
We originally examined the power of studying high risk samples
(offpspring of one or more affected parents who are at risk for developing
illness) in order tc confirm that potential biological trait markers are not
a consequence of the ill state. We originally calculated the power of this
method for a simple normally distributed trait. We have extended these
studies to consider how powerful the high risk method is when a trait is
dichotomous or when there are large differences in the variance of a
quantitative trait between ill and well individuals. We have shown how
these determinations depend on underlying assumptions about the mode of
inheritance of the trait and its relationship to illness susceptibility.
For example, using the genetic model hypothesized by Matthysse et al.
to account for the familial association between schizophrenia and deviant
eye tracking, we calculated that this association could be confirmed in
a high risk study if about 50 individuals/group were studied (i.e.,
50 offspring of one affected parent and 50 controls). To examine the
effect of different variances in patients and controls, we simulated high
risk samples based on the published findings of melatonin supression in
response to light in patients with affective disorders and controls. That
is, we assumed that the high risk population was a mixture of individuals
having different susceptibilities. Under these assumptions, we determined
that 25-30 individuals/group are needed to confirm the presence of the
abnormality in a high risk sample. For any trait, studying offspring of
two affected individuals is substantially more powerful than studying
offspring of one affected individual.
Significance to Biomedical Research and the Program of the Institute
For the long-term goal of identifying genes that cause psychiatric
disorders, our present results are important milestones. The family study
of schizophrenia provides definitions of affected and unaffected to be used
in genetic linkage studies and inherited risk factor studies. The cultured
cell lines provide the biological substrate for the linkage studies with
recombinant DNA and protein markers. The mathematical analyses have
defined the most powerful sampling methods to use to test linkage and candidate
gene hypotheses, and provided an impetus to the study of inherited risk factors
in well relatives of patients.
Proposed Course of Project
We plan to continue to investigate the biology and genetics of characteris-
tics that may be implicated in the genetics of affective disorders, as described
above. The molecular genetics approach of interindividual differences in
neuropeptides and other substance will be pursued. Establishing a library of
DNA and living cells from entire pedigrees is a major priority. Study of
relatives at risk for affective disorders and schizophrenia will proceed.
Mathematical methodology for clinical investigation will continue to be
studied.
389
ZOl MH 00084-13 CNG
Several projects have been spun off from this one: ZOl MH 02236-03 CNG,
Schizophrenia Studies, ZOl MH 02237-03 CNG, Molecular Genetics of Neuropsychia-
tric Disorders and ZOl MH 00086-11 CNG, Outpatient Clinic for Genetic and
Pharmacological Studies of Affective Disorders, ZOl MH 0085-11 CNG, Pharmaco-
genetics of Psychoactive Drugs. This project will be continued as the
main biologic-genetic project of the Section, and the start-up of new
initiatives will be within this project.
References
Berrettini, W.H. , Bardakjian, J., Barnett, A.L. , Nurnberger, J.I. , Jr., and
Gershon, E.S.: Beta-adrenergic receptor function in human adult skin
fibroblasts: A Study of manic-depressive Illness. In Porter, R. (Eds.):
Antidepressants and Receptor Function. New York, John Wiley and Sons,
1986, pp. 30-42.
Gershon, E.S., Nurnberger, J.I., Jr., and Sitaram, N. : Recent genetic
findings in mood disorders. In Hippius, H. Klerman, G.L. , and Matussek,
N. (Eds.): New Results in Depression Research. Berlin, Springer-Verlag
Publishers, 1986, pp. 79-89.
Gershon, E.S. and Goldln, L.R. : Clinical methods in psychiatric genetics
I. Robustness of genetic marker investigative strategies. Acta Psych.
Scand. 74: 113-118, 1986.
Goldin, L.R. , Nurnberger, J.I., Jr., and Gershon, E.S.: Clinical methods
In psychiatric genetics II. A high risk approach. Acta Psych. Scand.
74: 119-128, 1986.
Clerget-Darpoux, F. , Goldin, L.R. , Gershon, E.S.: Clinical methods in
psychiatric genetics III. Environmental stratification may simulate a
genetic effect in adoption studies. Acta Psych. Scand. 74: 305-311, 1986.
Gershon, E.S.: Forward. In Vandenberg, S.A. and Pauls, D. (Eds.):
The Heredity of Behavior Disorders in Adults and Children. New York,
Plenum Medical Book Company, 1986.
Nurnberger, J.I. , Jr., Goldin, L.R. , Gershon, E.S.: Genetics of
psychiatric disorders. In Winokur, G. and Clayton, P. (Eds.): Medical
Basis of Psychiatry. Philadelphia, W.B. Saunders Co, 1986, pp. 486-521.
Gershon, E.S., Welssman, M.M. , Guroff, J.J. , Prusoff, B.A. and Leckman, J.F. :
Validation of criteria for major depression through controlled family study.
J. Aff. Dis. 11(2): 125-131, 1986.
Baumgold, J.: Effects of verapamil on the binding characteristics of
muscarinic receptor subtypes. Eur. J. Pharm. 126: 151-154, 1986.
390
ZOl MH 00084-13 CNG
Baumgold, J.: Muscarinic receptors in brain from four strains of inbred
mice. Neurochem. Res. 12(11), 1987, in press.
Baumgold, J., Merril, C.R. , Gershon, E.S.: Loss of pirenzepine regional
selectivity following solubilization and partial purification of the
putative Mj and M2 muscarinic receptor subtypes. Mol. Brain Res. 2: 7-14, 1987,
Gershon, E.S., Hamovit, J.H. , Guroff, J.J. and Nurnberger, J.I., Jr.:
Birth cohort changes in manic and depressive disorders in relatives of
Bipolar and Schizoaffective patients. Arch. Gen. Psychiatry 44: 314-
319, 1987.
Gershon, E.S.: Discovering biologically specific risk factors and
genetic linkage markers in affective disorders. In Dunner D.L. ,
Gershon, E.S. and Barrett, J. (Eds.): Relatives at Risk For Mental
Disorders. New York, Raven Press, in press.
Editors: Dunner D.L., Gershon, E.S. and Barrett, J.: Relatives at Risk
For Mental Disorders. New York, Raven Press, in press.
Gershon, E.S.: Genetic perspectives on manic-depressive illness. In
Goodwin, F.K. and Jamison, K.R. (Eds.): Manic-Depressive Illness.
New York, Oxford University Press, in press.
Gershon, E.S.: Single locus markers in affective disorders. In
Lerer, B. and Gershon, E.S. (Eds.): New Directions in Affective Disorders.
New York, Springer-Verlag , in press.
Editors: Lerer, B. and Gershon, E.S.: New Directions In Affective Disorders.
New York, Springer-Verlag, in press.
Gershon, E.S. , Berrettini, W.H. , Goldin, L.R. : Affective disorders:
Genetics. In Kaplan, H.I. and Saddock, B.J. (Eds.): Comprehensive
Textbook of Psychiatry/V Fifth Edition. Baltimore, MD, Williams &
Wilkins, 1987, in press.
Gershon, E.S., Berrettini, W.H. , Nurnberger, J.I., Jr., and Goldin, L.R. :
Genetics of affective illness. In Meltzer, H.Y. (Ed.): Psychopharmacology;
A Third Generation of Progress. New York, Raven Press, 1987, pp. 481-491,
in press.
Gershon, E.S. and Goldin, L.R.: The outlook for linkage research in
psychiatric disorders. J. Psych. Res. , in press.
Gershon, E.S. , Merril, C.R. , Goldin, L.R. , DeLisi, L.E. , Berrettini, W.H. ,
and Nurnberger, J.I., Jr.: The role of molecular genetics in psychiatry.
Biol. Psychiatry, in press.
391
ZOl MH 00084-13 CNG
Goldln, L.R. , DeLisi, L.E. and Gershon, E.S.: Unravelling the relationship
between genetic and environmental risk, factors in psychiatric disorders.
Brit. J. Psych. , in press.
Goldin, L.R. , DeLisi, L.E. , Gershon, E.S.: Genetic aspects to the biology
of schizophrenia. In Henn, F.A. and DeLisi, L.E. (Eds.): Handbook of
Schizophrenia, Vol. 2, Amsterdam, Elsevier Science Publishers, in press.
Goldin, L.R. and Gershon, E.S.: The power of the affected-sib-pair method
for heterogeneous disorders. Gen. Epidem. , in press.
Gershon, E.S., Cloninger, C.R. Propping, P., DeLisi, L.E. , Hippius, H. ,
and Bondy, B. (Eds.): Special issue of J. Psych. Res. , Genetic Research
in Psychiatry, in press.
Berrettini, W.H. , Bardakjian, J., Cappellari, C.B. , Barnett, A.L. , Jr.,
Albright, A., Nurnberger, J.I., Jr., and Gershon, E.S.: Skin fibroblast beta
adrenergic receptor function in manic-depressive illness. Biol. Psychiatry,
in press.
Berrettini, W.H. , Cappellari, C.B., Nurnberger, J.I., Jr., and Gershon, E.S.:
beta adrenergic receptors on lymphoblasts: a study of manic-derpessive
illness. Neuropsychobiology 76: in press.
Gershon, E.S. , DeLisi, L.E. , Maxwell, M.E. , Nurnberger, J.I., Jr., Hamovit, J.,
Schreiber, J., Dauphinais, D. , Dingman, C.W. , Guroff, J.J.: A controlled family
study of chronic psychoses: schizophrenia and schizoaffective disorder.
Arch. Gen. Psychiatry, in press^
392
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00086 U-CNG
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one lir)e between tfte borders.)
Outpatient Clinic for Genetic and Pharmacologic Studies of Affective Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aftitiation)
PI: W.H. Berrettini
Others: L. DeLisi
E.S. Gershon
S. Simmons-Ailing
J.R. Hamovit
M.E. Maxwell
Medical Officer
Asst. Professor
Chief
Clinical Nurse Expert
Research Social Worker
Research Social Worker
CNG, NIMH
SUNY at Stony Brook
CNG, NIMH
CC, NIH
CNG, NIMH
CNG, NIMH
COOPERATING UNITS (if any)
CC, NIH: CHP, BPB, LPP, LCS, NPB, LCS, NIMH: Catholic University: NSB, NIAA:
University of Pittsburgh: Karolinska Institute: LSN, NIA: Indiana University
LAB/BRANCH
Clinical Neurogenetics Branch
SECTION
Section on Clinical Genetics
INSTITUTE AND LOCATION
NIMH, Bethesda, MD 20892
TOTAL MAN-YEARS:
3.8
PROFESSIONAL:
1.5
OTHER:
2.3
CHECK APPROPRIATE BOX(ES)
C (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The high-risk study of healthy adolescent offspring of bipolar parents has
identified a potential vulnerability marker in the at-risk population.
The high-risk group shows supersensitivity to the inhibitory effects of light
on plasma melatonin, suggesting that this finding may represent a genetic
vulnerability marker for affective disorder.
We have completed a study of cholinergic Induction of rapid eye movement (REM)
sleep in euthymic unmedicated bipolars. The bipolars showed more rapid
iaductiou of REM sleep than did matched controls, contirming previous
observations and providing additional evidence that cholinergic
supersensitivity may be a genetic vulnerability marker for affective dlsorrler.
Studies of the growth hormone response to clonidine in euthymic unmedicated
bipolars suggest that this response is blunted in bipolar subjects compared to
controls. The possibility that this represents a genetic vulnerability marker
is currently being evaluated.
Studies of CSF neuropeptides in normal monozygotic and dizygotic twins have
shown that neuropeptide Y levels are heritable , are high in underweight
anorectics and are normal in depressives. Additionally, the related
neuropeptide, peptide YY, is elevated in CSF from bulimic subjects. Galanin
levels in CSF are normal in Alzheimer's Disease, depression and eating
disorders.
Acute IV administration of physostigmine produces increases in CSF neuropeptide
Y, in both monkeys and man, indicating that pharmacologic challenges may employ
CSF neuropeptide levels in assessing the drug effect on various CNS
neurotransmitters. 393
PHS 6040 (Rev. 1/84)
GPO si4-«ie
ZOl MH 00086-11 CNG
Investigators (Continued);
J. Gelernter
L. Goldin
L. Sapin
W.H. Kaye
D. Pellegrini
T. Zahn
T. Sunderland
N. Garrick
D. Murphy
D. Pickar
0. Wolkowitz
M. Linnoila
P. Gold
C. May
L. Tamarkin
D. Rubinow
G. Oxenstierna
G. Sedvall
Medical Staff Fellow
Senior Staff Fellow
Guest Researcher
Assoc. Prof, of Psychiatry
Asst. Prof, of Psychology
Research Scientist
Staff Fellow
Research Scientist
Branch Chief
Section Chief
Clinical Associate
Clinical Director
Section Chief
Senior Staff Fellow
Clinical Associate
Unit Chief
Staff Psychiatrist
Professor of Psychiatry
CNG, NIMH
CNG, NIMH
CNG, NIMH
U. of Pitts.
Catholic Univ.
LPP, NIMH
LCS, NIMH
LCS, NIMH
LCS, NIMH
NSB, NIMH
NSB, NIMH
NIAAA
BPB, NIMH
LNS, NIA
CPB, NIMH
BPB, NIMH
Karolinska Inst.
Karolinska Inst.
Project Description
We maintain an outpatient clinic of approximately 100 bipolar subjects
for the purpose of: 1) identifying potential markers of genetic vulnerability
to affective disorder; and 2) studying the onset and course of illness of
bipolar disease. In addition to the bipolar subjects themselves, we are
conducting a study of the healthy adolescent children of bipolar parents,
children at "high risk" for the development of affective disorder.
We study euthymlc (well-state) bipolar subjects to determine those
abnormalities which are most likely to be inherited characteristics of the
Illness. Additionally, the study of children at high risk for affective
illness is useful in avoiding drug effects or secondary effects of the illness
which may persist even in the euthymic state.
For comparison purposes we maintain a pool of approximately 50
well-screened normal volunteers.
Lastly, this project includes an active laboratory devoted to the study
of CSF neuropeptides in psychiatric diseases. This laboratory has collaborated
with clinical investigators, attempting to elucidate the roles of various
neuropeptides in eating disorders, affective illness, Alzheimer's disease and
schizophrenia.
Each aspect of the project is described below.
394
ZOl MH 00086-11 CNG
1. HIGH RISK STUDY
We are continuing a prospective study of the healthy adolescent (age
15-25) children (N=61) of bipolar parents and age-matched children (N=44) of
control parents. These high-risk adolescents are being studied prior to onset
of affective illness, and it is expected that 30% of them will eventually
become ill. By conducting this prospective study, we hope to identify
biological or psychological variables that will predict who will become ill.
Subjects are interviewed annually for evidence of affective disorder. To date
six children of bipolar parents have become ill (1 bipolar and 5 unipolar
cases), while one control child has developed affective disorder during the
first three years of the study.
One potential vulnerability marker has been identified. Unmedicated
bipolar subjects, independent of mood state, show supersensitivity to the
inhibitory effects of light on plasma melatonin. To evaluate this potential
marker, we studied the high risk population and the control group using this
paradigm in collaboration with Dr. Larry Tamarkin. Ten of 23 at risk subjects
were supersensitive, compared to three of 22 controls. This results suggests
that the supers ens tivity to light may be a genetic vulnerability marker for
affective disorder. However, studies on the heritability of this response and
segregation studies in pedigrees are necesary to confirm this putative marker.
2. CHOLINERGIC REM INDUCTION
We have confirmed our previous observation that euthymic unmedicated
bipolars have more rapid induction of REM sleep by the cholinergic agonist,
arecoline. While the difference between the bipolar and control groups was not
as great as that observed in a previous study, it remained statistically
significant (p<.05, N=18 in each group). These results suggest that
cholinergic supersensitivity may be a genetic vulnerability marker in affective
illness. However, due to the great overlap between patients and controls, this
particular paradigm is not useful for diagnosis, but may be appropriate for
segregation studies.
3. CSF NEUROPEPTIDES
Disease-related studies:
We have continued to study CSF neuropeptides in psychiatric disorders,
to determine the extent to which CSF levels of a neuropeptide can reflect
abnormal behaviors or reveal aspect of pathophysiology. For example, we have
developed two new assays for measurement of galanin and peptide YY in CSF.
These substances are potent stimulators of feeding behavior in rats, and so
we measured their CSF levels in anorexia nervosa and bulimia (Dr. Walter Kaye).
While galanin did not differentiate eating disorder subjects from controls,
CSF peptide YY levels were elevated in bulimic subjects who had abstained from
bingeing and purging behavior for 30 days, compared to anorectics or controls.
395
ZOl MH 00086-11 CNG
A third potent stimulator of feeding behavior in rats is neuropeptide Y (NPY)
which was also measured in CSF from eating disorder patients. NPY was elevated
in the underweight anorectics and returned to normal with weight recovery.
CSF NPY levels in bulimics were not different from those of controls.
Galanin is found within 40% of the cholinergic neurons of the basal
forebrain (the cells which degenerate in Alzheimer's disease). We studied CSF
galanin levels in subjects with Alzheimer's disease and age-matched controls
and elderly depressives (Drs. Trey Sunderland and Conrad May). No group
differences were found.
Heritability studies:
To determine whether CSF neuropeptide levels are heritable we studied
CSF NPY, CRF and GHRF in monzygotic and dizygotic twins and in brothers (Drs.
Gabriella Oxienstierna and Goran Sedvall). NPY levels were determined by
additive genetic factors while CRF and GHRF levels were influenced more by
environmental factors, as shown in the table below.
Heritability of CSF Neuropeptides
MZ TWINS
DZ TWINS
BROTHERS
(N=16)
(N=12)
(N=ll)
NPY^
0.59*
0.26
0.18
CRF
0.40
0.59
0.13
GHRF
0.27
0.31
0.11
""'Jensen's Heritability Index=0.66
*intraclass correlation coefficient
CSF pharmacologic challenges:
While basal CSF levels of a neuropeptide may be unchanged in a certain
disease state, if a methodology could be developed to study stimulated release
of a peptide from brain into CSF, abnormalities undetectable in the basal state
could become apparent. We have attempted to develop such a methodology, by
using IV physostigmine (up to 15 ug/kg) to provoke release of neuropeptides
into CSF. Monkey experiments (Drs. Nancy Garrick and Dennis Murphy) and human
studies have indicated that CSF NPY increases after 15 ug/kg IV physostigmine
and that this increase can be blocked by atropine. Neostigmine is without
effect. No changes were seen for somatostatin (SRIF), CRF, GHRF, vasopressin,
beta-lipotropin (LPH) or vasoactive intestinal peptide (VIP). Smaller doses of
physostigmine are not effective in increasing CSF NPY.
V)(.
ZOl MH 00086-11 CNG
CSF Correlations:
We have observed a set of highly significant positive correlations
between concentrations of unrelated neuropeptides in CSF (as shown below).
The origin of these correlations is obscure, but factor analysis suggests that
a single factor is responsible for 50% of the variance observed for each
peptide. This observation is intriguing and deserves further study.
Correlations among CSF Neuropeptides
LPH CRF SRIF ACTH
VIP .69* .57 .68 .73
.003+ .02 .003 .001
LPH .66
.63
.62
.006
.008
.008
CRF
.53
.71
.03
.001
SRIF
.59
.01
*correlation coefficient
+significance level
N=16
$. GROWTH HORMONE RESPONSE TO CLONIDINE
The growth hormone response to clonidine has been reported to be blunted in
subjects with affective disorder. To determine whether this is a
state-independent phenomenon, we are studying unmedicated euthymic bipolars and
controls. To date 12 bipolars and 15 controls have been studied. The bipolars
do show a significantly blunted growth hormone response (p<.04). We are
currently attemping to study the same patients during lithium treatment to
assess the effect of lithium on this putative marker. This measure may
represent a clinically available test of alpha adrenergic function in affective
disorder. Moreover, this test may be a useful method of selecting pedigrees to
search for linkage to restriction fragment length polymorphisms of the alpha
adrenergic receptor gene, a project which will begin shortly.
Significance to Biomedical Research and the Program of the Institute
The high-risk study has identified a putative vulnerability marker
for affective illness. As a result of the annual follow-up, we will be able to
determine the effect of psychosocial and biological variables on the
^)7
ZOl MH 00086-1 1 CNG
development of affective disorder. Additionally, through annual follow-up, we
may be able to Identify, early in the course of illness, subjects with
affective disorder, and we may be able to delineate carefully subclinical
antecedents.
The finding of elevated peptide YY in abstinent bulimics is intriguing,
and is congruous with the hypothesis that bulimics binge to correct a
central peptide YY abnormality, as their CSF levels are normal when they are
actively bingeing. In light of the potent stimulation of feeding behavior in
rats exerted by peptide YY, this finding may prove to be of etiological
significance in bulimia.
The fact that an acute IV dose of physostigmine can provoke release of
NPY into CSF suggests that pharmacologic challenges can employ CSF neuropeptide
measurements as endpolnts in assessing brain neurotransmitter systems. This
methodology may be applicable to numerous drugs and different CSF
neuropeptides. Measurement of a CSF endpoint may be a more useful strategy in
pharmacologic challenges, which have tradionally relied on plasma, behavioral
or cardiovascular measurements.
The observation that many unrelated CSF neuropeptide levels are highly
positively correlated with one another indicates that a major determinant of
CSF concentrations of various neuropeptides remains to be discovered. This
determinant may be related to the rate of formation or absorption of CSF or the
rate at which neuropeptides disappear from CSF. Because multiple studies of
CSF neuropeptides are being conducted in neuropsychiatrlc disease, delineation
of the determinant (s) would be a major contribution to our understanding of the
significance of CSF neuropeptide concentrations.
The clonldine studies confirm previous reports of blunted growth
hormone responses in affective disorder. The persistence of this finding in
the euthymic state suggests that this may be a state-independent marker for
affective illness.
Proposed Course of Study
The high-risk study will continue as planned with annual follow-up of
the high-risk and control groups. It is anticipated that additional subjects
will be studied for melatonin inhibition induced by light. In addition, we
will begin studies of bipolar subjects using Psoralen, an agent which provokes
release of melatonin from the pineal gland.
We plan to continue to study CSF neuropeptides in neuropsychiatrlc
diseases. One approach which avoids the problem of spinal cord contributions
to CSF levels of a peptide is to obtain ventricular CSF at autopsy. We plan to
collaborate with Neal Cutler, MD , who will send us ventricular fluid from
398
ZOl MH 00086-11 CNG
Alzheimer's disease victims and suitable controls. We plan to measure CRF
SRIF, NPY and galanin. We plan to expand the study of peptide YY in bulimia
by including plasma peptide YY measures in bulimics as well. Additional
subjects may be studied in collaboration with David Jlmerson, MD.
We intend to continue to study the increase in CSF NPY evoked by
physostigmine. If we can make reliable measurements of the increase, we plan
to study this phenomenon in Alzheimer's Disease.
We plan to expand the number of observations in the clonidine study.
Additionally, we propose to study the ill relatives of patients who show the
marker, to determine whether the blunted response segregates with illness in
pedigrees. Lastly, we hope to use a cDNA probe for the alpha adrenergic
receptor to evaluate linkage in some affective disorder pedigrees, using
Southern blotting techniques.
References:
Berrettini W.H. , Nurnberger J.I. , Jr., Simmons -Ailing, S. : Growth hormone
releasing factor in human cerebrospinal fluid. Psychiatry Res. , in press.
Berrettini, W.H. , Doran, A.R. , Kelsoe, J., Roy, A., Pickar, D. : Cerebro-
spinal fluid neuropeptide Y in depression and schizophrenia. Neuropsychopharm.
in press.
Sapin, L.R. , Berrettini, W.H. , Nurnberger, J.I. , Jr., Rothblat, L.A.:
Mediational factors underlying cognitive change and laterality in affective
illness. Biol Psychiatry, in press.
Berrettini W.H. , Simmons -Ailing S. , Nurnberger, J.I. , Jr.: Epidural blood
patch does not prevent headache after lumbar puncture. Lancet i: 856-857, 1987.
Berrettini, W.H. , Nurnberger, J.I. , Jr., Narrow, W. , Simmons -Ailing, S. ,
Gershon, E.S.: Cerebrospinal fluid studies of bipolar patients with and
without a history of suicide. Ann. NY Acad. Scl. 487: 197-201, 1986.
Berrettini W.H. , Nurnberger J.I. , Jr., Hare T.A. , Simmons-Ailing S. ,
Gershon E.S.: CSF GABA in euthymic manic-depressive patients and controls.
Biol. Psychiatry 21: 842-844, 1986.
Berrettini, W.H. , Nurnberger, J.I. , Jr., Zerbe, R.L. , Gold, P.W. , Chrousos ,
G.P. , Tomai, T.: CSF neuropeptides in euthymic bipolar patients
and controls. Brit. J. Psychiatry 150: 208-212, 1987.
Berrettini, W.H. , Nurnberger, J.I. , Jr., DiMaggio, D.A.: Neuropeptide Y
immunoreactlvlty in human cerebrospinal fluid. Peptides 7:455-458, 1986.
Nurnberger, J.I. , Jr., Hamovit, J., Hibbs, E. , Pellegrini, D. , Guroff, J.J. ,
Maxwell, M.E. , Smith, A., and Gershon, E.S.: A high-risk study of primary
affective disorder: Selection of subjects, initial assessment, and 1-2
year follow-up. In Dunner, D.L. , Gershon, E.S. and Barrett, J. (Eds.):
Relatives at Risk for Mental Disorders. New York, Raven Press, in press.
399
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02236-03 CNG
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line betweer) the borders.)
Schizophrenia Studies
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.:
Others;
L.E. DeLisi
E.S. Gershon
L.R. Goldin
C.W. Dlngman
I.D. Dauphinais
M.E. Maxwell
J.R. Hamovit
Staff Psychiatrist
Chief
Senior Staff Fellow
Staff Psychiatrist
NRSA Fellow
Research Social Worker
Research Social Worker
CNG, NIMH
CNG, NIMH
CNG, NIMH
Chestnut Lodge
CNG, NIMH
CNG, NIMH
CNG, NIMH
COOPERATING UNITS (if any)
Springfield Hospital; Chestnut Lodge; Clinical Center, NIH
LAB/BRANCH
Clinical Neurogenetics Branch
SECTION
Section on Clinical Genetics
INSTITUTE AND LOCATION
NIMH, Bethesda, MD 20892
PROFESSIONAL:
1.4
OTHER:
0.8
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
n (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
A study of clinical variables in multiplex families with schizophrenia was
completed. In 53 of these families at least 2 siblings satisfied Research
Diagnostic Criteria (RDC) for schizophrenia.
Brain imaging study: preliminary analysis of Magnetic Resonance Imaging (MRI)
scans of these siblings revealed signficantly reduced hippocampal area in
schizophrenics as compared with related controls, suggesting that this may be
a familial vulnerability factor.
Genetic marker studies: no association of Restriction Fragment Length
Polymorphisms were found in genomic DNA from schizophrenics using several
neuropeptide probes and probes for chromosome 11 (see ZOl MH 02237-03 CNG).
Protein polymorphisms in CSF were examined in 14 unrelated schizophrenics
with a family history of schizophrenia. No abnormalities were found.
401
PHS 6040 (Rev. 1/84)
GPO 91 4-018
ZOl MH 02236-03 CNG
Project Description:
We maintain a structured evaluation system to screen referrals for chronic
schizophrenia and to examine family pedigrees. We have recruited
for multiplex families (families with more than one schizophrenic) throughout
the United States with the help of the National Alliance for the Mentally 111
(NAMI).
Outpatient Clinic for the Treatment of Schizophrenia;
An outpatient clinic was established for pharmacologic treatment of schizo-
phrenic patients who are members of multiplex families. Ten patients presently
receive treatment as part of this clinic. Evaluations of other potential
participants and their families continue.
Genetic Marker Studies:
Studies of potential protein markers in cerebrospinal fluid (CSF)
have yielded negative results. Protein polymorphisms linkage analysis in
lymphoblasts is in progress with Dr. Carl Merril.
A cellular collection of affected-sib-pairs and pedigrees of schizo-
phrenics has been established for DNA marker studies (see ZOl MH 00084-13 CNG).
Brain Imaging Studies:
Our latest brain imaging studies are with the use of the MRI. Twenty-
four schizophrenic patients (11 sibling pairs and 2 unrelated siblings of
schizophrenics) had signficantly reduced bilateral medial limbic area compared
with controls (N = 18).
Schizophrenics Controls T S
(N = 24) (N = 18)
Total
Anterior Limbic Complex
(% Head Size)
Left
1.47 +
.31
1.67 +
.33
1.96
.058
Right
1.41 +
.33
1.63 +
.36
2.01
.05
Posterior Limbic Complex
Left
1.16 +
.27
1.44 +
.47
2.28
.03
Right
1.09 +
.24
1.32 +
.33
2.64
.01
Further analysis of these scans are in progress.
402
ZOl MH 02236-03 CNG
Significance to Biomedical Research and the Program of the Institute
Results from family, twin and adoption studies have strongly suggested
a genetic component to the etiology of schizophrenia. In addition,
environmental factors such as obstetrical complications, seasonality of
birth, viral illness and a history of drug abuse have been implicated with
the later development of schizophrenia. The exact nature of the Inherited
vulnerability and the Interactions with environmental Insults to the etiology
of schizophrenia remains uncertain. Our registry of multiplex families is
an Important resource from which we can continue to study risk factors,
and to evaluate the association of risk factors with biological markers that
might be present in members of one family who have become ill with schizo-
phrenia.
Our MRI study, now well underway, will be an important tool to further
study the structural abnormalities demonstrated in postmortem studies of the
brain in schizophrenia. In addition, the scanning of well siblings from the
same families may establish these structural abnormalities as genetic
vulnerability markers for the development of schizophrenia within these
families.
The value of studying these families Is further enhanced by our maintaining
a collection of lymphoblast cultures for molecular genetic studies, linkage
analysis and RFLP studies.
Proposed Course of the Project
We plan to perform MRI brain scans on well siblings In these familes
and to continue our evaluation of structural abnormalities in brain regions
of interest (limbic regions, basal ganglia, temporal and frontal cortex)
by computer analysis. We will continue to Investigate the significance
of risk factors in this population and our efforts to recruit new families
Into our program with emphasis on large pedigrees for molecular genetic
studies.
References:
DeLisi, L.E. , Nurnberger, J.I. , Jr., Goldin, L.R. , Simmons -Ailing, S. ,
Gershon, E.S.: Epstein-Barr virus and depression. Arch. Gen. Psychiatry
(letter) 43: 815-816, 1986.
DeLisi, L.E. and Crow, T.J.: Viruses and Immune dysfunction in schizophrenia.
In Meltzer, H. (Ed.): Psychopharmacology , the Third Generation of
Progress , New York, Raven Press, in press.
DeLisi, L.E. , Smith, S.B. , Hamovit, J.R. , Maxwell, M.E. , Goldin, L.R. ,
Dingman, C.W. , Gershon, E.S.: Herpes simplex (HSV-1 , HSV-2), cytomegalovirus
(CMV) and Epsteln-Barr (EBV) viral antibody titres in sera from schizophrenics
patients. Psycholog. Med. 16:757-763, 1986.
403
ZOl MH 02236-03 CNG
Goldin, L.R. , DeLisi, L.E. , Gershon, E.S.: The relationship of HLA to
schizophrenia in 10 nuclear families. Psychiatry Res. 20: 69-77, 1987.
DeLisi, L.E. The III Bi-Annual Winter Workshop on Schizophrenia. (Meeting
report). Comment, Arch. Gen. Psychiatry 43: 706-711, 1986.
Goldin, L.R. , DeLisi, L.E. , Gershon, E.S.: Genetic aspects to the biology
of schizophrenia. In: Handbook of Schizophrenia, Vol. 2, Neurochemistry and
Neuropharmacology. Henn, F.A. , DeLisi, L.E. (eds), Elsevier Science Publishing
Co., Amsterdam, August 1987, in press.
DeLisi, L.E. , Buchsbaum, M.S. and Holcomb, H.H.: Increased temperal lobe
glucose utilization in schizophrenia. Biol. Psych. , in press.
Buchsbaum, M.S., Wu , J., DeLisi, L.E. , Holcomb, H.H. , Kessler, R. ,
King, E.G., Hazlett, E. , Post, R.M.: Frontal cortex and basal ganglia
metabolic rates assessed by PET with 18-F-2-DG in affective illness.
J. Aff. Dis. 10: 137-152, 1986.
Buchsbaum, M.S., DeLisi, L.E. , Holcomb, H.H. , Wu , J., Hazlett, E. ,
Cohen, R.M. , Langston, K. , Kessler, R. Comparison of neuroleptic drug
affects and differences between normal controls and schizophrenic patients
with Positron Emission Tomography. Biol. Psychiatry, in press.
Goldin, L.R. , DeLisi, L.E. , Gershon, E.S.: HLA antigens and schizophrenia.
Psychiatry Res. 20: 69-77, 1987.
DeLisi, L.E. and Goldin, L.R. : Small heads and schizophrenia? Arch.
Gen. Psychiatry (letter) 44: 672-673, 1987,
404
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02237-03 CNG
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)
Molecular Genetics of Neuropsychlatric Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
P.I.: S. Detera-Wadlelgh
Others: E.S. Gershon
C. de Miguel
B. SenGupta
W.H. Berrettlnl
L.E. DeLlsl
L.R. Goldln
Senior Staff Fellow
Chief
Visiting Fellow
Guest Researcher
Staff Psychiatrist
Staff Psychiatrist
Senior Staff Fellow
CNG, NIMH
CNG, NIMH
CNG, NIMH
CNG, NIMH
CNG, NIMH
CNG, NIMH
CNG, NIMH
COOPERATING UNITS (if any)
Howard University
LAB/BRANCH
Clinical Neurogenetics Branch
SECTION
Section on Clinical Genetics
INSTITUTE AND LOCATION
NIMH, Bethesda, MD 20892
TOTAL MAN-YEARS,
4.2
PROFESSIONAL:
2.3
OTHER:
1.9
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
n (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
Genetic linkage of affective disorders (AD) to c-Harvey-ras-1 (HRAS 1) and
in insulin (INS) genes on chromosome 11 was examined using three pedigrees
with bipolar illness. Our results indicate the absence of linkage from
0 to 15% recombination which contrasts with the finding of another group who
found linkage to these markers in a large Old Order Amlsh pedigree. The
reason for this discrepancy is not clear. Conceivably these contrasting
findings favor etiological heterogeneity in AD.
Linkage to the color-blindness region, Xq28, was found to be absent in
these same pedigrees using the Stl4 probe-
New restriction fragment polymorphisms (RFLP's) were detected at the gene
regions encoding the gastrin releasing peptide (GRP), substance P, muscarinic
receptor M-1 , muscarinic receptor, M-4 and beta-adrenergic receptor. Analysis
of the linkage relationships between AD and these genes are in progress.
New full length calmodulin cDNA for both human and rat have been isolated and
sequenced. The nucleotide structures of these cDNAs are substantially different
from those that have been reported. These results provide evidence for the
presence of at least two actively transcribed calmodulin genes in these species.
A cDNA clone specific for the 87 kPa substrate of protein kinase C (PKCS-87) has
been isolated from a rat brain expression library using a polyclonal antibody.
405
PHS 6040 (Rev 1/84)
ZOl MH 02237-03 CNG
Other Investigators:
J.
Gelernter
p.
Gejman
T.
Bonner
J.
Patel
D.
Kligman
S.
Anderson
Molecular Genetics
Medical Staff Fellow CNG, NIMH
Visiting Fellow CNG, NIMH
Senior Staff Fellow LCB, NIMH
Visiting Associate BPB , NIMH
Staff Fellow LCB, NIMH
Medical Student Univ. of Connecticut
I. DNA Polymorphisms in Neuropsychiatric Diseases
Objectives
a. To screen for restriction fragment length polymorphisms (RFLPs) at the
loci of proteins with recognized neuroblological importance such as neuro-
peptides, receptors, enzymes involved in neurotransmitter synthesis and
degradation and other proteins required for normal brain function.
b. To use RFLPs in mapping the putative loci for affective illness and
schizophrenia by linkage analysis.
c. To search for association of polymorphic alleles of genes for DNA segments
on the genome with schizophrenia and affective disorder.
Methods Employed
Genomic DNA is extracted and purified from lymphoblast cell lines or
blood samples derived from patients, members of the patients' extended
family and normal (control) individuals using standard procedures. The DNA
is digested using a variety of restriction enzymes, the fragments fractionated
on an agarose gel and eventually transferred to a nylon membrane by Southern
blotting. Hybridization of the DNA with a radioactively labelled cDNA
probe is done and the membrane Is washed. The restriction pattern is
revealed by autoradiography.
Major Findings
la. The possibility of linkage of HRAS 1 and INS genes on the short
arm of chromosome 11 to an AD locus was investigated in three North American
pedigrees that have been collected by the Clinical Neurogenetics Branch.
Haplotypes were derived from the HRAS 1 and INS alleles and because of the
high polymorphism information content (PIC) value of these markers, all
pedigrees were informative. Upon calculation of lod scores using a model
of a dominant Inheritance with high penetrance negative values lower than
-2 were obtained at 0 to 15% recombination. These results Indicate absence
of linkage which is unlike the positive linkage obtained in a large Old
406
ZOl MH 02237-03 CNG
Order Amish pedigree by other investigators. This suggests that there is
more than one gene involved in the etiology of AD. A cDNA probe for the
tyrosine hydroxylase (TH) gene, which also maps to the HRAS 1 region of
chromosome 11 was used to analyze the same AD pedigrees.
lb. Using the same 3 AD pedigrees and Stl4 (a generous gift of
Dr. J.L. Mandel) as probe, linkage of the color-blindess region on xq28 was
examined also. Linkage was negative in this region of the genome.
Ic. The region of chromosome 7q22 to 7q35 was also studied for linkage
to AD using the same 3 CNG pedigrees. Probes to the met oncogene and the
gene for the Beta subunit of the T-cell receptor were employed. So far
the results are indeterminate therefore more pedigrees need to be
examined .
2. Two new RFLPs were identified at the substance P gene. The alleles
found in all samples were analyzed to determine whether any of them is
associated with either AD or schizophrenia. No association was detected,
however, in the manic-depressive and schizophrenic patients studied
although there is a tendency for homozygosity in both patient samples
compared to normals. Similar results were found using the newly identified
GRP RFLP. This tendency for homozygosity although not significant is
intriguing.
3. Using genomic clones for the Ml and M4 muscarinic receptor genes
which were isolated and characterized by Dr. T. Bonner, new RFLPs have been
detected. Polymorphisms were also identified at the beta adrenergic receptor
gene, a clone of which was kindly provided by Dr. Lefkowitz. Highly
polymorphic minisatellite probes are currently being used to search for
linkage in affective disorder and schizophrenia. These probes supplied by
Dr. Jeffreys define approximately 40 loci in human genome, the location
of which are not at present defined. Because they define so many loci
simultaneously, these probes are valuable in the search for linkage.
II. Molecular Cloning of Human and Rat Calmodulin Genes
This project is being done in collaboration with Drs. Banani SenGupta
and Felix Frledberg, Department of Biochemistry, Howard University.
Objectives
1. To determine the structure and organization of human calmodulin
genes.
2. To determine the role of each of the active calmodulin genes
in cellular regulation.
407
ZOl MH 02237-03 CNG
3. To examine the calmodulin locus for the presence of RFLPs and use
these for association and linkage studies in schizophrenia and affective
disorder.
4. To correlate the expression of calmodulin gene in various parts of
the rat brain with development and aging.
Methods Employed
A rat brain lambda gtll cDNA libary kindly provided by Drs. A. Dawsett,
L. Fritz and N. Davidson, California Institute of Technology, was screened
for positively hydridizing plagues using Xenopus laevis calmodulin cDNA
probe from Dr. Igor Dawid , National Institute of Child Health and Human
Development following standard procedures. Clone lambda rCBl was isolated
and purified. It was then digested with EcoRI and subcloned in pUC9 and
M13 using standard methods. Sequencing was done following Sanger's
dideoxynucleotide termination method.
A lambda gtll cDNA library constructed using mRNA from human terato-
carcinoma cell line, NTera2Dl, was kindly supplied by Drs. J. Skowronski ,
and M. Singer, National Cancer Institute. This human libary was screened
with the large EcoRI segment of lambda rCBl in order to isolate calmodulin
hybridizing plagues. One positive plague was purified, subcloned in
M13mp9 and sequenced by Sanger's method. Standard methods were followed
in all the above procedures.
Major Findings:
A cDNA clone, lambda rCBl , encoding calmodulin was isolated from a rat
expression library. The sequence was determined and compared to the
structures of the previously described rat gene, lambda SC4 and lambda SC8.
Faithful sequence conservation is observed in the coding regions of lambda
rCBl and lambda SC4, the bona fide gene. Both cDNAs encode identical
amino acid sequence. Very limited sequence homology, however, is noted
in the 3' untranslated segments of these clones. Surprisingly, when the
lambda rCBl nucleotide structure is compared to the processed itronless
gene, lambda SC8, extensive sequence homology is found both in the coding
and noncoding regions. The inferred protein sequences of lambda SC8 and
lambda rCBl , however, are divergent. Using a fragment of lambda rCBl to
screen an expression library derived from a human embryonic cell line,
calmodulin cDNA, lambda hCEl was cloned and characterized. Comparison of
the sequence of lambda hCEl to calmodulin cDNA from human liver, hCWP ,
reveals substantial structural divergence. Strikingly poor homology is
seen in the 5' and 3' noncoding regions but the coding portions were 85%
homologous. Both lambda hCEl and hCWP encode proteins of identical
primary structure which is equivalent to the protein sequence deduced
from lambda rCBl and lambda SC4. Taken together these results suggest
the existence of an additional actively transcribed calmodulin gene, not
408
ZOl MH 02237-03 CNG
previously Identified, In each of the human and rat genomes. Transcripts
of lambda rCBl and lambda hCEl were observed in all tissues examined
indicating the absence of tissue specific expression. Calmodulin gene
polymorphisms were detected using Taq I, Hind III and Mspl.
III. Molecular Cloning of the Rat 87kDa Substrate of Protein Kinase C
This project is being done in collaboration with Drs. J. Patel and
D. Kligman.
Objectives :
1. To determine the primary structure of PKCS-87.
2. To determine the gene organization of PKCS-87.
3. To study PKCS-87 cDNA in brain development and cellular transformation.
4. To isolate human PKCS-87 cDNA and genomic clones. To use these clones
to search for RFLPs which will be employed in linkage mapping studies.
5. To determine the exact function of PKCS-87 in brain.
Methods Employed:
A rat brain lambda gtll-cDNA library, kindly provided by Drs. A. Dowsett,
L. Fritz and N. Davidson, was screened with a PKCS-87 specific antiserum
after induction of expression by IPTG. After several rounds of screening
a single plague was purified, amplified, subcloned and sequenced by the
Sanger dideoxytermination method.
Subsequent screenings of the library were performed using oligonucleo-
tides prepared on the basis of partial amino acid sequence of several
tryptic peptides.
Major Findings
1. A cDNA clone containing a 600 bp insert was isolated using the
antibody screening method from a rat brain expression library. Upon
subcloning and sequencing the DNA segment was found to encode a peptide
that is rich in alanine residues. This is an Indication that the clone
is authentic because PKCS-87 is composed of approximately 30% alanine.
In order to obtain the full-length sequence other clones are being isolated
using oligonucleotides as probes for screening.
409
ZOl-MH 02237-03 CNG
Significance to Biochemical Research and the Program of the Institute:
Recent linkage studies on an Amish pedigree with bipolar Illness have
provided convincing evidence for the existence of a gene on chromosome 11
that is involved in the etiology of AD. This is a very important finding
because the possibility of identifying a biochemical marker for AD is no
longer remote. Our data as well as those of another group, however,
suggest that in other pedigrees with AD other loci or genes different
from the Amish disease gene might be involved. In view of this, one
universal AD marker might not exist therefore the search for other loci
should continue using more pedigrees and polymorphic probes. A similar
approach to the study of the molecular genetics of schizophrenia must be
undertaken since the underlying biochemical defect is not known in this
disease. A by-product of these studies is the systematic exclusion of
various regions on the human genome as potential genetic markers for
affective disorder and schizophrenia since different kinds of cDNA probes
will be used. Discovery of a DNA marker locus will have an Immense
impact on patient care as well as on the elucidation of the basic abnormality
in these neuropsychiatric disorders.
Calmodulin is a calcium-binding protein with myriad functions In the
cell. It is highly abundant in the brain. The molecule modifies the
activity of various enzymes and receptors which are Involved in neuro-
transmission, growth regulation and other basic functions of the cell.
Other investigators have shown that there are at least two calmodulln-
llke genes in vertebrates. We recently found evidence that in rat and
human at least two actively transcribed calmodulin genes exist which
encode the same protein sequence. In order to study the regulation of
these active genes we are isolating specific genomic clones. The genes
will be studied to determine unique structural differences. We will
attempt to ascertain whether one gene is involved in functions distinct
from that of the other gene. This series of investigations is pertinent
to the understanding of the role of calmodulin in development and
neurological diseases.
Another second messenger system is linked to the calcium/
diacylglycerol/ phospholipid dependent protein kinase C (PKC). This enzyme
is present in very high concentrations in the brain and plays a major role
in the regulation of neuronal excitability. The main substrate of
phosphorylation of PKC in the brain is PKCS-87 whose exact function is not
known. The tumor promoting phorbol esters stimulate the phosphorylation of
PKCS-87 at the same time influence the growth of neurons in culture,
synthesis and release of neurotransmitters and the movement of ions
across the membrane. PKC has been implicated also in the production of long
term potentiation in the hippocampus, a phenomena which is hypothesized to be
related to memory. Cloning of the cDNA and gene encoding PKCS-87 is
important in the elucidation of the role this molecule plays in
learning, memory and disease.
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ZOl-MH 02237-03 CNG
Proposed Course of Study:
In the following year we plan to screen other candidate genes for
new RFLPs. These polymorphisms will be used to study association and
linkage in schizophrenia and affective disorder. Genomic blots derived
from additional AD and schizophrenia pedigrees and sib-pairs collected by
the CNG Branch will be examined for linkage to the INS-HRASl-TH region.
Other probes with high PIC value will be used also. Preparation of
genomic DNA from the new collection of AD and schizophrenia pedigrees and
schizophrenia sib-pairs will be continued.
The structures of the actively transcribed calmodulin genes in humans
will be determined. Regulation of each gene will be studied as it relates
to development and the cell cycle. RFLPs will be determined at the
calmodulin loci so that it can be used as probe in linkage studies.
The full-length cDNA for the PKC substrate protein, PKCS-87, will be
derived from individual clones isolated using both antibody and oligo-
nucleotide screening methods. The expression of PKCS-87 will be examined
in the presence of cellular stimuli. Human PKCS-87 will be isolated also
for RFLP linkage studies.
References
SenGupta, B. , Friedberg, F. and Det era-Wad leigh, S.D.: Isolation and
characterization of calmodulin cDNA from rat brain. Fed. Proc. 45: 1693, 1986.
Detera-Wad leigh, S.D., Berrettini, W.H. , Goldin, L.R. , Boorman, D. ,
Anderson, S. , and Gershon, E.S.: Close linkage of c-Harvey-ras-1 and the
insulin gene to affective disorder is ruled out in three North American
pedigrees. Nature 325: 806-808, 1987.
Detera Wad leigh, S.D., Anderson, S. , and Spindel, E.R.: A frequent PvuII
RFLP of the human gastrin releasing peptide gene. Nuc. Acids Res. 15:
375, 1987.
SenGupta, B. , Friedberg, F. and Detera-Wadleigh, S.D.: Evidence for the
presence of multiple human calmodulin genes. Fed. Proc. 46: 2001, 1987.
SenGupta, B. , Friedberg, F. , and Detera-Wadleigh, S.D. Molecular analysis
of human and rat calmodulin cDNA clones: Evidence for additional active
genes in these species. J. Biol. Chem. , in press.
Detera-Wadleigh, S.D. , de Miguel, C. , Berrettini, W.H. , DeLisi, L.E. ,
Goldin, L.R. and Gershon, E.S.: Neuropeptide gene polymorphisms in affective
disorder and schizophrenia. J. Psych. Res. , in press. Also to appear in
Gershon, E.S. , Cloninger, C.R. , Propping, P., DeLisi, L.E. , Hippius, H. and
Bondy, B. (Eds.): Genetic Research in Psychiatry. Oxford, Pergamon Press,
in press.
411
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00935-20 CNG
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Studies of Plasmids and Small Genomes in Human Cells
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: C.R.Merril Chief, Biochemical Genetics Section CNG, NIMH
Others: L. Mitchell Staff Fellow CNG, NIMH
D. Rath Staff Biologist CNG, NIMH
B. Budowle Chief, Forensic Sci. Research FBI, Academy
COOPERATING UNITS (if any)
Forensic Science Research Group, FBI Academy, Quantico, Virginia
LAB/BRANCH
Clinical Neurogenetics Branch
SECTION
Biochemical Genetics Section
INSTITUTE AND LOCATION
NIMH, Bethesda, MP
20892
TOTAL MAN-YEARS;
2.25
PROFESSIONAL:
1.25
OTHER:
L.
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
^(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Numerous studies of the human mitochondrial genome have proven
the utility of this small, naturally occurring plasmid in
elucidating the relationship between populations. The unique
perspective provided by investigations of this genome depend on
the maternal inheritance pattern of this plasmid and its
relatively high rate of mutation. These characteristics of the
mit-genome have suggested that it may be useful in identifying
individuals and in providing information on the geographic
origin of the individuals maternal ancestors. A collaborative
study is underway with the FBI Forensic Science Research Group
to determine the feasibility of employing the mitochondrial
genome in forensic applications. The methods that are being
developed in this collaborative study are also being used to
examine the rate of mutation of the mit-genome in post-mitotic
tissues, such as the central nervous system. Additionally, these
methodologies are being used to study a group of diseases that
display non-Mendel ian maternal inheritance patterns, which may
be due to mutational events in the mitochondrial genome.
413
PHS 6040 (Rev. 1/84)
GPO B14-Sie
ZOl MH 00935-20 CNG
PROJECT DESCRIPTION
Mitochondrial DNA was initially discovered more than 20
years ago. Since then, it has been determined that this DNA
molecule represents a unique genome 16.5 kb in length which
exists in a closed circular structure. Additional research
has also shown that mitochondrial DNA (mit-DNA) is monoclonal
in origin and has a high mutation rate (5 to 10 times that of
comparable nuclear genes) . This mutation rate may be due to
the apparent lack of both replicative and post-replicative DNA
repair mechanisms in the mitochondria.
The lack of repair mechanisms suggested that the human
mitochondrial genome might serve as a good indicator
concerning the accumulation of mutational events in
postmitotic somatic tissues and in the germ line. Studies of
somatic mutational events may provide insights into
pathophysiological processes while germ line mitochondrial
mutational events have already proved useful in
anthropological studies and they may further our ability in
establishing forensic individuality.
The section has been developing methods to examine the
intra-individual somatic heterogeneity of the mitochondrial
genome in the central nervous system. The brain, a highly
aerobic tissue, relies heavily on its mitochondrial population
for critical metabolic processes. Many of these metabolic
processes create free radicals, which have the potential to
cause mutational events by their interactions with DNA. The
close proximity of the mitochondrial DNA to the sites of
production of free radicals, coupled with the apparent lack of
DNA repair mechanisms, in the organelle suggests that somatic
mutations should accumulate in postmitotic tissues with aging
and in certain disease processes.
Studies of intra-individual somatic cellular
mitochondrial heterogeneity have been initiated by purifying
mit-DNA from human brain tissue samples obtained at autopsy.
The mit-DNA has been digested with restriction endonucleases
and attempts have been made to clone a 63 6bp fragment from one
of the most mutational labile regions of the mitochondrial
genome, the hypervariable region of the D-loop. Despite
numerous attempts only one clone has been obtained which
contains the fragment of interest, despite several repetitions
of the protocol. To detect a reasonable mutation rate by the
RNA-DNA duplex procedure, it is necessary to screen 2 00+
independent clones for base pair mutations. Since this single
clone is inadequate for our study and due to the apparent
refractory nature of this area of the human mitochondrial
genome to cloning [Anderson et al. noted difficulties in
cloning this region during the first sequencing exercise of
the human mit-genome] , a second area, the cytochrome oxidase
414
ZOl MH 00935-20 CNG
subunit II, has been selected for investigation. The D-loop
clone has been stored for possible use in future studies.
The region coding for the cytochrome oxidase subunit II
has an intermediate degree of sequence variability compared to
the hypervariable region of the D-loop. The fragment selected
from the cytochrome oxidase region is 548bp in length and
encompasses 80% of the entire gene. At present, this 548bp
segment has been cloned into a pUC vector and then subcloned
into a pSP vector. The pSP vector contains a SP6 promoter
which allows for the synthesis of RNA probes homologous to the
cloned insert. These probes will then be hybridized to DNA
from 200+ individual clones obtained from the same tissue as
the original clone. If any base substitutions are present,
they will be detected by cleavage with ribonuclease A at the
mismatched regions in the RNA: DNA duplexes followed by
denaturing gel electrophoresis. This method will detect
greater than 70% of the mismatches present and can be used
more efficiently to examine the mitochondrial genome in an
individual for the accumulation of mutational events than
conventional sequencing technologies. In this manner, the
variations which may have accumulated through physiological or
pathophysiological events over the course of a lifetime as a
result of random mutations in a specific region of an
individual's brain can be evaluated.
The section is also developing procedures to permit the
direct sequencing of plasmid DNAs, such as the human mit-DNA.
These sequencing techniques will permit a detailed examination
of any mutational events discovered in the screening program
described above. Direct sequencing with elimination of the
need for cloning should save more than half the time currently
needed to determine a DNA sequence. Development of such
methods may aid in determining the degree nucleotide sequence
heterogeneity within and between individuals.
The section has concentrated its attention to three
regions; the D-loop which is considered to be the most
variable region in the entire human genome, an adjacent t-RNA
region which should be highly conserved, and the cytochrome
oxidase subunit II, which has an intermediate degree of
variability. Clones of these regions from a number of
individuals are being prepared for sequence analysis to verify
the levels of inter-individual variability previously reported
in the literature. These clones are also serving as the
initial source of DNA for the direct sequencing studies since
they provide control sites for the initiation of Sanger
dideoxy sequencing.
With the assistance of Dr. M. Brownstein, we have
acquired a number of synthetic DNA oligomers complementary to
the three previously mentioned mit-DNA regions;
5 ' -GATTCCTGCCTCATCCTATT-3 ' for the D-loop,
415
ZOl MH 00935-20 CNG
5'-TTGACTGTAATGTGCTATGT-3' for the D-loop and proline tRNA,
and 5'-ACAGCTCATGAGTGCAAGAC-3' for the cytochrome oxidase II
regions are representative examples. These oligomers are
being used as primers for the direct sequencing strategy.
Partial mit-DNA sequences have been produced with this
technique. A source of difficulty in achieving an operational
method with this approach has been attributed to the
ribonucleotide residues which are contained within the
mit-genome. Mitochondrial DNA contains an average of five
ribonucleotides per molecule and denaturation of these
molecules with alkali prior to the sequencing reaction often
breaks the molecules at the random locations of these
ribonucleotides, producing anomalies in the sequencing
process. A test concerning the effects of exposure to alkali
on mit-DNA has shown that exposure for periods as short as 3 0
seconds can result in the obliteration of an electrophoretic
mitochondrial band which is clearly present in the untreated
sample. Alternative means of producing single stranded
denatured DNA are being investigated to optimize the
conditions for the sequencing reactions.
Significance to Biomedical Research and the Program of the
Institute:
The mitochondria's relatively high mutation rate and
apparent lack of DNA repair mechanisms may result in the
accumulation of errors in the mitochondrial DNA which, in
turn, may be associated with degenerative alterations in
highly aerobic tissues. Additionally, the methods under
development may be useful in studying some of the maternally
inherited diseases, such as Leber's optic atrophy. Direct
sequence analysis may permit the identification of precise
nucleic acid mutations in the mitochondrial genome which may
be responsible for certain of these diseases.
Proposed Course of the Project;
The direct DNA sequencing and refined hybridization
methodologies which are being adapted to study the
mitochondrial genome should enable us to identify and
rapidly sequence portions of mitochondrial genomes which
demonstrate interesting variations. By examining large
numbers of independent clones from specific regions of the
mitochondrial genome it should be possible to determine
whether or not mutational events accumulate in this genome
with aging, or during disease processes, and whether such
changes are tissue specific. Furthermore, these techniques
should permit enhanced examinations of inter-individual
genomic variation. Such inter-individual genomic variations
may prove useful for the establishment of individuality and
they may also provide some clues as to the maternal
geographic origin of forensic samples. The section has
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ZOl MH 00935-20 CNG
established a collaboration with the Federal Bureau of
Investigation's Forensic Science Research Group to test the
feasibility of using mitochondrial and nuclear DNA's isolated
from forensic samples for identification purposes.
The direct mit-DNA sequencing technique will also be
utilized in studies of a group of diseases which are inherited
in a maternal non-mendellian pattern. Some of these diseases
may be due to mutational events in the mitochondrial genome.
We have initially chosen to study Leber's optic atrophy, a
hereditary blindness which is maternally inherited. Fifty
percent of affected females pass the disease to their children
and an additional 40% of the offspring are carriers. To date
there has not been a report of paternal transmission. In
collaboration with Dr. Kaiser of the NEI, we have obtained
platelets from a family with this disorder, including both
affected and normal individuals. The platelets were collected
by the NIH Blood Bank and we have isolated the mit-DNA from
them for future analysis.
Publications None
417
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00941-07 CNG
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Biochemical Genetics and metabolic diseases.
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
C.R.Merril Chief, Biochemical Genetics Sect. CNG, NIMH
M.G.Harrington Visiting Associate CNG, NIMH
S.Charya Staff Associate CNG, NIMH
COOPERATING UNITS f/r any; NIMH, NINCDS , USUHS , Vanderbilt University, Harvard
Medical School, California Institute of Technology, Baylor College
of Medicine, University of Goteborg Sweden, US NAMRU-11
(Philippines) .
LAB/BRANCH
Clinical Neurogenetics Branch
SECTION
Biochemical Genetics Section
INSTITUTE AND LOCATION
NIMH, Bethesda,
MD 20892
TOTAL MAN-YEARS:
3.5
PROFESSIONAL:
2.5
OTHER:
1
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
]S (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Disease- associated cerebrospinal fluid (CSF) protein changes that were previously identified
by this group have been further investigated: The presence of two 30,000 HW proteins in CSF of
Creutzfeldt-Jakob disease (CJD) patients have been studied prospectively for their usefulness in
the diagnosis of 70 nationally and internationally referred cases: no false positives or false
negatives were found in the 30 cases that have so far come to autopsy (this has included 3 cases
of in-vivo diagnosis of growth hormone transmitted CJD). Further characterization of abnormal CSF
proteins have led to purification of 8 proteins for structural analysis, and one of these, a
glycoprotein, has been partially sequenced. Four peptide fragments, obtained from a tryptic acid
digest, 8 to 18 amino acids in length have been sequenced from this protein. These sequences
represent a new protein that has not previously been characterized in tKe protein sequence data
banks. This glycoprotein has been shown to be quantitatively altered in schizophrenia.
Parkinson's Disease and multiple sclerosis.
Two dimensional electrophoretic survey studies of brain have been initiated with inbred
strains of mice, with a view to developing a protein reference map for both genetic murine
disorders and human brain disorders. Similar studies have led to the observation of protein
alterations in learning mutants of Drosophila. and T lymphocytes infected with the human
iitmunodef iciency virus.
Continued efforts to investigate protein detection methods has permitted us to develop a
physical explanation for the production of specific colors by protein stained with certain silver
stains. Electron microscopic studies coupled with computerized image analysis provided evidence
that the colors are formed by light scattering which is caused by the presence of silver grains
of specific sizes in the gel. Silver grains in yellow bands had an average size of 29.6nm while
those in blue bands were 71.6nm in diameter.
A 19
PHS WMO (Rev 1/84)
GPO Dl 4-01
Collaborators ;
L. DeLisi
D. Dauphonais
E.Gershon
T.Sunderland
D. Asher
D.C.Gajdusek
P . Brown
E.F.Torrey
R.S.Burns
A. Percy
B.Hagberg
G.Watt
T. Folks
I. Hay
R.Brady
L.Hood
S . Kent
R. Aebersold
R.Sidman
P.Neumann
A.Steven
Objectives;
ZOl MH 00941-07 CNG
Assoc. Professor SUNY, Stoneybrook
Staff Fellow CNG NIMH
Chief, Clinical Neurogenetics Branch NIMH
Staff Fellow LCS NIMH
Senior Staff CNSS NINCDS
Chief, Lab. of Central Nerv.Sys. Studies CNSS NINCDS
Neurologist NINCDS
Staff Psychiatrist St. Elizabeths Hosp. Wash.,D.C.
Assoc. Professor Vanderbilt Univ.
Assoc. Professor Baylor College of Med.
Chairman, Dept. of Ped. Univ. of Goteborg, Sweden
Med. Director Navy/ Army Med. Res. Unit 2, Philippines
LIG NIAID
USUHS
NINCDS
of Tech.
of Tech.
of Tech.
Senior Staff Fellow
Professor of Virology
Chief, DMN
Chairman, Biology Dept. California Inst
Scientist California Inst
Scientist California Inst
Prof .Neuropath Dept. Path. Harvard Med. School
Staff Fellow Dept. Path. Harvard Med. School
Senior Staff LPB NIAMS
Investigations of diseases which affect the central nervous
system, such as schizophrenia, would greatly benefit from a
knowledge of the genes and proteins that are active in the human
brain. The technologies that will permit the establishment of the
complex databases required for this task are now becoming
available. The development of high resolution protein separation
technologies [eg. two-dimensional electrophoresis], highly
sensitive protein detection methods [such as silver staining] and
microsequencing methods [eg. as those currently under development
in Leroy Hood's Cal-Tech laboratories] will provide the opportunity
of separating, visualizing, and identifying most of the protein
gene products present in human tissues, and body fluids. These
technologies will also permit studies involving global
relationships between these protein gene products. Furthermore, the
knowledge of even partial sequences of each of the proteins will
permit the interlinking of this protein database with the genomic
DNA databases which are currently being developed.
Within the next decade the complete human genome will be
mapped in some detail, and the efforts to completely sequence the
genome may also be well advanced. The establishment of protein
data-banks will complement the genomic mapping and sequencing
endeavors.
A20
ZOl MH 00941-07 CNG
The section has initiated a brain and spinal fluid protein
database program. Current technology permits the visualization of
1,000 proteins in 40ul of unconcentrated spinal fluid [CSF] . Of
these 1,000 proteins 80 have been identified by co-electrophoresis
and western blotting [immunological methods]. Partial amino acid
sequences have been determined, from proteins isolated from the
gels, for two of the CSF proteins. Of particular interest are 5 CSF
proteins which have only been observed in disease states. The
immediate strategy is to obtain partial sequences for these disease
specific proteins, and to construct synthetic peptides to raise
antibodies for immunological assays, and synthetic oligonucleotides
to screen DNA libraries for the origin of these proteins.
The construction of a mammalian brain protein databank has
been
advanced by the section's studies of protein changes in the nervous
system of mutant mouse stocks. These studies may provide
identifying homologs of human diseases, and they may also help us
achieve a greater understanding of the molecular processes of the
nervous system. From a baseline of normal proteins of regions of
brain, spinal cord and peripheral nerve, identification of
mutant-specific protein changes can be made by a combination of
protein biochemistry and classical mouse genetics/breeding systems.
The section also continues to invest in the development of
methods to separate, visualize and identify proteins. The
introduction of silver-staining by this section, to detect
electrophoretically separated proteins in polyacrylamide gels has
provided a method that, with the most responsive proteins, is more
sensitive by a factor of -100 than Coomassie Blue, the most
commonly used organic stain. With silver staining, most proteins
take on a brownish hue. However, under appropriate conditions,
certain proteins have been found to exhibit distinct and vivid
colors. Yellow, blue, red and green bands have all been observed.
Colorability is a property with considerable analytical potential,
in that it may become possible to infer chemical properties of
proteins on the basis of their propensities for coloration upon
silver-staining. Such information would considerably enhance the
analytical capabilities of gel electrophoresis, which for the most
part have been restricted to estimates of molecular weiights and
isoelectric points. To help realize this potential, we have
investigated the physical basis of the colorability of proteins.
Subjects:
All the serum, tissue and spinal fluid samples were obtained
from patients who were diagnosed by our clinical collaborators with
nationally recognized diagnostic criteria. Inbred mouse and
Drosophila strains were obtained from our collaborators at the
Harvard Medical School.
421
ZOl MH 00941-07 CNG
Laboratory procedures:
The laboratory employs numerous protein purification
procedures, including chromatography, and electrophoresis
methodologies. The electrophoretic methods include both one and two
dimensional electrophoresis. Proteins are detected by silver, dye
and immunological staining.
Progress in the computer-assisted analysis and image
processing of 2D gels has been enhanced by the establishment of a
collaborative effort with Mark Miller and his co-workers in the
NCI. The Image Analytics Corporation (lAC) [Delaware] computerized
laser gel scanner has proven to be fairly reliable and it has
provided linear quantitative data for gel analysis. The
collaborative arrangement with the NCI is providing higher level
gel matching programs then lAC scanner could achieve without their
support .
Electron microscopic analysis of colored silver stained gels
was performed in collaboration with Alasdair Stevens and his
colleagues in the NIAMSD. Silver stained bands were dissected from
gels and examined with a Philips EM300 transmission electron
microscope at a magnification of 16,000X.
MAJOR FINDINGS:
1. CSF protein studies:
(a) . Studies in recent years with our colleagues in the NINCDS have
resulted in the identification of two abnormal 30,000 MW CSF
proteins in patients with Creutzfeldt-Jakob disease. This year, we
initiated a prospective differential diagnosis of study of patients
with suspected CJD dementia. CSF from 70 national and international
cases of suspected CJD have been referred to us at various stages
of their illness, and at present, autopsies have been obtained in
3 0 of these patients. The two abnormal proteins have been
identified in all patients in this study that have been found to
have pathology consistent with CJD. No false positives or
negatives have occurred so far. Three atypical cases of CJD
originating in the recipients of cadaver-derived human pituitary
growth hormone were diagnosed in-vivo, two of whom have been
pathologically verified.
The two abnormal CJD diagnostic proteins (numbered 130 and
131) have been purified by two dimensional electrophoresis. The
purified proteins have been analyzed, following tryptic digestion,
for their peptide patterns. Separation of the tryptic digested
products by HPLC produced similar chromatographic profiles for each
of the two abnormal proteins, suggesting that proteins 13 0 and 131
are very similar. They may only differ by one or a few amino acid
substitutions or by a post-translational modification. Additional
quantities of these proteins are being purified to extend these
studies to obtain partial amino acid sequences.
422
ZOl MH 00941-07 CNG
(b) . In conjunction with our collaborators in NIMH, NINCDS, NIAID,
USUHS, NAMRU 11, Baylor College of Medicine, Got eborg/ Sweden, and
Vanderbilt University, we continue to characterize CSF proteins
that have been identified as being altered in diseases of the
nervous system. The work is at the stage where many of these
proteins are in varying stages of purification. One protein
(25,000 MW) is present in varying degrees of glycosylation in the
normal CSF, and is quantitatively altered from normal in several
diseases studied: Parkinson's disease, schizophrenia and multiple
sclerosis. After purification and tryptic digestion, the amino
acid sequence of four peptide fragments from this protein have been
obtained in collaboration with our collaborators in Leroy Hood's
laboratory at Cal Tech. The sequences range from 8 to 18 amino
acids in length. They form part of the 25,000 MW protein and are
different from all known sequences in the protein sequence data
banks. Studies are in progress to synthesize peptides and
oligonucleotide probes to further characterize this major CSF
protein (circa 5% of the total CSF protein) .
2 . Studies on genetic models of disease;
(a) . In collaboration with Dr. Brady (NINCDS) , we have identified
protein changes in the fly heads of a number of learning mutants of
Drosophila melanogaster. These proteins are being further
characterized .
(b) . In collaboration with Dr. Folks (NIAID) , we have
characterized, in preliminary studies, many protein alterations in
T lymphocytes infected with the human immunodeficiency virus
compared to uninfected cells. We are pursuing this to identify
perturbations in the cell metabolism that are caused by the virus
as compared to both normal cells and other retroviruses.
(c) . In collaboration with Sidman and Neumann (Harvard Medical
School) , we have initiated experiments on protein patterns in the
nervous system of inbred strains of mice. The strategy is to
create a baseline of normal proteins in different brain regions
under different preparative conditions: a protein brain map. This
will allow a future comparison with protein patterns in mutant
mice, and will afford an animal model to study technical questions
that may be relevent to human genetic diseases affecting the CNS.
3. Detection of Proteins
A priori, two mechanisms appeared most likely to be
responsible for color specificity of silver stains observed with
certain proteins: (i) the development of microscopic silver grains
whose size dictates the color; and (ii) the bonding of silver atoms
to functional groups on the proteins to form specific complexes.
In the former case, color would be produced by dif tractive
scattering of light, and in the latter case, by conjugate bond
systems analagous to those in naturally-occurring colored proteins.
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ZOl MH 00941-07 CNG
To distinguish between these hypotheses, we have compared the size
distributions of silver particles visualized in thin section
prepared from protein gel bands of different colors. Mechanism (i)
predicts a pronounced correlation between visible color and the
diameters of the silver grains, whereas, according to mechanism
(ii) , the silver-containing entities responsible for color
generation would always be on the sub-molecular scale (i.e. <lnm) .
Samplings of the silver grains typical of a yellow-staining protein
(human serum albumin) and a blue-staining protein (a minor
satellite band associated with apoliprotein A) demonstrated grains
that were bigger than the 5-15nm micro-grains found sparsely
distributed throughout the (brownish) background areas of the gel.
In the yellow band, these grains are markedly smaller in diameter
(20-40nm, u=29.6nm) and more homogeneous than those in the blue
band (40-lOOnm, u=71.6nm).
Based on these and other observations, we envisage the
following mechanism for colored silver-staining of proteins: the
presence of certain functional sites or groups will, under
appropriate development conditions, nucleate globular silver grains
that grow to sizes whereby their light-scattering properties
emphasize specific colors. Within this overall scenario, the
precise nature of the nucleation event (s) as well as of other
aspects of the process have still to be determined, although it has
been established that a high incidence of certain amino-acids in a
protein - notably basic residues or cysteines - will strongly
influence its colorability.
It is intriguing that, in contrast to the familiar sheen of
bulk metallic silver, colloidal suspensions of microscopic silver
particles may produce many different colors. In this respect,
colored silver-staining of proteins appears to be broadly analagous
to silver-based color photography.
Significance to Biomedical Research and to the Program of the
Institute;
The development of protein databases based on their
electrophoretic separation with two-dimensional electrophoresis
[which potentially provides information on their mass, charge and
quantitation] could facilitate research on the pathophysiology of
diseases of interest to the NIMH, such as schizophrenia.
The section has initiated efforts, including a number of pilot
experiments, to develop a brain database. We have, in collaboration
with David Jacobowitz, examined protein patterns in 25 different
regions of the rodent brain. In these studies, we were able to
demonstrate brain region-specific protein patterns. We have also
found disease specific proteins in human spinal fluid and are
currently collaborating with Dr. Hood's Cal Tech. group to sequence
these proteins so that immunological and DNA probes can be
constructed. These probes will serve as useful diagnostic aids and
will help to futher our understanding of these diseases.
424
ZOl MH 00941-07 CNG
The section's continued efforts to improve protein separation
and detection methods has led to an enhanced understanding of why
certain proteins produce specific colors with silver staining.
Color is a property with considerable analytical potential. As we
learn more about the chemistry of silver staining it may become
possible to determine chemical properties of proteins based on the
proteins ability to produce a specific color or staining reaction
with silver. This type of information would considerably enhance
the capabilities of gel electrophoresis, both as an analytical and
diagnostic tool.
Proposed course of the Project;
The section plans further development of brain and spinal
fluid protein databases based on electrophoretic separation
technologies and protein detection by silver staining. The
section's
computerized microdensitometry equipment, has proven insufficient
for the analysis of the large amount of data that is currently
being collected for these databanks. It is hoped that the current
up-grading of the section's computer facilities coupled with the
collaborative arrangement that has been established with image
processing groups in the NCI will alleviate these problems. Brain
proteins that display quantitative or qualitative alterations after
viral infection or after the administration of certain drugs, such
as the neuroleptic (antischizophrenic) drugs, chlorpromazine and
haloperidol, may warrant detailed investigations. Knowledge of
region-specific proteins could also permit molecular investigations
that would complement the current PET scan studies of abnormal
brain metabolism.
Microsequencing of disease specific proteins and
physiologically important proteins will be conducted in
collaboration with Leroy Hood's Cal Tech. laboratory. The sequences
obtained will be used to determine whether the disease specific
proteins are endogenous or exogenous [possible viral origin] . They
will also provide information for the construction of peptides to
stimulate antibody production [for diagnostic assays] and DNA
oligonucleotide probes [to determine their origin] . Furthermore,
the sequences of the proteins of interest will permit
cross-referencing with the genomic DNA databases.
The section plans to continue to develop methods to enhance
our ability to resolve and identify individual proteins from
tissues and body fluids. Currently, it is often possible to
separate and visualize up to 5,000 proteins on a single
electrophoretic gel from a tissue sample with high resolution 2DE
and silver staining.
425
ZOl MH 00941-07 CNG
Publications;
Merril,C.R. and Pratt,M. : A rapid sensitive protein silver stain
and assay system for proteins on membranes. Anal .Biochem. 156;
96-110, 1986.
Harrington,M.G. , Merril,C.R., Asher,D.M., and Gajdusek,D.C. ;
Cerebrospinal fluid proteins distinguish Creutzfeldt-Jakob disease
from other human dementias. New England Journal of Medicine, 315;
279-283, 1986.
Merril,C.R.; Recent advances and applications in the art and
science of detecting Proteins and Nucleic acids by Silver Staining.
Electrophoresis ^86 (ed. Dunn, M. J.) Verlagsgesellschaft, Weinheim
West Germany; 273-290, 1986.
Merril,C.R. Harasewych, M.G. and Harrington, M. G. ; Protein staining
and Detection Methods. Analytical Gel Electrophoresis of
Proteins, (ed. Dunn, M.J.) Wright, Bristol: 323-362, 1986.
Merril,C. R. ; Development and Mechanisms of Silver Stains for
Electrophoresis. Acta Histochem. Cytochem. 19; 655-667, 1986.
Merril,C.R.; Silver Stain Detection of Proteins Separated by
Polyacrylamide Gel Electrophoresis. In; New Directions in
Electrophoretic Methods, ACS Symposium Series 335, (ed.
Jorgenson, J.W. , and Phillips, M. ) , American Chemical Society,
Washington, D.C.: 74-90, 1987.
Baumgold,J., Merril,C.R., and Gershon,E.S. ; Loss of pirenzepine
regional selectivity following solubilization and partial
purification of the putative M and M muscarinic receptor
subtypes. Molecular Brain Research 2; 7-14, 1987.
Merril,C.R.; Detection of Proteins by staining. Advances in
Electrophoresis . In Press.
Goldman, D., and Merril,C.R.; Protein Polymorphisms Detected by Two
Dimensional electrophoresis; An Analysis of Overall Informativeness
of a Panel of Linkage Markers. In Press.
Merril,C.R., and Harrington, M. G. ; Two-Dimensional Protein Maps in
Studies of schizophrenia. Schizophrenia Bulletin, National
Institute of Mental Health. In Press.
Gershon, E.S., Merril,C.R., Goldin,L.R., Delisis,L.E. ,
Berrettini,W.H. , and Nurnberger, J. I . Jr. ; The Role of Molecular
Genetics In Psychiatry. Biological Psychiatry. In Press.
Merril,C.R., Bisher,M.E., Harrington,M. , and Steven, A. C;
Observations Concerning the Colors of Silver-Stained Protein Bands
in Polyacrylamide Gels and the Development of Silver Grains of
Different Sizes. Proc. Natl. Acad. Sci. U.S.. In press.
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ZOl MH 00941-07 CNG
Harrington, M.G. and Kennedy, P. G. E. : The use of Cerebrospinal Fluid
Protein Studies in Demyelinating Diseases. Postgrad. Med. Jour. .
In Press.
Croxson,M. , Brown, P., Synek,B., Harrington,M.G. , Merril,C.R.,
Firth, R., Clover, G., Wilson, J. and Gajdusek,D.C. : A new case of
Creutzf eld-Jakob Disease Associated with Human Growth Hormone
Therapy in New Zealand. Neurology. In Press.
Marzewski,D. , Tofigi,R., Harrington,M.G. , Merril,C.R., and
Brown, P.: Creutzf eld-Jakob Disease Following Pituitary-Derived
Human Growth Hormone: A New American Case. Neurology. In Press.
Patent Pending:
NIH # E-191-87 diagnostic test for Creutzf eld- Jakob Disease.
427
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01836-09 NS
PERIOD COVERED
October 1, 1986 to September 30, 1987
TTTLE OF PROJECT (80 charmcfn or haa. TMa muat Htooonalint (M(H««n tfM bonHn.)
GABA/Receptors in the Central Nervous System: Biochemistry to Behavior
PRINCIPAL INVESTIQATOR (Utt othtr prolaasional panonnal bttow tfM Principal krvsttgrnlor.) (Nam*. ttOa. laboratory, and kamuta aimatlon)
PI: S.M. Paul, Chief, NS, NIMH
Others: J. Crawley Sr Staff Fellow NS, NIMH
S. Cottingham PRAT Fellow/Guest Researcher NS, NIMH
P. Montpied Visiting Fellow NS, NIMH
A. Lingford-Hughes Visiting Fellow - NS, NIMH
P. Suzdak PRAT Fellow NIGMS, NIMH
A. Morrow PRAT Fellow NIGMS, NIMH
S. Deutsch PRAT Fellow NIGMS, NIMH
COOPERATING UNITS (it any)
Laboratory of Bioorganic Chemistry, NIADDK; Section on Molecular Pharmacology, NS,
NIMH; Unit on Molecular Neurogenetics, NS, NIMH; Pharmacology Research Associate
Training Program, NIGMS; LSU School of Medicine, New Orleans, LA
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Preclinical Studies
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
4.0
PROFESSIONAL
3.5
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
n (a2) Interviews
D (b) Human tissues U (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the apace provided.)
Benzodiazepines interact with a specific neuronal membrane receptor to initiate a
series of neuronal events resulting in an enhancement of GABA-mediated chloride
permeability. The latter results behaviorally in the major pharmacological
actions of benzodiazepines, namely their anxiolytic, anticonvulsant, hypnotic and
muscle relaxant actions. In addition to benzodiazepines, a variety of
sedative/hypnotic agents of the minor tranquilizer class (e.g. the barbiturates)
appear to interact with one or more components of the benzodiazepine/GABA receptor
complex, and thus the latter has been proposed as a common site of minor
tranquilizer action. Several aspects of the benzodiazepine/GABA receptor complex
are currently being studies. Recent work has employed an in vitro system for
measuring GABA receptor-effector coupling in a subcellular preparation from rat
brain (the synaptoneurosome) . This technique has greatly facilitated studies on
the regulation of the GABA receptor-coupled chloride ion channel. Using this
method, we have studied the interaction of ethanol with the GABA receptor complex
and have found that ethanol, related short-chain alcohols and several anesthetic
agents are capable of stimulating this receptor and at pharmacologically-relevant
concentrations. In related studies we have identified a novel
imidazobenzodiazepine, Rol5-4513, wfeich blocks both the in vitro effects of
ethanol on GABA receptor-mediated CI" uptake as well as many of the behavioral
effects of ethanol. Chronic administration of ethanol to rats results in a
decrease in GABA receptor-mediated CI' uptake in synaptoneurosomes, an effect
that is reversible since it is not observed after the ethanol withdrawal syndrome.
In other studies we have examined the use of the radiolabelled benzodiazepine
receptor antagonist Rol5-1788 for measuring benzodiazepine receptors in vivo. Our
results have validated the suitability of this technique and have demonstrated
significant effects of barbiturates, naturally-occurring steroid hormones, ethanol
and "stress" on benzodiazepine receptors invivo.
PHS 6040 (Rov 1/84) CP0..4HI..
ZOl MH 01836-09 NS
PROFESSIONAL PERSONNEL:
A. Weizman Guest Researcher NS, NIMH
R. Weizman Visiting Scientist NS, NIMH
F. Vocci Guest Researcher NS, NIMH
B. Martin Visiting Scientist NS, NIMH
E. Ginns Neurologist/Biochemist NS, NIMH
P. Skolnick Pharmacologist LBC, NIADDK
L. Miller LSU
PROJECT DESCRIPTION
Objectives:
1. To characterize the interaction of anxiogenic and anxiolytic compounds with
the benzodiazepine/GABA receptor complex at the molecular/cellular,
neurophysiologic and behavioral levels.
2. To understand the mechanism{s) responsible for the stress-induced
"sensitization" and drug-induced desensitization of this receptor complex
(including the possible involvement of steroid hormones); and to unravel whether
changes in the ionophore underlie the development of tolerance to
sedative/hypnotic/anxiolytic drugs.
3. To use the GABA receptor-coupled CT ion channel complex as a model of
other neurotransmitter-gated ion channels.
4. To explore basic neurochemical mechanisms of anxiety, fear and stress as
they relate to the many clinical and medical problems associated with stress.
Methods Employed:
(See: 1986 Annual Report, Project Number ZOl MH 01836-08 NS, GABA/Receptors in
the Central Nervous System: Biochemistry of Behavior)
Ma.ior Findings:
Agents that perturb one or more of the components of the benzodiazepine-GABA
receptor chloride ionophore complex (e.g., benzodiazepines, GABA and GABAmimetics
like muscimol and barbiturates) have been shown to increase chloride conductance
in both electrophysiological preparations and intact cells. Attempts to develop
a quantitive measurement of chloride flux in a "cell-free" preparation (in order
to explore the functional relationships between the binding sites associated with
the receptor complex have generally been unsuccessful. We have developed a
method for measuring CI' flux in "filtered synaptoneurosomes. " The filtered
synaptoneurosome preparation was employed because it has been shown to contain
both presynaptic nerve endings and attached postsynaptic densities. We have
previously shown that barbiturates, including pentobarbital, cause a
concentration dependent increase in the efflux and uptake of CI' efflux from
synaptoneurosomes which is reversed by the chloride ionophore antagonist.
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ZOl MH 01836-09 NS
picrotoxin. A good correlation (r = 0.90, p<0.01) was observed between the
potencies of a series of barbiturates in increasing CI' efflux from preloaded
synaptoneurosomes and their anesthetic potencies in mice. Barbiturates also
potentiate the stimulation of CI" uptake induced by GABA agonists such as
muscimol. In addition to barbiturates, a number of naturally-occurring
substances including the A ring reduced metabolites of progesterone and
deoxycorticosterone (3 alpha 5 alpha dihydroprogesterone and
tetrahydrodeoxycorticosterone) have been found to be potent barbiturate-like
modulators of the GABA receptor-gated chloride ion channel. The latter have also
been shown to have anxiolytic and hypnotic effects in rats and mice.
Work over the past several years has documented that both benzodiazepines and
barbiturates stimulate CT efflux or uptake via a pharmacologically-relevant
GABA/\ receptor. Since the latter drugs show cross-tolerance and cross-dependence
with alcohol we have further examined the effects of ethanol (the most commonly
used anxiolytic/hypnotic/intoxicant) on the GABA receptor coupled CI' ion
channel. Our data demonstrate that ethanol at concentrations from 20 to 100 mM
stimulate CT uptake via the GABA/\ receptor, since this effect is blocked by
the GABA;\ receptor antagonists bicuculline and picrotoxin and not by a variety of
other neurotransmitter receptor antagonists. In addition to ethanol, many short-
chain alcohol and anesthetic agents tested stimulate CT uptake. These data
suggest that ethanol (and related alcohols) produce at least some of their
behavioral effects via an interaction with the GABA receptor-coupled CT ion
channel and most likely by altering the membrane (1 ipid/protein) microenvironment
of the receptor, rather than directly binding to the receptor protein itself. In
related experiments we have found that the imidazobenzodiazepine Rol5-4513, which
is a derivative of thfigbenzodiazepine antagonist Rol5-1788, blocks the ability of
ethanol to stimulate CT uptake in vitro. Moreover, Rol5-4513 fails to block
either muscimol- or pentobarbital -stimulated CT uptake at concentrations <1
uM. In behavioral studies, Rol5-4513 also blocks the effects of low (1 g/kg) as
well as moderate (2 g/kg) doses of ethanol in rodents; but not higher (>4 g/kg)
doses of ethanol. The effects of Rol5-4513 in blocking both ethanol -stimulated
CT uptake in vitro and ethanol -induced intoxication in vivo were not mimicked
by the full inverse agonist BCCE or the partial inverse agonist FG-7142. The
latter finding suggests that Rol5-4513 has "selective" anti -alcohol actions
heretofore not observed with other benzodiazepine receptor inverse agonists.
Together, these data support the hypothesis that many of the neuropharmacological
effects of low to moderate doses of alcohol are mediated via augmentation of
central GABAergic neurotransmission. Chronic administration or treatment with
sedative/hypnotic drugs (barbiturates, benzodiazepines, ethanol) is generally
associated with the development of pharmacodynamic tolerance. However, the
mechanisms for this tolerance are obscure. Recently, we have shown that chronic
barbiturate or ethanol administration-to rats results in a 25-35% decrease in the
apparent V of muscimol -stimulated CT uptake in cerebral cortical
synaptoneurosomes. Following chronic ethanol administration, this
"subsensitivity" in GABA receptor function is highly correlated to the blood
ethanol level and with the development of the withdrawal syndrome.
Despite strong evidence that suggests benzodiazepine receptors mediate the
antianxiety (anxiolytic) actions of the benzodiazepine (e.g., diazepam,
431
ZOl MH 01836-09 NS
chlordiazepoxide) and related compounds, the physiological function(s) of these
sites is unclear. Several lines of evidence now suggest that these receptors may
play a role in the control of arousal and the central "stress" response. We have
recently demonstrated that several stressors (forced ambient temperature swim,
brief immersion in ice water, and food deprivation for the first three hours of
the dark cycle) elicit a rapid and robust change in [-^HJbenzodiazepine binding
that is observed only in the presence of Eccles' permeable anions (e.g.,
chloride, iodide and bromide ions). These differences are manifest as an
increase in the apparent affinity of [-^Hjflunitrazepam, with no significant
differences in the maximum number of binding sites (B ) between the groups.
Both an increase in the maximum enhancement of [-^Hlflunttrazepam binding in
response to optimum concentrations of halide ions (Emay) s^d an increased
sensitivity to halide ions (reduced (EC50) were observed in response to stress.
These results suggest that acute exposure to stress effects either the coupling
of the chloride ionophore and benzodiazepine receptor, or that the chloride
ionophore 4iself in some way modified. Further, the ability of muscimol to
stimulate CI" uptake in vitro is enhanced in synaptoneurosomes prepared from
"stressed" rats when compared to stress-habituated controls. The effects of swim
stress in potentiating muscimol -stimulated CI" uptake were attenuated in
adrenalectomized animals suggesting a "permissive" role for glucocorticoids in
mediating this stress response. Recently, using an in vivo technique for
labelling the benzodiazepine receptor (using [-^HlRolS-nSS) we have observed
biphasic effects of swim stress on benzodiazepine receptor occupancy. Acute swim
stress increases specific [3h]Ro15-1788 binding immediately (< 1 hr) after stress
whereas specific binding is decreased in many brain regions 24 hours after
stress. Similar acute effects were observed after "defeat stress" another form
of social stress. The possible role of adrenal steroid secretion in mediating
the "delayed" effects of acute or chronic stress are currently being
investigated.
Many membrane associated receptors have been shown to be sensitive to alterations
in their lipid milieu. Changes in membrane lipids induced by activation of
phospholipase A? (PLA2) (an endogenous constituent of membranes) has been
proposed as a physiologic mechanism for regulating receptor function. We have
shown a differential sensitivity of "peripheral" and "central" benzodiazepine
receptors to this enzyme. Phospholipase A2 slightly increased the apparent
affinity of the central benzodiazepine receptor ligands [-^Hlflunitrazepam and
[^H]3-carboethyoxy-B-carboline, with no concomitant change in the B of these
ligands. In contrast, GABA enhanced [^HJflunitrazepam was unaffected by PLA2.
Both pyrazolopyridine and barbiturate enhanced [-^Hlflunitrazepam binding were,
however, reduced by very low (0.002 U/ml ) concentrations of PLA2. Since both
pyrazolopyridines and barbiturates bind to sites at or near the chloride
ionophore. we examined the effects of PLA2 on the specific chloride ionophore
ligand ['^^Sit-butylbicyclophosporothionate (TBPS). It was found that PLA2
inhibited [ S]TBPS binding at the same concentrations needed to disrupt
barbiturate and^pyrazolopyridine enhanced [^HJflunitrazepam binding. The
inhibition of [ S]TBPS binding by PLA2 was manifest as a reduction in the B^
of this ligand with no change in the apparent affinity. PLA2 was also found 10
reduce the apparent affinity of [2h]Ro5-4854 to "peripheral" benzodiazepine
receptors and the reduction in the apparent affinity of [•^H]Ro5-4864 was
432
ZOl MH 01836-09 NS
independent of the tissue source (e.g. the same reduction in apparent affinity
was found in heart, kidney and brain membranes).
On screening extracts of various tissues for inhibitors of [^H]Ro5-4864 binding
we isolated and purified a 14 kDa protein "antral in" which was subsequently shown
to have PLA2 activity. Recently, "antral in" has been purified to homogeneity and
sequenced using automative Edmann analysis of tryptic fragments. Antralin was
shown to be a member of the PLA2 family based on its amino acid sequence.
Substrate and Ca dependency supported its PLA2-like activity. Antibodies
raised against antralin also cross-react with purified porcine pancreatic PLA2.
Studies on the possible physiological role of this protein in peripheral organs
(stomach) and brain are currently underway.
Recently, the nucleotide sequence (along with the deduced amino acid sequence) of
both the alpha and beta subunits of the bovine GABA/^ receptor have been
established through classic cloning techniques. Using this sequence we
synthesized several oligonucleotide probes for both the alpha and beta subunit
and screened a GT,, cDNA library prepared from human brain. Several near full
length cDNA clones for the alpha subunit have been isolated and have been used as
probes to study the expression of receptor protein during or following various
pharmacological and environmental conditions.
PUBLICATIONS:
1. Arendt RM, Greenblatt DJ, Liebisch DC, Luu MD, Paul SM. Determinants of
benzodiazepine brain uptake: lipophilicity versus binding affinity.
Psvchopharmacoloqy 1987;93:72-6.
2. Arora PK, Hanna EE, Paul SM, Skolnick P: Suppression ofthe immune response
by benzodiazepine receptor inverse agonists. J Neuroimmunol , in press.
3. Havoundjian H, Reed GF, Paul SM, Skolnick P: Protection against the lethal
effects of pentobarbital in mice by a benzodiazepine receptor inverse
agonist, 6,7,-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline. J Clin
Investigation, 1987;79:473-7.
4. Hoffman PL, Tabakoff B, Szabo G, Suzdak PD, Paul SM: Effect of an
imidazobenzodiazepine, Rol5-4513, on the incoordination and hypothermia
produced by ethanol and pentobarbital. Life Sci 1987;41:611-9.
5. Mantione CR, Goldman ME, Bolger GT, Lueddens HWM, Paul SM, Skolnick P:
Purification and characterization of an endogenous protein modulator of
radioligand binding to "peripheral -type" benzodiazepine receptors and
dihydropyridine Ca++-channel antagonist binding sites. Biochem Pharmacol ,
in press.
5. Miller LG, Greenblatt DJ, Abernethy DR, Friedman H, Luu MD, Paul SM, Shader
RI: Kinetics, brain uptake, and receptor binding characteristics of
flurazepam and its metabolites. Psvchopharmacoloqy. in press.
433
ZOl MH 01836-09 NS
7. Miller LG, Greenblatt DJ, Barnhill JG, Deutsch SI, Shader RI, Paul SM.
Benzodiazepine receptor binding of triazolobenzodiazepines in vivo:
increased receptor number with low-dose alprazolam. J Neurochem
1987;49{5):1595-1601.
8. Moody EJ, Suzdak PD, Paul SM, Skolnick P: Inhalation anesthetics increase
CI "uptake into rat brain synaptoneurosomes. Eur J Pharm, in press.
9. Morrow AL, Paul SM: Benzodiazepine enhancement of gamma-aminobutyric acid
mediated CI' ion flux in rat brain synaptoneurosomes. J Neurochem, in
press.
10. Morrow AL, Suzdak PD, Karanian JW, Paul SM: Chronic ethanol administration
alters gamma-aminobutyric acid, pentobarbital and ethanol -mediated CI"
uptake in cerebral cortical synaptoneurosomes. J Pharmacol Exp Ther. in
press.
11. Morrow AL, Suzdak PD, Paul SM: Steroid hormone metabolites potentiate GABA
receptor-mediated chloride ion flux with nanomolar potency. Eur J Pharmacol
1987;142:483-5.
12. Paul SM, Crawley JN, Skolnick P: The neurobiology of anxiety: the role of
the GABA/benzodiazepine receptor complex. In: American Handbook of
Psychiatry, in press.
13. Schwartz RD, Skolnick P, Paul SM: Regulation of GABA/barbiturate receptor-
gated chloride ion flux in brain vesicles by phosphol ipase A2: Possible
role of oxygen radicals. J Neurochem, in press.
14. Schwartz RD, Seale TW, Skolnick P, Paul SM: Differential seizure
sensitivities to picrotoxinin in two inbred strains of mice (DBA/2J and
BALB/c ByJ): Parallel changes in GABA receptor-mediated chloride flux and
receptor binding. J Neurochem, in press.
15. Schwartz RD, Wess MJ, Labarca R, Skolnick P, Paul SM: Acute stress enhances
the activity of the GABA receptor-gated chloride ion channel in brain.
Brain Res 1987; :411 :151-5.
16. Skolnick P, Havoundjian H, Paul SM: Benzodiazepines and their receptors in
anxiety disorders. In: Receptors and Liqands in Psychiatry and Neurology,
Sen AK, Lee T (eds). Cambridge, Cambridge University Press, in press.
17. Skolnick P, Paul SM: Benzodiazepines and nonbenzodiazepines. In: O'Brier
RA (ed). Receptor Binding in Drug Research. New York, Marcel Dekker, Inc.,
1987, pp 53-75.
18. Suzdak PD, Glowa JR, Crawley JN, Schwartz RD, Skolnick P, Paul SM: A
selective imidazobenzodiazepine antagonist of ethanol in the rat. Science
1986;234:1243-7.
434
ZOl MH 01835-09 NS
19. Suzdak PD, Schwartz RD, Skolnick P, Paul SM: Alcohols stimulate gamma-
aminobutyric acid receptor-mediated chloride uptake in brain vesicles:
correlation with intoxication potency. Brain Res, in press.
20. Suzdak PD, Paul SM, Crawley JN: Effects of Rol5-4513 and other
benzodiazepine receptor inverse agonists on alcohol -induced intoxication in
the rat. J Pharmacol Exp Ther. in press.
435
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02186-05 NS
PERIOQ COVERED
October 1, 1986 to September 30, 1987
^ OF PPOJECr (80 chvuctars or leas. THh must a on onalnab»twMnth»bonian.) Brd I fl ReCOgn 1 LI Oil SILeS foT
imulants and Antidepressants: Relationship to Pharmacological Activity
^r
PfUNCIPAL INVESTlQATOfl (tM oOm pnjlouion^ pamnnal btlow th» Principal InyuMgtor.} (Nam; Oh. labontory, and InaOuta aimaOon)
Ki: S.M. Paul ' Chi'^ef
Other: J.N. Crawley Senior Staff Fellow
E. Lestringant Visiting Associate
G. Muscettola Visiting Scientist
P. Skolnick Pharmacologist
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
LBC, NIADDK
COOPERATING UNITS (» any)
Laboratory of Bioorganic Chemistry, NIADDK; Section on Molecular Pharmacology, NS,
NIMH
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Preclinical Studies
INSTITUTE ANO LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.0
PROFESSIONAL
3.5
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
ID (b) Human tissues S (c) Neither
SUMMARY OF WORK (Use standanl unraducad type. Do nor exceed the space pmvkied.)
Recognition sites for a variety of psychotherapeutic drugs have been identified in
the central nervous system. Over the past several years we have attempted to
identify recognition sites for other common psychotropic drugs including tricyclic
antidepressants and the psychomotor stimulants, amphetamine and methylphenidate.
In each case saturable, and stereospecific binding sites have been delineated; and
for amphetamine and methylphenidate relatively good correlations have been
observed between the affinities of a series of analogues in vitro and at least
some of the pharmacological properties of these agents. Recent work has shown
that the [3H] (+)-amphetamine binding site in hypothalamic membranes is sensitive
to circulating levels of blood glucose. Hypoglycemia decreases, and hyperglycemia
increases, the number of [3H] (+)-amphetamine binding sites in hypothalamic
membranes respectively. Furthermore, these changes seemed to be coupled to th#
activity of (Na+ K+) (ATPase; and there is a good correlation between the changes
in [3H] (+)-amphetamine and [3H]-ouabain binding both in vivo and in vitro. More
recent studies have shown that [3H]-mazindol a chemically unrelated
anorectic/psychostimulant also can be used to label the [#H] (+)-amphetamine
cognition site and that there is a good correlation between the inhibition of
[3H]-mazindol binding by a series of phenylethylamines and their anorectic
otencies in rats. These data suggest the existence of a membrane-bound receptor
complex capable of "sensing" circulating glucose concentration and in regulating
both glucostatic ingestive behavior and perhaps some aspects of the central
regulation of energy metabolism. Recent work has demonstrated that genetically
obese mice (ob/ob) have an abnormal i#y in this system and fail respond to
glucoprivic feeding signals. Over the past year, we have developed a method for
measuring oubain-sensitivew °°Rb uptake into synaptoneurosomes and have used this
method to measure "sodium pump" activity after treatment with anorectic drugs and
in genetically obese rodents.
437
PHS 6040 (Rev. 1/84)
CPO 91 4-«1
ZOl MH 02186-05 NS
Pro.iect Description:
Objectives:
1. To elucidate the mechanisms of action of important psychotropic drugs such
as as the psychomotor stimulants, anorectic agents and antidepressants.
2. To understand the neurochemical change associated with animal models of
hyperphagia and obesity.
Methods Employed:
(See: 1984 Annual Report, pp 707-712 Project Number ZOl MH 02816-02 NS, Brain
Recognition Sites for Stimulants and Antidepressants: Relationship to
Pharmacological Activity.)
Ma.ior Findings:
High affinity, stereospecific binding sites for [3H] mazindol have been
previously described in rodent brain. The highest density of these sites are
found in the synaptosomal fraction of brainstem and hypothalamus. A good
correlation has been demonstrated between the ability of a series of
phenylethylamine derivates in displacing [3H] mazindol binding and their
potencies as anorectic agents. These observations suggest that the [3H]-
mazindol binding site (anorectic drug recognition site, ADRS) may be involved in
the appetite suppressant actions of chemically unrelated anorectic agents. The
ADRS has also been studied in genetically obese mice (ob/ob). In these animals
the density of hypothalamic ADRS is greater than in lean litter mates.
Furthermore, food deprivation of rats (24-72 hours) results in a dramatic (35-
50%) reduction in the density of hypothalamic ADRS. Refeeding food-deprived
animals for a four hour period (or allowing access to a 10% glucose solution)
results in a return of ADRS density to control values. These data suggest that
the ADRS may be intimately involved in the regulation of feeding behavior. In
more recent experiments the changes in ADRS during food deprivation and refeeding
have been localized to discrete hypothalamic and brainstem nuclei
(paraventricular nucleus and nucleus tractus solitarius).
Since the ADRS in hypothalamus is decreased following 24 hours of food
deprivation (30% reduction in (Bmax), and the site density restored to control
levels if the animals are permitted to refeed for four hours we have examined the
factors responsible for this rapid modulation in site number. Good correlations
between circulating glucose concentration and the cnanges in ADRS during food
deprivation and refeeding suggest that glucose (or a metabolite) regulate the
density of ADRS.
Injection of 2-deoxy-D-glucose also elicits a significant increase in ADRS in the
hypothalamus and brainstem of rats and mice. However, this treatment did not
alter ADRS in other brain regions. Further, injection of 2-deoxy-D-glucose
elicited increases in ADRS density that were again confined to the PVN and NTS.
Interestingly, if animals are permitted access to food during the four hour
A38
ZOl MH 02186-05 NS
interval following injection of 2-deoxy-D-glucose, no increase in ADRS is
observed. These observations suggest that the ADRS in hypothalamus are coupled
to glucose utilization and "hunger". The good correlation previously reported
between the ability of a number of phenethylamines to inhibit ADRS and their
potencies as anorectics may thus link the anorectic actions of phenethylamines
with their ability to effect glucose-responsive neurons in the hypothalamus.
Although the density of ADRS in hypothalamus is increased in genetically obese
(ob/ob) mice the latter fail to increase food intake following administration of
2-deoxy-D-glucose. Further no further increase in hypothalamic ADRS is observed
in ob/ob mice following 2-deoxy-D-glucose administration.
In related experiments the regulation of ADRS in hypothalamic tissue slices in
vitro have confirmed that glucose plays a major role in determining the density
of ADRS. Moreover, the ability of glucose to stimulate ADRS in hypothalamic
slices in vitro is blocked bv ouabain and correlated with similar increases in
[3H]ouabain binding and Na+K+ ATPase activity. These data suggest a close
functional coupling between the ADRS and Na+K+ ATPase. In related studies we
have found that the genetically obese mouse has not only an increased density of
ADRS, but of [3H]ouabain binding and Na ATPase activity in several brain regions.
Together, with the lack of hyperphagic response to 2-deoxy-D-glucose, these data
suggest that the genetically obese mouse has an altered glucostatic satiety
signal .
Previous studies in our laboratory have demonstrated the presence of high
affinity, stereospecific binding sites for [3H] (+) threo-methylphenidate in the
striatum and brainstem of the rat. Subsequent studies demonstrated that the
binding of [3H]-methylphenidate is localized to synaptosomes, and that the
binding is dependent on the presence of sodium. Intraventricular administration
of 6-hydroxydopamine or medial forebrain bundle lesions. results in a signficant
loss of [3H]-methylphenidate binding in striatum which Is highly correlated with
a loss in the capacity of this tissue to take up [3H]-dopamine. Structure-
activity studies suggest that this site is associated with a dopamine transport
system since a good correlation (r=0.88, p<.001) was found between the potencies
of a series of compounds to inhibit [3H]-dopamine uptake and [3H]-
methylphenidate binding. These findings suggest that the methylphenidate binding
site may be part of a dopamine "transporter". In a related series of experiments
several diphenyl -substituted piperazines (GBR-12935, GBR-12921) have been tested
for their selectivity in inhibiting dopamine uptake. The marked specificity of
these compounds in inhibiting dopamine uptake has prompted the radioactive
labeling of GBR-12935. [3H]-GBR-12935 appears to be a "super high affinity"
ligand for the dopamine uptake site and may be useful for in vivo imaging of
dopamine-containing neurons. Studies with postmortem human brain have further
documented the association of [3H] GBR-12935 binding to dopamine neurons since we
observed significant decreases in [3H] GBR-12935 binding in striatal tissue from
Parkinson's patients.
Significance to Biomedical Research and Program of the Institute:
All of the drugs under investigation have important psychotropic and behavioral
actions and are either of therapeutic benefit or reliably mimic various
439
ZOl MH 02186-05 NS
behavioral states. Thus, an understanding of their mechanisms of actions should
be of value to understanding the behavioral and psychopathological states
responsive to treatment with these agents.
Proposed Course:
Studies will continue on the various recognition sites described above to more
fully elucidate their pharmacological as well as physiological significance. A
major emphasis will be placed on defining the alterations in ADRS density that
occur in vivo during various manipulations of "appetite" and "satiety", in order
to test the hypothesis that these sites are coupled to a physiological mechanism
regulating good intake (particularly carbohydrate intake) in animals. The
relationship between ADRS and the neuronal form of ^^+^+ ATPase will also be
investigated using both binding, enzyme assay, and °°Rb flux measurements.
Emphasis will be placed on whether these binding sites label some novel
postsynaptic effector system and whether conventional neurotransmitters such as
dopamine and serotonin regulate these sites in vitro.
Publ ications
1. Hauger, R.L., Skolnick, P. and Paul, S.M.: Brain recognition sites for
typical and atypical antidepressants. In: Advances in Human
PsYchopharmacoloqy, Vol. IV, G.D. Burrows and J.S. Werry (Eds.), Pergamon Press,
New York, in press.
2. Angel, I, Kiss, A., Stivers, J. A., Skirboll, L. Crawley, J.N. and Paul,
S.M.: Regulation of [3H] Mazindol binding to subhypothalamic areas: involvement
in glucoprivic feeding. Brain Res. Bull.. Vol. 17, 873-877, 1986.
3. Angel, I., Luu, M.D. and Paul, S.M.: Characterization of [3H] mazindol
binding in rat brain: sodium-sensitive binding correlates with the anorectic
otencies of phenyl ethyl amines. J. Neurochem., Vol. 48, 491-497, 1987.
4. Janowsky, A., Vocci, P., Berger, P., Angel, I., Zelnik, N., Kleinman, J.E.,
Skolnick, P. and Paul, S.M.: [3H] GBR-12935 Binding to the dopamine transporter
is decreased in the caudate nucleus in Parkinson's Disease. J. Neurochem., Vol.
29, 617-621, 1987.
5. Labarca, R., Janowsky, A. and Paul, S.M.: Neurotransmitter-Stimulated
inositol phosphate accumulation in hippocampal slices. In: Conn PM and Means AR
(eds). Methods in Enzymoloqy, Vol 141. Orlando, Academic Press, ppl92-201, 1987.
6. Skolnick, P., Schweri , M.M., Rafferty, M.F., Rice, K.C., Janowsky, A.J. and
Paul, S.M. [3H]-threo=(+)-Methylphenidate binding in neuronal dopamine uptake
sites in corpus striatum: correlation with the stimulant properties of ritalinic
acid esters. J. Neurochem, in press.
7. Hauger, R.L., Rehavi, M., Angel, I., Janowsky, A., Skolnick, P. and Paul,
S.M. Receptor-mediated mechanisms of antidepressant drug action. Forthcoming in
Psychiatry, Vol 3, Section 2: Psychobiological Foundations of Clinical Psychiatry
440
ZOl MH 02186-05 NS
(L.J. Judd and P.M. Groves, section editors), J.B. Lippincott Company,
Philadelphia, PA, 1987
441
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 MH 02340-02 NS
PERIOO COVERED
October 1, 1986 to September 30, 1987
TTTLE OF PROJECT (SO ctwmctan or lass. Vth must fir on on* Srw (s«Mwan ITw t>ordan.t BlOChemiCa I and C I 1 fliCa I
Studies of Gaucher Disease and Other Neurogenetic Disorders
PRINCIPAL INVESTIGATOR (Ust othar pnjtoutanaf punonnal bakm 0M PrInciQuI InvsUgator.) (Nun; Oh. labontmy. and kutltuta attKatton)
PI: E.I. Ginns, Head, Molecular Neurogenetics Unit, NS, NIMH
Others: W. Eliason Guest Researcher
M. LaMarca Guest Researcher
B. Martin Visiting Scientist
B. Martin Guest Researcher
K. Maysak Guest Researcher
B. Stubblefield Biologist
S. Winfield Microbiologist
NS,
NIMH
NS,
NIMH
NS,
NIMH
NS,
NIMH
NS,
NIMH
NS,
NIMH
NS,
NIMH
COOPERATING UNITS (it any)
Human Genetics Branch, National Institute of Child Health and Human Development;
Interinstitute Genetics Program, NIH; Arthritis Service, Hospital for Joint
Diseases, New York, NY
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Molecular Neurogenetics Unit, Preclinical Studies Section
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.6
PROFESSIONAL
0.7
±^
CHECK APPROPRIATE BOX(ES)
[^ (a) Human subjects
H (a1) Minors
D (a2) Interviews
SI (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The cl inical study of neurogenetic diseases provides the foundation for the
development of techniques for improved diagnosis and strategies for therapy. This
goal is greatly facilitated by having a comprehensive knowledge of the
biochemistry and clinical heterogeneity of the disorder. Gaucher disease, the
most common sphingol ipidosis, has a high priority as a model for gaining insight
into this group of neurogenetic disorders because of the occurrence of both
neuronopathic and non-neuronopathic phenotypes as well as the broad spectrum of
clinical diversity within the major types of the disorder. Once the
pathophysiologic mechanisms of systemic involvement are understood, the therapy of
nervous system dysfunction may be more rationally approached. Basic research on
qlucocerebrosidase. the enzyme deficient in Gaucher disease, has generated a more
detailed understanding of the structure, .biosynthesis, intracellular routing, and
turnover of the enzyme. These studies will complement other studies within our
branch focusing on the investigation of the potential and efficacy of gene
transfer as a therapeutic approach. The use of recombinant DNA technologies to
produce large amounts of both normal and mutant human proteins is being pursued.
A.'. 3
PHS 6040 (Rev 1/84)
.;>>o •i«-«i«
ZOl MH 02340-02 NS
OTHERS:
S. Tsuji Visiting Fellow NS, NIMH
Various Genetic Fellows IGP, NIH
J. Sidbury Chief, Section on Human Biochem Genetics HGB, NICHD
S. Stuchin Orthopedic Hospital, Hospital for Joint Diseases, NY
PROJECT DESCRIPTION
Biochemical : From our progress in the study of the molecular biology of Gaucher
disease we plan to: purify mutant enzymes; characterize the primary amino acid
sequences and post-translational processing of the mutant enzymes; correlate the
structural mutations of the protein with the observed clinical heterogeneity;
characterize the structure, orgatiization, and regulation of expression of the
normal mutant gl ucocerebrosidase genes; investigate the production of large
quantities of protein using recombinant DNA methodologies; develop diagnostically
useful recombinant DNA tests (i.e., RFLPs); and evaluate the potential of somatic
cell gene therapy for Gaucher disease.
CI inical : Using Gaucher disease as a prototype of inherited disorders having
both neurologic and non-neurologic phenotypes, clinical evaluations are
undertaken in an attempt to correlate the clinical heterogeneity with both
biochemical and genetic data. The role of the macrophage in pathogenesis of the
numerous clinical manifestations of this disorder will be studied. Specifically,
approaches to therapeutic intervention for Gaucher disease focus on the
hypothesis that this disorder is iiiacrophage-mediated. The involvement of
hematopoietic derived cells in thn pathogenesis of this disorder is crucial to
the applicability of somatic cell gene therapy as a potential therapeutic
strategy.
MAJOR FINDINGS
1. Recombinant normal glucocerebrosidase was purified on a pilot scale.
2. Rabbit polyclonal and mouse monoclonal antibodies were used for studying
the glycosylation of recombinant glucocerebrosidase using Western blot
analysi s .
3. Amino acid sequencing was performed on recombinant glucocerebrosidase using
protein immobilized on teflon membranes.
4. The post-translation processing of glucocerebrosidase in Type 1, 2 and 3
Gaucher disease was further characterized.
5. The normal glucocerebrosidase gene was sequenced.
6. The pseudogene for glucocerebrosidase was sequenced.
7. All the exons splice junctions and flanking regions of a genomic clone from
a type 1 patient were sequenced and a single base change occurring in high
frequency among non-neuronopathic phenotype (type 1) of Gaucher disease was
identified.
444
ZOl MH 02340-02 NS
8. A useful diagnostic test based on hybridization of an oligonucleotide
probe, for the identification of the mutation in type 1 phenotypes of
Gaucher disease was developed.
9. The expression by DNA mediated gene transfer of active human
glucocerebrosidase in heterologous mammalian host cell lines was extended
to include high-level production in the baculovirus expression system.
10. The correction of the enzyme deficiency in type 2 Gaucher fibroblasts in
culture by retroviral mediated gene transfer was accomplished with cDNA
retroviral constructs. Experiments with genomic constructs were begun.
11. The spectrum of symptoms of patients having Gaucher disease was further
studied and correlated with RFLP analysis.
SIGNIFICANCE TO BIOMEDICAL RESEARCH
Gaucher disease is useful as a prototype disorder for furthering our
understanding of the mechanisms responsible for the clinical heterogeneity seen
within many of the neurogenetic disorders. It is the most common
sphingolipidoses and many patients could benefit from the development of an
efficacious therapy. The techniques and information obtained from the study of
the protein processing and gene in Gaucher disease should be useful and helpful
to formulating strategies for understanding the biochemical and genetic bases of
other neuropsychiatric disorders.
PROPOSED COURSE
Patients having type 1, 2 or 3 Gaucher disease will be studied to further define
the biochemical and genetic mechanisms responsible for the clinical heterogeneity
within this disorder. The involvement of hematopoietic stem cell derived
macrophages in the pathogenesis of symptoms makes type 1 Gaucher disease an
attractive candidate for somatic cell gene therapy.
PUBLICATIONS
Barranger JA and Ginns EI: Glucosylceramide lipidoses: Gaucher disease. In:
Scriver CR, Beaudet AL, Sly WS, Valle D (eds), The Metabolic Basis of Inherited
Disease. New York, McGraw-Hill (in press).
Ginns EI: Molecular biology of inherited metabolic disorders. In: Handbook of
Immunoblottinq. CRC Press (in press).
Tsuji S, Choudary PV, Martin BM, Barranger JA, Stubblefield BK, Mayor JA, Ginns
EI: A mutation in he human glucocerebrosidase gene in neuronopathic Gaucher
disease. N Engl J Med 361:570-575, 1987.
van Dongen JM, Willemsen R, Ginns EI, Sips HJ, Tager JM, Barranger JA, Reuser
AJJ: Subcellular localization of soluble and membrane-bound lysosomal enzymes in
I-cell fibroblasts: A comparative imiiiunocytochemical study. Eur J Cell Biol (in
press) .
445
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PU8UC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl
02341-02 NS
PERIOO COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 chtmctan or hsa. Wa most tit on ona in* tMtwan tht tnnlft.)
Correction of Inherited Enzyme Deficiencies by Gene Transfer
PRINOPAL INVESTIQATOR (Ust othtr prolaaaional pertonoal batow 0M Principal tnvatOgator.f (Nama, Mto. laboratory, and InatHuta amMaOon)
PI: E.I. Ginns, Head, Molecular NfMirogenetics Unit, NS, NIMH
Martin Visiting Scientist NS, N
Tsuji Visiting Fellow NS, N
Stubblefield Biologist NS, N
LaMarca Guest Researcher NS, N
Winfield Microbiologist NS, N
Others: B
W
Martin
El iason
Guest Researcher
Guest Researcher
NS, N
NS. N
MH
MH
MH
MH
MH
MH
MH
COOPERATING UNITS (it any)
Center for Cancer Research, MIT and Whitehead Institute for Biomedical Research,
Boston, ,MA
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Molecular Neurogenetics Unit. Preclinical Studies Section
iNsrrruTE and location
NIMH. NIH. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
1,
PROFESSIONAL
0-7
XX
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Usa standard unmducad type. Do not axcaod ffie spaca providad.)
The isolation and characterization of proteins involved in the pathogenesis of
neurogenetic disorders has permitted the isolation of cDNA and genomic DNA that
can be used to investigate the correction of inherited enzyme deficiencies using
recombinant DNA techniques, specifically somatic cell gene transfer. Particular!}
suited for initial attempts at gene therapy are those disorders (such as Gaucher
disease, the most common sphinqolipidosis) in which the manifestations of the
disorder are due to abnormalities of hematopoietic cells, in this case, the
macrophage. In this instance the transfer of normal genes to bone marrow
progenitor cells is a rationale theiapnutic approach. Using the lysosomal
disorder Gaucher disease as a model, wo have been successful in utilizing
retroviral vectors to transfer and express human glucocerebrosidase in host mouse
and Gaucher cell lines. The complete correction of glucocerebrosidase activity in
Type 2 Gaucher fibroblasts in culture has provided the impetus for evaluation of
retroviral mediated somatic cell gene transfer of the glucocerebrosidase gene into
mice by bone marrow transplantation. The initial goal of this research is the
application of these recombinant DNA therapeutic strategies to the non-
neuronopathic phenotypes. When our understanding of the pathogenetic mechanisms
of inherited neurological and psychiatric diseases improves and when retroviral
mediated expression of genes in specific tissues and cells become more
predictable, we can begin to invest igate the potential usefulness of gene therapy
i for treatment of selected nervous system disorders.
4A7
PHS 60*0 (Rev 1/84)
OfO 91 4-«(
ZOl MH 02341-02 NS
OTHERS:
K. Maysak Guest Researcher NS, NIMH
R. Mulligan Center for Cancer Research, MIT and Whitehead Institute
for Biomedical Research, Boston, MA
B. Guild Center for Cancer Research, MIT and Whitehead Institute
for Biomedical Research, Boston, MA
E. Dzierzak Center for Cancer Research MIT and Whitehead Institute
for Biomedical Research, Boston, MA
PROJECT DESCRIPTION
The isolation of cDNA and genomic DMA encoding specific proteins involved in
neurogenetic disorders permits the application of recombinant DNA technologies as
therapeutic approaches to the correction of these inherited enzyme deficiencies.
Using the lysosomal storage disorder Gaucher disease as a prototype we are
investigating the efficacy of retroviral mediated gene transfer, first applied in
tissue culture and then in small animals.
MAJOR FINDINGS
1. Complementary DNA and genomic DNA clones encoding human glucocerebrosidase
have been isolated and secjuenced.
2. Clones for both the functional genomic DNA and a pseudogene for human
glucocerebrosidase have been isolated.
3. Active human glucocerebrosidase has been transferred to both mouse and
monkey cell lines using eukaryotic shuttle vectors.
4. The enzyme deficiency in a Type 2 Gaucher cell line has been corrected by
transfer of the normal human glucocerebrosidase cDNA to these cells in
culture.
5. Recombinant retrovirus containing human glueocerebrosidase cDNA has been
used to infect mouse bone marrow cells and obtain reconstituted mice have
provirus in their blood cells.
SIGNIFICANCE TO BIOMEDICAL RESEARCH
The isolation of the cDNA and genomic DNA for human glucocerebrosidase and the
transfer of this gene by retroviral vectors suggests that such recombinant DNA
approaches may be useful therapeutic strategies for Gaucher disease and other
selected genetic disorders.
PROPOSED COURSE
The project initially focuses on the DNA mediated-transfer of normal, human
glucocerebrosidase to rodent and human cell lines in culture. Once the efficacy
of gene transfer (particularly retroviral -mediated) is demonstrated, the approach
4A8
ZOl MH 02341-02 NS
will be applied to appropriate human subjects with specific inherited disorders
(such as Gaucher disease).
PUBLICATIONS
Choudary PV, Tsuji S, Martin BM, Guild BC, Mulligan RC, Murray GJ, Barranger JA,
Ginns EI: The molecular biology of Gaucher disease and potential for gene
therapy. Cold Spring Harbor Symposia 51 on Molecular Biology of Homosapiens.
1986, pp 1047-1052.
Martin BM, Tsuji S, LaMarca ME, Maysak K, Eliason W, Ginns EI: Molecular biology
of Gaucher disease: Therapeutic strategies utilizing recombinant DNA
technologies, in NATO Advanced Research Workshop and Inserm Symposium: Lipid
Storage Disorders (Biological and Medical Aspects). Toulousse, 1987, in press.
449
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02342-02 NS
PERIOO COVERED
October 1, 1986 to September 30, 1987
TTTIE OF PROJECT (BO chmncimn or hsa. TM» must SI on on* frw twdvaon tfw boidtn.)
Gene Regulation within the Nervous System
PRINCIPAL INVESTIOATOR (Uat othor pratasitana/ panonnti iMfow Om Phndpal knmtligalor.) (Nam*. Ml*. ItMntorf. and kutlMa aimaOon)
PI: E.I
Others:
Ginns, Head, Molecular Neurogenetics Unit, NS, NIMH
Martin Visiting Scientist NS, NIMH
S. Tsuji Visiting Fellow NS, NIMH
S. Winfield Microbiologist NS, NIMH
P. Marangos Unit on Neurochemistry BPB, NIMH
D. Schmeckel Neurology Department VAMC
J. Polak Royal Post-Graduate Medical School, London
J. Hozier Medical Genetics FIT
COOPERATING UNITS (if any)
Unit on Neurochemistry, Biological Psychiatry Branch, NIMH; Veterans
Administration Medical Center, Durham, NC; Royal Post-Graduate Medici School,
London, England; Florida Institute of Technology, Melbourne. FL
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Molecular Neurogenetics Unit. Preclinical Studies Section
INSTITUTE AND LOCATION
NIMH. NIH. Bethesda. Maryland 20892
TOTAL MAN- YEARS:
0.8
PROFESSIONAL-
AJl
JLA.
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
n (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not excood tha tpaca proMad.)
We approached the cell -specific and developmental ly regulated expression of
proteins within the nervous system using the neuron specific (NSE) and non-
neuronal (NNE) enolase isozymes as a model. Human brain cDNA and genomic DNA
1 ibraries were constructed so that the genes for these and other brain specific
proteins could be isolated and characterized. Using both antibodies and
oligonucleotide probes, cDNAs for both human NSE and NNE have been isolated and
sequenced. Employing unique regions of these cDNA clones as probes, the
developmentally and cell -specific regulated appearance of mRNA for each of these
proteins can be investigated using in-situ hydridization. The human chromosome
loci for each of these isozymes will be identified. In addition, the isolation of
human genomic clones for each of these proteins .should provide information on the
regulation of expression of neuron and gl ial .specific proteins during cell
differentiation of the human nervous system in normal and disease states. The
normal specificity of NSE for neural derived cell lines and the availability of
specific DNA probes for NSE should provide a useful approach to the
characterization of neural derived normal and tumor cell lineages.
451
PHS 6040 (Rev. W84)
cpo at*-*!*
ZOl MH 02342-02 NS
PROJECT DESCRIPTION
The enolase isozymes will be used as a model for studying the transcriptional and
translational control of develoixnentally regulated genes within the human nervous
system. The genomic organization and regulation of expression of these genes
will be investigated.
MAJOR FINDINGS
1. Human brain cDNA and genomic DNA libraries have been constructed.
2. Complementary DNA clones for human neuron specific and non-neuronal
enolases have been isolated and sequenced.
3. Specific cDNA probes for NSE and NNE have been demonstrated to be useful
for high resolution in-situ chromosome localization and tissue
hybridization studies.
SIGNIFICANCE TO BIOMEDICAL RESEARCH
A description of the transcriptional and translational control mechanisms for
neuron specific and non-neuronal enolases should provide a more detailed
understanding of the mechanisms involved in regulation of gene expression in
neurons and glia within the human nervous system during development and
di fferentiation.
PROPOSED COURSE
The project will focus on the description of the genomic organization and control
mechanisms of expression of NSE and NNE genes. The involvement of mutations in
the genes for these proteins in the pathogenesis of neuropsychiatric disorders
will be investigated.
PUBLICATION
Schmechel DE, Marangos PJ, Martin BM, Winfield S, Burkhart DS, Roses AD, Ginns
EI: Localization of neuron-specific enolase (NSE) mRNA in human brain. Neurosci
Lett 76:233-238, 1987.
452
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 MH 02343-02 NS
PEHIOO COVERED
October 1, 1985 to September 30, 1987
TITLE OF PROJECT (60 chancfn or hsa. Tttto imat tit on on* Sna (miwvm tfw turdtrs.)
Molecular Genetics of Inherited Neurologic and Psychiatric Disorders
PRINaPAL INVESTIQATOn (IM othar prohsalonal partonnti tMtow 0m Ptktdp^ knrntOgtor.} (Nam; tiUa. laboratory, and ktstUuta amHaHon)
PI: E.I. Ginns, Head, Molecular Neurogenetics Unit, NS, NIMH
Others: S.M. Paul Chief, Clinical Neuroscience Branch NS, NIMH
D. Pickar Chief, Section on Clinical Studies NS, NIMH
J. Kelsoe Medical Staff Fellow NS, NIMH
B. Martin Visiting Scientist NS, NIMH
B. Stubblefield Biologist NS, NIMH
S. Winfield Microbiologist NS, NIMH
COOPERATING UNPTS (if any)
Pediatrics Department, Johns Hopkins School of Medicine, Baltimore, MD; Florida
Institute of Technology, Melbourne, FL; Dept of Anatomy & Neurobiology, Washington
Univ School of Medicine
LAB/BRANCH
Clinical Neuroscience Branch
Molecular Neurogenetics Unit, Preclinical Studies Section
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.8
PROFESSIONAL:
0.9
0.9
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
[x| (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We approached the characterization of the mutations responsible for inherited
neurological or psychiatric disorders by studying the gene organization of
specific proteins that might have a role in the pathogenesis of the cl inical
manifestations. Using the inherited lysosomal storage disorders, Gaucher disease
and Fabry disease, as models, we demonstrated that the phenotypic heterogeneity
seen within these inherited disorders is a consequence of different mutations,
each affecting protein activity and influencing the processing,
compartmental ization and/or stabil ity of the protein. Similar approaches are
being used to investigate the involvement of genes on chromosome II in mania-
depression and other psychiatric disorders. Recombinant DNA techniques have been
used to elucidate the structure of the gene for the proteins (enzymes and
receptors) involved in these and other n^urop.sychiatric disorders. Restriction
fragment length polymorphisms (RFIPs) have been identified that are useful for the
identification of mutations in Gaucher disease that frequently occur in both non
neuronopathic and neuronopathic pheiioLypes. Northern blot analysis provides
further details of the structure of the normal and mutant genes. The molecular
mechanisms leading to nervous system involvement in these disorders have also been
investigated. The results of this research should provide a more rational
foundation for the diagnosis and formulation of therapeutic strategies for these
inherited disorders. Genes on chromosome 11 specific for neurotransmitter
biosynthesis have been isolated (for example, human tyrosine hydroxylase and
tryptophan hydroxylase). Comparison of normal gene sequence to the gene sequence
in Amish manic-depressive patients is in progress. Recombinant DNA approaches
have been used to produce large amounts of tyrosine hydroxylase isozyme for
structural and biochemical studies.
453
PHS 6040 (Rev 1/84)
ZOl MH 02343-02 NS
OTHERS
B. Migeon Pediatrics Department Johns Hopkins
J. Hozier Medical Genetics FIT
K. O'Malley Anatomy & Neurobiology, Washington Univ School Med
PROJECT DESCRIPTION
The diversity in the presentation of neurogenetic disorders may be a consequence
of multiple allelic mutations. The understanding of the mechanisms of this
phenotypic heterogeneity will be derived from genetic and biochemical analyses.
Recombinant DNA techniques are used to isolate and characterize the genes for
specific proteins. The study of mutations should elucidate the structural
abnormalities and consequences of the abnormal biosynthesis and post-
translational processing of the mutant proteins. The identification of RFLPs
associated with clinical manifestations is studied using cDNA and genomic DNA
probes. The comparison of gene expression for proteins in neural and non-neural
tissues should extend our understanding of protein regulation.
MAJOR FINDINGS
1. Complementary DNA and genomic libraries were constructed from human brain
tissue.
2. Human cDNA clones encoding active tyrosine hydroxylase have been isolated
and sequenced.
3. A single base mutation has been identified within the coding regions of a
type 1 (non-neuronopathic) Gaucher genomic DNA clone. This mutation
occurs in high frequency in patients having non-neuronopathic Gaucher
disease.
4. Chromosome 11 specific probes (globin, harvey ras, tyrosine hydroxylase)
have been obtained for RFLP analysis of patient's having bipolar affective
illness.
5. The normal gene for human tyrosine hydroxylase was isolated and sequenced.
6. Active recombinant human tyrosine hydroxylase has been produced.
SIGNIFICANCE TO BIOMEDICAL RESEARCH
The description of the molecular basis for neuropsychiatric disorders should
provide a more rational basis for the development of diagnostic and therapeutic
strategies.
PROPOSED COURSE
The project will focus on the biochemistry and genetics of these disorders to
obtain a more complete understanding of the mechanisms responsible for the
454
ZOl MH 02343-02 NS
clinical manifestations of these inherited disorders. This information will be
used to develop diagnostic and therapeutic strategies.
PUBLICATIONS
Willemsen R, Van Dongen JM, Ginns EI, Sips HJ, Schram AW, Tager JM, Barranger
JA, Reuser AJJ: Ultrastructural localization of glucocerebrosidase in cultured
Gaucher's disease fibroblasts by immunocytochemistry. J Neurol 234:44-51, 1986.
Ginns EI: Application of immunoblotting to the study of the molecular biology
of inherited metabolic disorders. In: Handbook of Immunoblotting (Chap 9.5).
CRC Press (in press).
Jonsseon LMV, Murray GJ, Sorrell S, Strijland A, Aerts JFGM, Ginns EI, Barranger
JA, Tager JM, Schram AW: Biosynthesis and maturation of glucocerebrosidase in
Gaucher fibroblasts. Eur J Biochem 164:171-179, 1987.
O'Malley KL, Anhalt M, Martin BM, Kelsoe JR, Winfield SL, Ginns EI: Isolation
and characterization of the human tyrosine hydroxylase gene: Identification of
5' alternative splice sites responsible for multiple mRNAs. Biochemistry (in
press) .
Tsuji S, Martin BM, Kaslow D, Migeon BR, Choudary PV, Stubblefield BK, Mayor JQ,
Murray GJ, Barranger JA, Ginns EI: Signal sequence and DNA mediated expression
of human lysosomal alpha-galactosidase A. Eur J Biochem 165:275-280, 1987.
455
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02344-02 NS
PERKX} COVERED
October 1, 1986 to September 30, 1987
TTLE Of PROJECT (BO ctmrmcttra or hsa. TMe must Stonona in* btwtn ttta boidun.)
Neuropsychiatric Disorders: Protein Structure-Activity Studies
PraNOPAL INVESnOATOR (Utt oOmr protesitomtf panonnal below ffw Principal ImtOgtor.) (Nun*. ttOt, Uttonloty, and ttsMuta aimtlon)
PI: B. Martin, Visiting Scientist, NS, NIMH
Others: E.I. Ginns Head, Molecular Neurogenetics Unit NS, NIMH
W. Eliason Guest Researcher NS, NIMH
K. Maysak Guest Researcher NS, NIMH
L. Possani Free University of Mexico, Mexico
COOPERATING UNITS (U any)
Free University of Mexico, Mexico
LAB/BRANCH
Clinical Neuroscience Branch
Molecular Neurogenetics Unit, Preclinical Studies Section
iNsrrruTE and location
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.6
PROFESSIONAL
0.5
1.1
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed Itte apace provided.)
This research is part of an effort to better understand the molecular mechanisms
underlying human nervous system development and function, as well as the
pathogenesis of certain neurogenetic disorders. Our studies have focused on
structural and active site properties of the human non-neuronal and neuron
specific enolases, lysosomal hydrolase (glucocerebrosidase and alpha-
qalactosidase A) . other enzymes (particularly those peptides and proteins that
interact with excitable membranes), and venom toxins. Proteins are purified from
both human and animal tissues using affinity chromatography, electrophoretic
separation, and high performance liquid chromatography. Using microsequencinq
techniques, the complete amino acid sequence of glucocerebrosidase, antral in and
major portions of sequences for the neuronal and non-neuronal enolases, venom
toxins, and alpha-galactosidase A have been obtained. Peptide maps of both normal
and mutant proteins are generated using chemical (cyanogen bromide) and enzymatic
(trypsin, thermolysin, V8 protease) cleavage. The identification of carbohydrate
attachment sites, sulfhydryl residues, and intra-chain disulfide residues is used
to predict protein structure. Alkylating agents and enzyme inhibitors are used to
define active sites. From the primary protein sequence, hydrophobic and
hydrophil ic domains of the protein are identified.
Information obtained from these protein structure studies permits the design of
oligonucleotides and peptides that are synthesized for collaborative research (see
ZOl MH 02343-02 NS) involving antibody production, cDNA cloning, DNA sequence
analysis and in vitro mutagenesis.
457
PHS 6040 (Rev WS4)
CPO •! 4.«1
ZOl NM 02344-02 NS
PROJECT DESCRIPTION
A goal of this project includes the identification of primary and tertiary
structure of the proteins. Once these aspects of protein structure are
elucidated, a three-dimensional model can be constructed using the secondary
structure data obtained from computer modeling. This information will be useful
in defining the hydrophilic and membrane domains of the protein. Active sites
are identified using sulfhydryl reagents and specific inhibitors and activators.
This information is used to design synthetic oligonucleotides and peptides for
collaborative research.
MAJOR FINDINGS
1. The first complete amino acid sequence of a human lysosomal enzyme
(glucocerebrosidase) was determined, as well as identification of the four
carbohydrate attachment sites and all three disulfide bridges in this
protein.
2. Biochemical characterization, including partial amino acid sequence, of a
novel calcium binding protein from snake venom.
3. Determination of the amino acid sequence of antral in, an unusual
phosphol ipase A2 from rat stomach.
SIGNIFICANCE TO BIOMEDICAL RESEARCH
The neuron and non-neuronal enolases are used as a model system to investigate
the developmentally controlled expression of specific proteins within the nervous
system. The lysosomal hydrolases are used as prototypes for understanding the
phenotypic heterogeneity within the neurogenetic disorders. Studies of venom
toxins should provide information on the structure of ion channels within the
nervous system. Together these studies further our understanding of the
structure-function relationships of nervous system proteins.
PROPOSED COURSE
The project will focus on the proteins described above as well as other proteins
involved in lysosomal and neuropsychiatric disorders.
PUBLICATIONS
Alagon AC, Guzmcin HS, Martin BM, Ramirez AN, Carbone E, Possani LD: Isolation
and characterization of two toxins from the Mexican scorpion Centruroides
limpidus karsch. Comp Biocheni Physiol (in press).
Mussar KJ, Murray GJ, Martin RM, Viswanatha T: A rapid chromatographic assay
procedure for peptide: N-glycos idase activity. J Ciiromat (in press).
Zasloff M, Martin BM, Chen ll-C: AiiLi -microbial polypeptides from X laevis skin:
sequencing and synthesis of the Maciaini family. Proc Natl Acad Sci (in press).
458
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00112-10 NS
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (90 chimdan or lata. THh imat Ht on <vw Cm bMw—n tfM berdtrt.)
Endorphin Research in Mental Illness
PRlNaPAL INVESTIQATOR (Ust otfw pfolnaloni ptnomtl blow 0M Principal ImtUgMor.i (Nanm. tUa. laboialoty, and kaaula aHmaOon)
PI: D. Pickar, Chief, Section on Clinical Studies NS, NIMH
COOPERATING UNITS (» any)
LAB/BRANCH
INSTITUTE AND LOCATION
TOTAL MAN-YEARS:
PROFESSIONAL;
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not axcoed the spaca pnrMad.)
INCORPORATED UNDER PROJECTS ZOl MH 02181-05 NS AND ZOl
02184-05 NS IN 1987
459
PHS 6040 (Rev. 1/M)
CPO •I4-«I«
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 MH 02181-05 NS
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 chanctan or lets. TWa must lit on ona lina batwaan tha bordafs.)
Neurobi ology of Schizophrenia
PRINCIPAL INVESTIGATOR (List othar pmtassional personnal below tha Principal InvasOgator.) (Nama, UHa, laboratory, and instltuta alfillation)
PI: D. Pickar Chief, Section on Clinical Studies NS, NIMH
Others: S.M. Paul Chief NS, NIMH
A.F. Breier Medical Staff Fellow NS, NIMH
J.R. Kelsoe Medical Staff Fellow NS, NIMH
P.B. Lucas NRSA Fellow NS, NIMH
C.N. Pato NRSA Fellow NS, NIMH
M.H. Rapaport Medical Staff Fellow NS, NIMH
J.L. Schreiber Social Worker NS, NIMH
COOPERATING UNITS (if any)
Laboratory of Psychology and Psychopathology, NIMH; Neuropsychiatry Branch, St.
Elizabeths Hospital, NIMH
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Clinical Studies
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
7.0
PROFESSIONAL:
5.0
2.0
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects D (b) Human tissues D (c) Neither
D (a1) IVlinors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed tha space provided.)
This project studies the psvchobioloqy of schizophrenia and attempts to develop
improved strategies for its treatment. An important goal is the understanding of
the mechanism of action of neuroleptic drugs. We have observed that neuroleptic-
induced time-dependent decrease in levels of plasma homovanillic acid (HVA), a
major dopamine metabolite, correlates with antipsychotic drug response, suggesting
that slow to develop changes in dopamine turnover may underlie the antipsychotic
action of neuroleptics. This clinically relevant dopamine marker is further
studied using a strategy in which peripherally derived HVA is reduced by the
administration of debrisoquin, a MAO inhibitor which does not enter the CNS. In a
double-blind, treatment trial, we have observed significant reduction in psychosis
in some patients when alprazolam is added to neuroleptic treatment; the use of
benzodiazepines to augment neuroleptic response is under investigation in
outpatient trials. These data contrast with the negative results found when the
calcium channel blocker, verapamil , was administered to neuroleptic-free
schizophrenic patients. In a recently completed study using magnetic resonance
imaging (MRI) performed in collaboration with the Clinical Brain Disorders Branch,
NIMH, compelling evidence was gained which supports enlargement of lateral and 3rd
ventricular volumes in schizophrenic patients. A follow-up study of previous
inpatients from our program has recently been completed and holds promise for
delineating biological correlates of outcome in schizophrenia. A controlled study
of expressed emotion in families of schizophrenic patients is in progress. The
proposed course involves the further development of an outpatient research program
which includes controlled pharmacologic treatment studies and the evaluation of
family schizophrenia pedigrees for collaboration with the Clinical Neuroscience
Molecular Biology Laboratory. Collaboration with the NIMH positron emission
tomography (PET) program focusing on the mechanism of action of typical and
atypical neuroleptics using glucose utilization studies and the development of new
PET ligands will hp pursued. ^l61
PHS 6040 (Rev. 1/84)
CPO 91 4-«ia
ZOl MH 02181-05 NS
OTHER PROFESSIONAL PERSONNEL
O.M. Wolkowitz Medical Staff Fellow NS, NIMH
R.M. Cohen Chief, Clinical Brain Imaging Section LPP, NIMH
D. Weinberger Chief, Section on Clinical Neuropsychiatry NPB, NIMH
PROJECT DESCRIPTION
This project is part of the research program of the Section on Clinical Studies
of the Clinical Neuroscience Branch. This section conducts clinical research on
the 4-East Nursing Unit and the ACRE of the Clinical Center.
Despite enormous research and clinical efforts to alleviate symptoms of
schizophrenia, the group of drugs known as neuroleptics have remained the
principal pharmacologic agents for treatment of this illness. Moreover, the
effects of neuroleptics on CNS dopamine systems also represent the cornerstone
for the dopamine hypothesis of schizophrenia. An important facet of our research
program is the pharmacotherapy of schizophrenia, including studies of the
mechanism of action of neuroleptic drugs.
We have performed longitudinal clinical studies in which neuroleptic-induced
alterations in dopamine function are assessed using levels of plasma homovanillic
acid (HVA). Patterns of neuroleptic-induced alteration in plasma HVA level are
examined as predictors of clinical response. Administration of the MAO-A
inhibitor, debrisoquin, is used to enhance the relative contribution of CNS
derived HVA to levels which circulate in plasma. Positron emission tomographic
(PET) studies of typical and atypical neuroleptic drugs in which regional brain
glucose utilization is examined are currently in progress.
Preclinical pharmacology of dopamine neurons, including new developments in the
understanding of mesocortical dopamine neuronal function, has prompted studies of
the clinical effects of the triazolobenzodiazepine, alprazolam, as an additive
treatment to neuroleptics in schizophrenic patients. Other aspects of our
research program include efforts to identify structural abnormalities in the
brains of schizophrenic patients using CT and MRI techniques as well as
pathological mechanisms, e.g., autoimmune phenomena, which may underlie these
structural abnormalities.
An outpatient program in schizophrenia research has recently been initiated. We
have completed a follow-up study of patients who had been discharged from our
research program at least two years previously. The goal of this work is to
examine the course of illness and to identify predictors of outcome. Pilot
outpatient investigation of neuroleptic augmenting effects of the
benzodiazepines, lorazepam and alprazolam, are in progress.
METHODOLOGY
(See: 1985 Annual Report, pp 471-474, Project Number ZOl MH 02181-03 NS.
Neurobiology of Schizophrenia)
We have administered the non-CNS active MAO inhibitor, debrisoquin, to reduce the
non-CNS contribution of HVA to levels which circulate in plasma. This
462
ZOl MH 02181-05 NS
pharmacologic strategy is intended to enable better assessment of CNS dopamine
function through plasma sampling and is applied to longitudinal studies of
neuroleptic treatment.
A follow-up study of schizophrenic patients who had previously participated in
our 4-east research program has been performed. All patients discharged at least
2 years prior to this study were contacted for follow-up interviews. Data
focused on levels of social, work and personal function, on current
symptomatology and overall illness course. Data from the index hospitalization
is then examined for correlation with outcome.
The effect of metabolic stress produced by the infusion of 2-deoxy-D-glucose (2-
DG) (50 mg/kg) in neuroleptic-treated schizophrenic patients and controls has
been performed. Behavioral and neurochemical response is studied.
A family study in which expressed emotion response to a schizophrenic family
member as compared with the expressed emotion response to a non-ill sibling
undergoing a stressful life period has now been completed. This study examines
the specificity of the expressed emotion response seen in families of
schizophrenic patients.
MAJOR FINDINGS
1. Expanded data support our initial findings that neuroleptic treatment
produces time-dependent decreases in plasma levels of the dopamine metabolite,
HVA, and that neuroleptic-induced changes in levels of plasma HVA correlate with
clinical response. Preliminary data using the debrisoquin strategy show similar
time-dependent decreases in plasma HVA levels, supporting the notion that this
pattern reflects similar changes in CNS dopamine systems. Preliminary analysis
of longitudinal MHPG and HVA data indicate that changes in MHPG and HVA together
provide the best information regarding negative symptoms, whereas HVA alone is
the best predictor of positive symptoms.
A particularly interesting aspect to this metabolite work is the contrast between
CSF and plasma HVA response to neuroleptic treatment. In contrast to the
neuroleptic-induced reduction seen in plasma HVA, CSF HVA levels tend to
increase, and remain increased, in response to neuroleptic treatment. This
discrepancy may relate to particularly prominent effects of mesocortical dopamine
neurons in determining levels of CSF HVA.
2. We have demonstrated that the relative balance between positive and negative
symptoms in schizophrenia are state- (e.g., drug-treated vs drug-free) rather
than trait-dependent. These findings have important implications with regard to
classifying schizophrenic patients into positive and negative "types."
3. We find no consistent relationship between negative and positive symptom
profiles and CT abnormalities including generalized and frontal cortical atrophy
nor lateral or third ventricular enlargement. We have observed that both medical
and schizophrenic patients share significant enlargement in lateral ventricular
size in comparison to normal controls. However, an abnormality unique to the
schizophrenic patient is CT scan changes consistent with prefrontal, but not
generalized, atrophy. Results from our completed MRI study provide additional
463
ZOl MH 02181-05 NS
support for the notion of structural brain changes in schizophrenic patients.
Specifically, enlargement of the cerebral ventricular system is found.
4. In a completed double-blind controlled investigation, we have observed that
the calcium channel blocker, verapamil, is without therapeutic effect in
neuroleptic-free patients. Significant increases in levels of plasma in CSF HVA
were, however, observed.
5. In a completed study of twelve patients, we have observed significant
improvement in psychosis ratings when alprazolam is added to a stabilized regimen
of neuroleptic treatment. These clinical effects appear to be divisible into
responders (5 of 12 patients) and nonresponders (5 of 12 patients; two partial
responders) categories. In comparison with nonresponders, responders have
greater prefrontal atrophy as seen on CT scan and greater alprazolam-induced
decreases in levels of plasma HVA than nonresponders.
6. Analysis of data from the follow up study of schizophrenic patients is in
progress.
7. 2-DG produces marked metabolic stress in normal subjects and in schizophrenic
patients. The response is characterized by increased levels of Cortisol and
ACTH. Although plasma levels of HVA were significantly increased in both
patients and controls, schizophrenic patients showed a significantly greater 2-DG
stimulated increase in plasma HVA, despite neuroleptic treatment, than did
controls.
8. The expressed emotion study is currently undergoing data analysis.
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
Schizophrenia is a major public health problem in the United States. This
project attempts to study possible etiologic factors in schizophrenia, to develop
a better understanding of mechanisms underlying current pharmacologic treatments
and to improve the overall current pharmacotherapy of schizophrenia.
Our recent findings with regard to change in levels of plasma HVA during
neuroleptic treatment and their demonstrated relationship to clinical response
are important to the field as they suggest the possibility that monitoring levels
of plasma HVA may be useful as a marker for the antipsychotic effects of
neuroleptics. This work, focusing on change in dopamine system activity during
neuroleptic treatment, is being pursued further using debrisoquin administration
strategies to enhance the CNS HVA "signal." The indication that the
antipsychotic process may involve a step-wise process, rather than a direct
relationship with dopamine receptor blockade, provides new opportunity for
strategies to augment neuroleptic response.
The augmentation of antipsychotic effects by the addition of alprazolam
represents a potentially important new pharmacotherapy for schizophrenia. The
elucidation of the mechanism by which this clinical response occurs is an
important goal for future research.
464
ZOl MH 02181-05 NS
Our findings from CT studies are important as they add continued support for the
notion of structural pathology in the frontal cortex of schizophrenic patients.
Confirmation of ventricular enlargement by MRI is an important contribution to
the field. Our inability to demonstrate the trait nature of the balance between
positive and negative symptoms in schizophrenia and their lack of relationship to
abnormality by CT scan raise into question the notion that structural brain
abnormality is associated with specific types of schizophrenic symptoms.
New treatment strategies which develop from this work would have considerable
importance to the field of psychiatry and to the estimated 2 million patients
suffering from schizophrenia in the United States. Our research ward is well-
suited to studying the clinical and biological affects of pharmacotherapy of
schizophrenia; we have had success in longitudinal studies of levels of plasma
HVA and in the demonstration of the augmentation of neuroleptic antipsychotic
effects by alprazolam.
The follow-up study of schizophrenic patients holds promise for developing
correlates of outcome in schizophrenia. The question of the natural course of
schizophrenia is also addressed in this study.
Paradigms for studying stress response of schizophrenic patients are important
because of the role of stress in the clinical course of schizophrenia.
PROPOSED COURSE
1. We will continue to study the implications of our plasma HVA data with regard
to predictors of antipsychotic response in patients with schizophrenia. Using
the debrisoquin technique and comparisons of plasma and CSF levels of HVA, we
hope to develop a more clear understanding of neuroleptic/dopamine system
interactions.
2. We will continue to assess the efficacy and mechanism of action of alprazolam
as an additive treatment to neuroleptics in schizophrenia. Moreover, we will
extend this work to include outpatient studies. An outpatient study addressing
this issue is currently in progress.
3. The analysis of data from our follow-up study will help to orient our thinking
about factors relating to the course of schizophrenia. This study has already
proven the feasibility of follow up studies at the NIMH and suggests the need for
continued longitudinal study.
4. Collaboration with the NIMH PET group has increased over the past year and
holds promise for developing new insights into not only the pathophysiology of
schizophrenia but also the mechanism of action of neuroleptic drugs.
PUBLICATIONS
Barbaccia, M.L., Costa, E., Ferrero, P., Guidotti, A., Roy, A., Sunderland, T.,
Pickar, D., Paul, S.M., and Goodwin, F.K.: Diazepam binding inhibitor, a brain
neuropeptide present in human spinal fluid: studies in depression, schizophrenia
and Alzheimer's disease. Arch. Gen. Psychiatry 43(12) :1143-1147, 1986.
465
ZOl MH 02181-05 NS
Breier, A., Arora, P.K., Pickar, D., Wolkowitz, O.M., and Paul, S.M.: Metabolic
stress produces rapid immunosuppression in man. Arch. Gen. Psychiatry (in
press) .
Breier, A., Wolkowitz, O.M., Doran, A.R., Roy, A., Boronow, J., Hommer, D.W., and
Pickar, D.: Neuroleptic responsivity of negative and positive symptoms in
schizophrenia. Am. J. Psychiatry (in press).
Cohen, R.M., Semple, W.E., Gross, M., Nordahl , T.E., DeLisi, L.E., Holcomb, H.H.,
King, A.C., Morihisa, J.M., and Pickar, D.: Dysfunction in a prefrontal
substrate of sustained attention in schizophrenia. Life Sci . 40:2031-2039, 1987.
Doran, A.R., Boronow, J., Weinberger, D.R., Wolkowitz, O.M., Breier, A., and
Pickar, D.: Structural brain pathology in schizophrenia revisited: prefrontal
cortex pathology is inversely correlated with CSF levels of homovanillic acid.
Neuropsychopharmacoloqy (in press).
Pickar, D.: Recent perspectives on the mechanism of action of neuroleptic drugs:
implications for a time-dependent model. Schizophr. Bull, (in press).
Pickar, D.: Pharmacotherapeutic approaches to schizophrenia. In: Jimerson,
D.C., Docherty, J. P. (eds), Psychopharmacology Consultation. Washington, DC, Am.
Psychiatric Press, 1986, pp 71-103.
Pickar, D., Breier, A., and Kelsoe, J.: Plasma homovanillic acid as an index of
central dopaminergic activity: studies in schizophrenic patients. NY Acad. Sci .
(in press) .
Pickar, D., Breier, A., Wolkowitz, O.M., and Doran, A.R.: The biochemical basis
for the antipsychotic effects of neuroleptics. Etiopathoqenic Hypotheses of
Schizophrenia. London, MPT Press (in press).
Pickar, D., Breier, A., Wolkowitz, O.M., and Pato, C: Profiles of the
pharmacologic response of positive and negative symptoms in schizophrenia.
Psychiatrie and Psychobioloqie (in press).
Pickar, D., Roy, A., Breier, A., Doran, A., Wolkowitz, 0., Colison, J., and
Agren, H.: Suicide and aggression in schizophrenia: neurobiologic correlates.
Ann. NY Acad. Sci. 487:189-196, 1986.
Pickar, D., Wolkowitz, O.M., Doran, A.R., Labarca, R., Roy, A., Breier, A., and
Narang, P.K.: Clinical and biochemical effects of verapamil administration to
schizophrenic patients. Arch. Gen. Psychiatry 44:113-118, 1987.
Pickar, D., Wolkowitz, O.M., Labarca, R., Doran, A.R., Breier, A., and Paul,
S.M.: Biochemical alterations produced by neuroleptics in man: studies of
plasma homovanillic acid in schizophrenic patients. In: Dahl , S.G., Gram, L.F.,
Paul, S.M., and Potter, W.Z. (eds). Clinical Pharmacology in Psychiatry.
Selectivity in Psychotropic Drug Action--Promises or Problems? (Vol 3). New
York, Springer-Verlag, 1986, pp 248-254.
466
ZOl MH 02181-05 NS
Roy, A., Pickar, D., Doran, A., Wolkowitz, 0., Gallucci, W., Chrousos, G., and
Gold, P.: The corticotropin-releasing hormone stimulation test in chronic
schizophrenia. Am. J. Psychiatry 143(11) :1393-1397, 1986.
Roy, A., Schreiber, J., Mazonson, A., and Pickar, D.
chronic schizophrenic patients: a follow-up study.
737-740, 1986.
: Suicidal behavior in
Can. J. Psychiatry 31(8):
Shelton, R.C., Doran, A.R., Pickar, D., and Weinberger, D.R.: Cerebral
structural pathology in schizophrenia: evidence for a selective prefrontal
cortical defect. Am. J. Psychiatry (in press).
467
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PEHIOO COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
Z01 MH 02184-05 NS
TTTLE OF PROJECT (80 charmctara or lass. 770s must tit on ona line balwaan Ota borOars.)
Neurobiology of Depression
PRINCIPAL INVESTIGATOR (IM other prolasaional paraonnal tMtow tfM Principal ktvastigator.) (Name, Wto. laboratory, and Inatltuta altmatlon)
PI: D. Pickar, Chief, Section on Clinical Studies NS, NIMH
Others:
S.M. Paul
A.F. Breier
C.N. Pato
M.H. Rapaport
O.M. Wolkowitz
G.A. Roy
Chief
Medical Staff Fellow
NRSA Fellow
Medical Staff Fellow
Medical Staff Fellow
Visiting Associate
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
LCS, NIAAA
COOPERATING UNITS (il any)
Laboratory of Clinical Studies, NIAAA; Laboratory of Clinical Science, Biological
Psychiatry Branch and Laboratory of Neurochemistry, NIMH
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Clinical Studies
INSTITUTE AND LOCATION
NIMH. NIH. Rethesda, Maryland 7089?
TOTAL MAN-YEARS:
AJL
PROFESSKDNAL
J^Jl.
_L_5-
CHECK APPROPRIATE BOX(ES)
^ (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The aim of this study is to investigate selected areas of the neurobiology of
depression and mania. In previous studies, we have observed that abnormal itv of
noradrenergic and HPA axis dysfunction occur together in seriously depressed
patients. We have pursued the study of the role of corticosteroids in depressive
illness by examining the effect of exogenous steroid administration. We have
found that orally administered dexamethasone produces selective effects on
catecholamine function in depressed patients; in contrast to normal controls,
depressed patients, particularly those with psychotic features, showed a
significant dexamethasone-induced increase in plasma MHPG and a decrease in plasma
HVA. These data suggesting abnormal corticosteroid -catechol amine interactions in
depression are consistent with the possibility that hypercortisolemia itself may
produce or enhance some of the biochemical changes and/or behavioral signs of
depression. In a double-blind study of orally administered prednisone (80 mg/day
X 5 days) to normal volunteers, we have further investigated steroid effects on
the central nervous system. The relationship between stress, steroids, and mood,
has been pursued in an experiment in which identical amounts of escapable and
inescapable aversive noise stimuli are presented to subjects. Preliminary results
suggest that inescapable but not escapable "stress" produces correlated mood and
neuroendocrine response. We have examined the effects of permanent separation
from one or both biological parent(s) between the ages of 2 and 17 years on the
development of adult psychopathology and observed that poorer quality of home 1 ife
and personal adaptation subsequent to parental loss was associated with
significantly higher incidence of ma.ior depression as adults and increased levels
of plasma Cortisol at the time of cross-sectional study. We are currently
investigating the therapeutic and biochemical effects of verapamil , a calcium
channel antagonist, in manic and hypomanic patients.
469
PHS 6040 (Rev. 1/84)
SPO ai4-9l«
ZOl MH 02184-05 NS
OTHER PROFESSIONAL PERSONNEL
W.Z. Potter Chief, Unit on Clinical Psychopharmacology LCS, NIMH
D. Rubinow Chief, Unit on Peptide Studies BPB, NIMH
PROJECT DESCRIPTION
The purpose of this project is to investigate selected aspects of the
neurobiology of depression and mania. Our studies have demonstrated the
association between hyperactivity of the noradrenergic system and hyperactivity
of the hypothalamic-pituitary-adrenal (HPA) axis in severely depressed patients.
In further studies of the HPA axis we have examined the effect of
corticosteroids, themselves, on behavior and neurotransmitter function. A
research project which examines the effects of "controllable" stress and
"uncontrollable" stress on behavior, HPA and catecholaminergic function has been
performed in normal volunteers and depressed patients. A study of the
development of depressive illness in adults who had experienced parental
separation during childhood has been accomplished.
METHODOLOGY (See: 1985 Annual Report, pp 475-477, Project Number
ZOl MH 02184 03 NS, Neurobiology of Depression)
Additional Methods:
1. The comparative behavioral and neuroendocrine effects of controllable and
uncontrollable aversive noise stress is studied in healthy volunteers, patients
with depression and patients with past histories of depression but in current
remission. The experiment consists of participation in two alternatively
assigned test days. On the controllable stress test day, aversive loud noise is
administered and can be terminated providing a simple button push sequence is
learned. On the uncontrollable stress test day the button responses are
independent of noise termination and the subjects are unable to stop the noise.
The amount of noise stress is held constant in both conditions by "yoking"
uncontrollable noise duration to controllable noise duration. Following the
noise stress, subjects are presented a mental task consisting of solving simple
anagrams under time pressure. Plasma levels of Cortisol and ACTH catecholamine
are measured throughout the experiment. Modifications of this protocol which
include mild electric shock in place of noise stress have been utilized in order
to enhance the stress response.
2. The effect of early parental loss on the development of adult psychopathology
was examined in a cohort of self-selected patients who had experienced permanent
separation from one parent between the years of 2 and 17 years of age. SADS-RDC
life time diagnoses were determined for all subject. Environmental factors
relating to personal and family adaptation to loss, family history and
neuroendocrine assessment at follow up were examined for relationship for the
development of adult psychopathology.
3. A prospective investigation of the clinical and therapeutic effects of the
calcium channel antagonist, verapamil, in patients with manic and hypomanic
symptoms is currently being performed on the 4-East Nursing Unit. Methodology is
the same as that already utilized to study the effects of verapamil in
470
ZOl MH 02184-05 NS
schizophrenic patients, and include longitudinal plasma amine metabolite sampling
and CSF assessment of neurotransmitter level.
MAJOR FINDINGS
1. Significant differences between plasma HVA and plasma MHPG response to
dexamethasone administration between depressed patients and controls was found.
In comparison to metabolite levels on the pre-dexamethasone control day, normal
controls showed a significant dexamethasone-induced increase in plasma levels of
HVA and a trend towards a decrease in plasma levels of MHPG. Conversely,
depressed patients, particularly those with psychotic features, showed a
significant dexamethasone-induced increase in plasma MHPG and a decrease in
plasma HVA relative to normal controls. Dexamethasone-induced increases in
plasma MHPG were directly correlated with the severity of depressive symptoms and
with post-dexamethasone Cortisol levels in the depressed patients.
2. The administration of 80 mg of prednisone to normal volunteers resulted in
subjective behavioral change in half of the subjects; behavioral effects
included depressive symptoms in some and mild elation in other subjects. In
addition to behavioral changes, prednisone produced a number of biological
effects including significant reductions in CSF levels of somatostatin, beta-
endorphin and beta-1 ipotropin.
3. In the studies of controllable and uncontrollable stress, despite the
duration of noise exposure being identical for each subject on both test days,
when exposed to uncontrollable stress subjects reported increased self-ratings of
helplessness, lack of control, attention, stress, unhappiness, anxiety and
depression as compared to either their baseline pre-stress ratings or following
exposure to controllable stress. The behavioral changes observed following
exposure to uncontrollable stress were also accompanied by increases in HPA axis
function as measured by increases in plasma ACTH; the latter were highly
correlated with increases in ratings of stress and tension and decrease in
ratings of happiness. In pilot data from depressed subjects, uncontrollable
stress appears to produce particularly pronounced increases in HPA axis function
and in behavioral response in comparison to controllable stress. Analysis of
catecholamine response (norepinephrine and epinephrine) is in progress.
4. The development of major depressive disorder in adulthood in patients who had
experienced permanent early parental separation was high. The best predictors of
adult depression was the quality of adjustment to the loss as determined by
Homelife and Personal Adaptation (HAPA) Scale developed by the investigators.
Plasma levels of Cortisol and ACTH were correlated positively with HAPA scores,
suggesting the possibility that early environmental trauma may be related to
enduring neuroendocrine traits.
5. To date three patients with manic-depressive illness have completed the
verapamil study. Verapamil appeared ineffective in either treating or preventing
mania in the two patients who had bipolar I type illness. In the third patient,
a rapidly cycling patient with bipolar II type illness, however, verapamil
produced mood stabilization. Further studies are in progress.
A7I
ZOl MH 02184-05 NS
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
In this project we have focused on selected areas of the neurobiology of
depression focusing on HPA function and stress. We have examined the CNS effects
of steroids on behavior and on catecholamine metabolites and peptide
transmitters. Results suggest abnormal corticosteroid-catecholamine interactions
in depressed patients, particularly in those with psychotic features. These data
are important as they contribute to the expanding body of data linking
catecholamine and HPA axis abnormalities in depression. The prospective
administration of oral prednisone to healthy volunteers has been a unique study
which contributes both to the understanding of the behavioral effects of
steroids, an important issue in medical practice, as well as steroidal
interactions with neurobiologic systems. Preliminary analysis suggests that
behavioral effects may be related to alterations of peptidergic systems.
The development of the controllable/uncontrollable stress paradigm is a unique
contribution to research in the area of depressive illness. Our preliminary
studies using this technique suggest that it may be an excellent tool for
examining the role of stress on mood and catechol aminergic and HPA axis function
in normal subjects. Its potential as a "model" of depression remains to be fully
explored.
Our study suggests that the association between early parental separation and the
development major depression in adulthood is critically dependent upon adjustment
to loss. This project provides extremely important information which may help in
adjusting children to parental loss. Moreover, findings suggest that adjustment
to parental loss in childhood may play a role in determining activity of the HPA
axis in adulthood.
The prospective evaluation of verapamil as a treatment for mania or hypomania
will provide important information pertaining to the treatment of bipolar illness
and provide excellent comparison for results already published from verapamil
administration to schizophrenic patients.
PROPOSED COURSE
We plan to pursue the use of the controllable/uncontrollable stress paradigm to
investigate the neurobiology of depression. We propose to investigate depressed
and currently euthymic unipolar and bipolar patients. We have developed a
modification of procedures to enhance the stress response. Pharmacologic
intervention which might modify the response to uncontrollable stress are being
evaluated.
PUBLICATIONS
Amsterdam, J.D., Henle, W., Winokur, A., Wolkowitz, O.M., Pickar, D., and Paul,
S.M.: Serum antibodies to Epstein-Barr virus in patients with major depressive
disorder. Am. J. Psychiatry 143(12) : 1593-1596. 1986.
Breier, A., Albus, M., Pickar, D., Zahn, T.P., Wolkowitz, O.M., and Paul, S.M.:
Controllable and uncontrollable stress in humans: alterations in mood,
neuroendocrine and psychophysiologic function. Am. J. Psychiatry (in press).
■'4 7 2
ZOl MH 02184-05 NS
Breier, A., Kelsoe, J.R., Kirwin, P.D., Beller, S.A., Wolkowitz, O.M., and
Pickar, D.: Early parental loss and the development of adult psychopathology.
Arch. Gen. Psychiatry (in press).
Roy, A. Five risk factors for depression. Br. J. Psychiatry 150:536-541, 1987.
Roy, A., Agren, H., Pickar, D., Linnoila, M., Doran, A. R., Cutler, N. R., and
Paul, S. M.: Reduced CSF concentrations of HVA and HVA to 5-HIAA ratios in
depressed patients: relationship to suicidal ity and dexamethasone
nonsuppression. Am. J. Psychiatry 143(12) :1539-1545, 1986.
Roy, A., Everett, D., Pickar, D., and Paul, S.M.: Platelet tritiated imipramine
binding and serotonin uptake in depressed patients and controls: relationship to
plasma Cortisol levels before and after dexamethasone administration. Arch. Gen.
Psychiatry 44:320-327, 1987.
Roy, A., Guthrie, S., Pickar, D., and Linnoila, M.: Plasma norepinephrine
responses to cold challenge in depressed patients and normal controls.
Psychiatry Res. 21:161-168, 1987.
Roy, A., Jimerson, D., Linnoila, M., Gold, P., and Pickar, D.: Premenopausal and
postmenopausal depressed patients. Aust. NZ J. Psychiatry 20:464-459, 1986.
Roy, A., Karoum, F., Linnoila, M., and Pickar, D.: Thyrotropin-releasing hormone
test in unipolar depressed patients and controls: relationship to clinical and
biologic variables. Acta Psychiatr. Scand. (in press).
Roy, A., Linnoila, M., Karoum, F., and Pickar, D.: Relative activity of
metabolic pathways for norepinephrine in endogenous depression. Acta Psychiatr.
Scand. 73:624-628, 1986.
Roy, A., Linnoila, M., IKaroum, F., and Pickar, D.: Urinary excretion of free
tyramine and norepinephrine and its metabolites in depression. Biol. Psychiatry
21:221-224, 1986.
Roy, A., Pickar, D., Douillet, P., Karoum, F., and Linnoila, M.: Urinary
monoamines and monoamine metabolites in subtypes of unipolar depressive disorder
and normal controls. Psychol. Med. 16(3) :541, 1986.
Roy, A., Pickar, D., Linnoila, M., Chrousos, G.P., and Gold, P.W.: Cerebrospinal
fluid corticotropin-releasing hormone in depression: relationship to
noradrenergic function. Psychiatry Res. 20:229-237, 1987.
Roy, A., Pickar, D., Paul, S., Doran, A., Chrousos, G.P., and Gold, P.W.: CSF
corticotropin-releasing hormone in depressed patients and normal control
subjects. Am. J. Psychiatry 144:641-645, 1987.
Wolkowitz, O.M., Doran, A.R., Breier, A., Roy, A., Jimerson, D.C., Sutton, M.E.,
Golden, R.N., Paul, S.M., and Pickar, D.: The effects of dexamethasone on plasma
homovanillic acid and 3-methoxy-4-hydroxyphenylglycol : evidence for abnormal
corticosteroid-catecholamine interactions in major depression. Arch. Gen.
Psychiatry 44:782-789, 1987.
473
ZOl MH 02184-05 NS
Wolkowitz, O.M., Rubinow, D.R., Breier, A., Doran, A.R., Davis, C, and Pickar,
D.: Prednisone decreases CSF somatostatin in healthy humans: implications for
neuropsychiatric illness. Life Sciences (in press).
474
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
-ZILL
n?i«7-n4 Ns
PERIOD COVERED
TITLE OF PROJECT (BO characters or less. Title must lit on one line tietween fhe borders.)
nifi7ppani Infusions as a Measure of Benzodiazepine Receptor Sensitivity 1n Humans
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atfillation)
PI: D.W. Hommer
Others: S.M. Paul
D. Pickar
A. Breier
H. Weingartner
Staff Psychiatrist NS, NIMH
Chief NS, NIMH
Chief, Section on Clinical Studies NS, NIMH
Medical Staff Fellow NS, NIMH
Chief, Section on Psychopathology LPP, NIMH
COOPERATING UNITS (it any)
Laboratory of Psychology and Psychopathology, NIMH; Alcohol Intramural Research
Program, National Institute of Alcohol Abuse and Alcoholism; Tufts University
^fftftf^l Neuroscience Branch
^^?8^ion on Clinical Studies
I'ffeS^iesda, Maryland 20205
TOTAL MAN-YEARS;
1.5
PROFESSIONAL;
1.0
0.5
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Increasing doses of
normal volunteers, d
subjects were pretre
Ro-15-1788. Followi
levels, plasma corti
anxiety and sedation
memory and attention
was quantified and d
curves provide a mea
as an evaluation of
benzodiazepines.
the benzodiazepine, diazepam,
rug-free alcoholic inpatients
ated with either placebo or t
ng each dose of drug saccadic
sol , and growth hormone were
were performed. After every
was performed. The effects
iazepam dose response curves
sure of benzodiazepine recept
the ability of specific antag
or placebo were administered to
In one series of experiments,
he benzodiazepine antagonist,
eye velocity, diazepam blood
"measured and self-ratings of
other dose cognitive testing of
of diazepam on these variables
constructed. These dose response
or sensitivity in humans, as well
onists to block the actions of
475
PHS 6040 (Rev 1/84)
OPo ai4-9ia
ZOl MH 02187-04 NS
OTHER PROFESSIONAL PERSONNEL
M. Linnoila Clinical Director ALC, NIAAA
D. Greenblatt Tufts University School of Medicine
PROJECT DESCRIPTION
We have been examining the ability of two BZ antagonists, the specific
antagonist Ro-15-1788 and the non-specific antagonist caffeine, to block the
effects of intravenous diazepam. Ro-15-1788 is given i.v., 0.035 mg/kg.
Caffeine is given orally either 3 or 10 mg/kg. Then diazepam is administered
intravenously in doses of 25, 25, 50, and 100 ug/kg at 15 minute intervals.
After each dose measurement of a variety biological and psychological variables
that are affected by benzodiazepines is made. These variables include: memory
and attention (done with Dr. Herbert Weingartner) , velocity of saccadic eye
movements growth hormone, Cortisol, and self-ratings of anxiety and sedation.
MAJOR FINDINGS
Plasma Cortisol, saccadic eye velocity, self-rated sedation and recent memory
all significantly decrease during diazepam administration while growth hormone
concentration is increased significantly in most, but not all, individuals.
From this data we constructed diazepam dose-response curves. These dose-
response curves provide an in vivo measure of BZ receptor sensitivity in humans.
Furthermore, we found extremely high correlations between all these variables
during diazepam administration suggesting that these disparate effects are all
mediated through the same class of BZ receptors as well as a high correlation
between changes in these variables and increasing plasma concentrations of
diazepam. Ro-15-1788 also blocks sedation, anxiety and decreases in saccadic
eye velocity. However, it failed to block diazepam-induced decrease Cortisol or
recent memory. In contrast Ro-15-1788 did block BZ induced changes in growth
hormone and attention.
In addition to studying BZ receptor sensitivity in normals we have also examined
BZ receptor sensitivity in 10 chronic alcoholic subjects who had been alcohol
and drug-free for one month at the time of the study. These subjects showed a
slightly greater sensitivity to diazepam than the normal controls.
476
ZOl MH 02187-04 NS
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
Benzodiazepines are the most commonly prescribed psychotropic drugs in the
world. The recent identification of stereospecif ic receptors for BZs in the
brain of both animals and humans raises several new and exciting avenues of
research not only into the mechanism of action of these anxiolytic and
anticonvulsant agents but also into the pathophysiology of anxiety itself. For
example, studies in rats have demonstrated that stress changes brain BZ
receptors. If a similar phenomena occurs in humans it may provide the
etiological link between stress and mental illnesses such as the anxiety,
depression and post-traumatic stress disorder. In order to study BZ receptor
sensitivity in psychiatric illness it is first necessary to develop a valid and
reliable measure of BZ receptor sensitivity in normal human subjects. This
project has provided such a measure of BZ receptor sensitivity and our
preliminary results suggest that BZ receptor sensitivity may be altered in
alcoholism.
PROPOSED COURSE
No further studies are planned since principal investigator is leaving NIMH.
PUBLICATIONS
Hommer, D. W., Matsuo, V., Wolkowitz, 0. M., Weingartner, H., and Paul, S. M.:
Pharmacodynamic approaches to benzodiazepine actions in man. Proceedings of the
4th International Meeting on Clinical Pharmacology in Psychiatry, Selectivity in
Psychotropic Drug Action — Promises or Problems, Vol. 3; Srpinger-Verlag, 1986,
pp. 52-61. Bethesda, MD (in press).
Weingartner, H., Thompson, K., Wolkowitz, 0., and Hommer, D. : Neuro-
pharmacological analysis of distinct types of memory processes.
Psychopharmacol ogy (in press).
Wolkowitz, 0. M., Weingartner, H., Thompson, K. , Pickar, D. , Paul, S. M., and
Hommer, D. W.: Diazepam-induced amnesia a neuropharmacological model of an
"organic amnestic syndrome." Am. J. Psychiatry 144:25-29, 1987.
477
PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
ZOl HH 02188-03 NS
TITLE OF PROJECT (80 characters or less. Title must fit on one line txtween the borders J
Biological Studies of Borderline Personality Disorder
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, latxtratorv, and institute affiliation)
PI: D.L. Gardner Staff Psychiatrist OCD, NIMH
Others: R.W. Cowdry Clinical Director NIMH
K.M. O'Leary Social Worker (Research) OCD, DIRP, NIMH
D. Pickar Chief, Sec. on Clinical Studies NS, NIMH
P. Lucas Clinical Fellow, NRSA Fellow NS, NIMH
D.L. Murphy Chief LCS, NIMH
COOPERATING UNITS (if any)
Office of the Director, Division of Intramural Research Program, NIMH;
Biological Psychiatry Branch, NIMH; Laboratory of Clinical Science, NIf
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Clinical Studies
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAL:
1.5
0.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues D (c) Neither
D (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been transferred to Office of the Clinical
Director (OCD).
479
PHS 6040 (Rev. 1/B4) SPO <M-9ia
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PflOjeCT NUMBER
ZOl MH 00179-06 NS
•"^c^o^^T, 1985 to September 30, 1986
TOU OF PfVMECT (SacAoractws or lM& M flwsf «.on
MorphoTogical Aspects of Peptides in Mammalian Brain
raiMCIPAL MVESTiaATpR.aM otfitr protantoMf ptnonntl bthwjht PilHOlptlJkunfilttirJ Pimm. tUt.
FT: L. SkirbolT Sr. Staff Fellow
NS, NIMH
COOPERATING UNITS (K any)
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Unit on Electrophysiology , Section on Molecular Pharmacology
INSTTTUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL-
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUUWMRY Of WORK (Usa standanl unraduc»d type. Do not 0xcaad lh» tpaem pmridad.)
PROJECT HAS BEEN DISCONTINUED DUE TO PRINCIPAL INVESTIGATOR'S REASSIGNMENT.
481
PMS 6040 (Rav. 1/84)
cfo •l4-«ta
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02177-05 NS
PERIOD COVERED
October 1, 1986 to September 30. 1987
TITLE OF PROJECT (80 Characters or lass. Title must fit on one line tMtween the borders.)
Behavioral Functions of Neuropeptides
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
D.M. Jacobowitz Section Chief LCS NIMH
PI:
J.N. Crawley
Senior Staff Fellow
Others:
L.R. Skirboll
Senior Staff Fellow
D.W. Hommer
Staff Psychiatrist
J. Mastropaolo
Staff Fellow
S.M. Paul
Chief
COOPERATING UNITS fff any; Unit on El ect rophysi ol ogy , Section on Molecular Pharmacology,
NS and Section on Histopharmacology, Laboratory of Clinical Science, NIMH
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Molecular Pharmacology
INSTITUTE AND LOCATION
NIMH, NIH. Bethesda. Maryland 20892
TOTAL MAN-YEAHS:
3.0
PROFESSIONAL:
_2^
AJL
CHECK APPROPRIATE BOX{ES)
□ (a) Human subjects
n (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The past decade has witnessed the discovery of forty or more peptides localized in
neurons of mammalian brain. Many cases of peptides coexisting in the same neuron
with classical transmitters have been described. Our laboratory is investigating
the functional significance of coexisting peptides and transmitters in the central
nervous system, using behavioral tools.
A) We previously showed that cholecystoi<inin (CCK) potentiates dopamine-
induced hyper! ocomotion in the nucleus accumbens, the terminal region of the
mesolimbic pathway where CCK and DA coexist. This year, we began to characterize
the role of CCK on DA autoreceptors in the ventral tegmentum (VTA), the cell body
region of the mesolimbic pathway where CCK and DA coexist. Preliminary data show
that CCK potentiates the hypol ocomotion induced by DA in the VTA, while having no
effect alone, i.e. the potentiating effect of CCK in the VTA was not blocked by
doses of proglumide which blocked the potentiating effect of CCK in the nucleus
accumbens. In addition, the unsulfated form of CCK-8 was also active in
potentiating DA-induced hypolocomotion in the VTA. These preliminary behavioral
data suggest that the pharmacology of CCK effects in the VTA match the "central-
type" CCK-receptor, as found by Skirboll and Hommer using electrophysiological
techniques. Conversely, the pharmacology of CCK effects in the nucleus accumbens
match the "peripheral -type" CCK receptor in our previous behavioral data.
j B) The nucleus basalis of Meynert lesion rat model of Alzeheimer's disease
'is now ongoing in our laboratory. We have completed one experiment showing that
! acetylcholine (ACH) can reverse the memory deficit in a T-maze task when given
I directly into the lateral ventricles, the first demonstration of a central site
of action for ACH. The second experiment found that atropine, but not
mecamylamine, blocked the ACH effect, suggesting the involvement of a muscarinic,
rather than a nicotinic cholinergic receptor. Tests of somatostatin and galanin,
alone and in combination with a submaximal dose of ACH, are in progress.
483
PHS 6040 (Rev 1/84)
CPO »\ 4-»\»
ZOl MH 02177-05 NS
OBJECTIVES
Behavioral analysis of the functions of neuropeptides, particularly the
functional significance of peptides coexisting with transmitters in mammalian
brain.
METHODS
(SEE: 1985 Annual Report, Project Number ZOl MH 02177-03 NS, pp 503-507)
Aseptic stereotaxic implanation of indwelling cannulae into brain nuclei of rats.
Microinjection of neurochemicals into discrete anatomical nuclei. Intra-
peritoneal injection of drugs. Behavioral analysis of neurochemical effects,
including locomotion, seizures, grooming, stereotyped motor behavior, and T-maze
alternation task. Histological verification of cannulae placement and injection
sites.
MAJOR FINDINGS
This year we initiated studies on the actions of cholecystokinin, (CCK) in the
ventral tegmental area (VTA) on the cell bodies of mesoljmbic neurons within
which CCK and dopamine (DA) coexist. CCK microinjected into the VTA, had no
effect alone on locomotion, at doses from lOOpg-lOOng. CCK significantly
potentiated the hypolocomotion into the VTA, at doses of lng-400ng CCK.
Unsul fated CCK, lOOng, also potentiated DA-induced hypolocomotion in the VTA.
Proglumide, lOmg/kg, did not block the ability of CCK, lOOng, to potentiate
DA-induced hypolocomotion. The potency of unsulfated CCK-8, and the lack of
action of proglumide, are consistent with the binding characteristics of the
"central -type" CCK receptors. These behavioral actions of CCK are consistent
with the electrophysiological data of Lana Skirboll and Dan Hommer, from
extracellular recording in the VTA. However, the behavioral profile of CCK in
the VTA differs from the behavioral profile of CCK in the nucleus accumbens,
where unsulfated CCK-8 Is inactive, and proglumide is an effective antagonist.
These preliminary data suggest that CCK modulates DA in a facilitatory manner, at
a "peripheral -type" receptor at the post -synaptic site in the nucleus accumbens,
and at a "central -type" receptor at the cell body autoreceptor site in the
ventral tegmentum.
The ability of oxytocin to stimulate grooming behaviors when microinjected into
the ventral tegmental area was characterized in terms of dose, time course,
influence of sex hormones, antagonism by an oxytocin antagonist, and antagonism
by dopamine receptor antagonists. In addition, doses of oxytocin which induced
grooming, considered a stress-related behavior, were found to have no effect on
locomotion. Most peptides affect locomotor activity when administered into the
VTA. Oxytocin may be a useful tool for delineating subpopulations of neurons
within the VTA with projections to brain regions other than the nucleus
accumbens, e.g. the prefrontal cortex.
486
ZOl MH 02177-05 NS
Several neuropeptides have been reported to decline in the brains of patient will
advanced Alzheimer's disease. The largest changes are in cerebral cortex regions
innervted by the nucleus basalis of Meynert, which degenerates in Alzheimer's
disease. To investigate the possible role of neuropeptides as potential
treatments for Alzheimer's disease, we are in the process of setting up the
nucleus basaslis of Merynert lesion model of Alzheimer's disease in rats. Dr.
John Mastropaolo joined our laboratory on July 7, 1986, to develop this project.
A standard t-maze alternation task was designed, built, and implemented. Dr.
Mastropaolo determined the best stereotoxic coordinates, the correct dose of
libotenic acid, the necessary number of lesion sites, and the minimum intertrial
delay, to optimize the difference in performance between lesioned and sham
control rats in the t-maze task. His first experiment showed that
intraventricular acetylcholine (ACH) could reverse the memory deficit created by
the nbM lesion, dose-response curve for ACH showed a maximal effect of lOug,
with doses of 20pg and above inducing seizures as we had previously reported for
carbachol. The restorative effects of ACH were blocked by atropine but not
mecamylamine, demonstrating that the ACH was acting through a muscarinic, rather
than a nicotinic, cholinergic receptor. These data are important for two
reasons: 1) they provide the first direct demonstration that ACH can reverse
memory deficits at a central site (all previous studies used systemic injections
of cholinergic drugs); 2) they provide the first direct demonstration of the
importance of the muscarinic receptor in memory processes after nbM lesions (most
previous studies of central cholinergic neurons in animal memory tasks implicated
nicotinic receptors). Current experiments are testing somatostatin and galanin,
alone and in combination with a submaximal dose of ACH, in this paradigm.
PROPOSED COURSE
All studies on peptide modulation of transmitter function will be continued, to
address questions of anatomical and neurochemical specificity, role of the
endogenous peptides, and testing of non-peptide analogs and antagonists as
putative new therapeutic drugs.
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
The basic research questions being answered center around the activity of
neuropeptides as either primary neurotransmitters or modulators of primary
neurotransmitters. The clinical applications of our results may be far-reaching.
If endogenous CCK is found to modulate endogenous dopamine in the mesolimbic
pathway, then new antipsychotic drugs could be developed from non-peptide
antagonists of CCK. Several drug companies, including Merck, Abbott, Cibu-Geigy
and Rotta are presently developing such antagonists. A combination of lower dose
of a DA antagonist, with a CCK antagonist, could reduce the symptoms of
schizophrenia without as great a risk for tardive dyskinesias. If peptides
reverse the memory deficits induced lesions of the nucleus basalis of Meynert,
then new treatments for Alzheimer's disease could be developed from non-peptide
analogs or antagonists.
487
ZOl MH 02177-05 NS
Blumstein, L. K. , Crawley, J. N., Davis, L. G. , and Baldino, F.: Neuropeptide
modulation of apomorphine-induced stereotyped behavior. Brain Res. 404:293-300
1987.
Crawley, J. N., Stivers, J. A., and Jacobowitz, D. M.: Neuropeptides modulate
carbachol -stimulated "boxing" behavior in the rat medial frontal cortex. In
Moody, T. W. (Ed.): Neural and Endocrine Peptides and Receptors, Plenum Press,
NY 1986.
Crawley, J. N.: Modulation of mesolimbic dopaminergic behaviors by
cholocyholecystokinin. The Mesocorticolimbic Dopamine System. Annals of the
York Academy of Sciences, in press.
Crawley, J. N.: Behavioral analysis of the functional significance of
peptide-transmitter coexistences. Receptor-Receptor Interactions, ed. K. Fuxe
and L. Agnati, in press.
Crawley, J. N., Hommer, D.W. and Skirboll, L.R.: Cholecystokinin-dopamine
interactions: electrophysiological and behavioral studies. Sixth International
Catecholamine Symposium, Alan R. Liss, Inc. NY, in press.
Hommer, D. W. , Stoner, 6., Crawley, J. N. , Paul, S. M. and Skirboll, L. R.:
Cholecystokinin-dopamine coexistence: electrophysiological actions corresponding
to cholecystokinin receptor subtype. J. Neurosci. 6:10:3039-3043, 1986.
Kaltwasser, M. T., Petrack, B. and Crawley, J. N.: Potency of CR 1409, a new
proglumide analog, on cholecystokinin-mediated behaviors and receptor binding.
Neurochemistry International, 10:4:547-553, 1987.
Kaltwasser, M. T. and Crawley, J. N.: Oxytocin and cholecystokinin induce
grooming behavior in the ventral tegmentum of the rat. Brain Research, (in
press).
Skirboll, L. R., Crawley, J. N., and Hommer, D. W.: Functional studies of CCK-
coexistence electrophysiology and behavior. In Hokfelt, T., Pernow, B., and Fuxe
K. (Eds.): Coexistence, Progress in Brain Research, volume 68, 1986.
Stivers, J. A. and Crawley, J. N.: Substance P antagonists block carbachol-
induced "boxing" behavior at a site of coexistence in the rat prefrontal cortex
Peptides, in press.
Stivers, J. A., Kaltwasser, M. T., Hill, P. S., Hruby, V. J. and Crawley, J. N.
Ventral tegmental oxytocin induces grooming. Peptides, in press.
Stivers, J. A., Skirboll, L. R., Long, R. and Crawley, J. N.: Anatomical
analysis of frontal cortex sites at which carbachol induces motor seizures in the
rat. Peptides, in press.
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1. 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 02178-04 NS
TITLE OF PROJECT (80 characters or less, me must fit on one line tietwaen the Ixyders.)
Pharmacol oq.y of Anxiety
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfillatlon)
PI: J.N. Crawley
R.C. Drugan
Others: P. Skolnick
Lesl ie Morrow
Steve Deutsch
Avi Weizman
Ronit Weizman
Steven M. Paul Chief
Senior Staff Fellow
NRSA Fellow
Pharmacologist
NS, NIMH
NS, NIHM
LBC, NIADDK
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
NS. NIMH
COOPERATING UNITS (it any)
Unit on Electrophysiology, Section on Molecular Pharmacology, NS, NIMH;
Laboratory on Bioorganic Chemistry, NIADDK.
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Molecular Pharmacology
INSTITUTE AND LOCATION
NTMH, RpthP-^Ha, Maryland 20892
TOTAL MAN-YEARS:
-UIL
PROFESSIONAL:
±JL
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (al) Minors
D (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Llse standard unreduced type. Do not exceed the space provided.)
Animal models of anxiety are being used to investigate the biological mechanisms
underlying anxiety-related behaviors. The anxiety component of the learned
helplessness model of depression was characterized using in vivo binding of
^H-Rol5-1788 and in assays of chloride flux in brain regions from rats
experiencing escapable versus inescapable stressors.
489
PHS 6040 (Rev 1/84)
SPO >i4-aia
ZOl MH 02178-04 NS
OBJECTIVES
Characterization of changes in neurotransmitters, receptors and effector system
induced by discrete behavioral events which may serve as models for human anxie
states.
METHODS
(SEE: 1985 Annual Report, Project Number ZOl MH 02178-03 NS, pp 509-511)
Mouse exploration test, rat learned helplessness test, receptor binding assays
for radiolabeled benzodiazepines, GABAergic drugs, and chloride channel ligands
Assays of radiolabeled chloride flux.
MAJOR FINDINGS
Dr Rob Drugan, a postdoctoral fellow in our Unit, has continued to analyze
neurochemical correlates of the learned helplessness paradigm. He has found a
large decrease in -^^s-tbRS binding in cerebral cortex and hippocampus of ra
who experienced inescapable tailshock but did not develop the syndrome,
suggesting that "coping" behavior requires changes in GABAergic function which
do not occur in "non-copers". These data also suggest that the chloride channe
is the most sensitive indicator for the benzodiazepine-GABA-chloride ionophore
receptor complex, since no changes in -^H-f lunitrazepam binding, and only mino
changes in -^H-muscimol binding, were seen in rats who showed large decreases
35s-TBPS binding. To further investigate the comparative indices of the
chloride channel versus the benzodiazepine binding site, two more powerful
techniques are presently being employed. In collaboration with Leslie Morrow,
Steven Paul 's lab, Rob is finding changes in chloride-18 flux in cerebral corte
from rats developing the learned helplessness syndrome. In collaboration with
Steve Deutsch, Ronit Weizman and Avi Weizman, in Steven Paul's lab, Rob is
finding significant changes in 3h-Ro-1788 binding when this radioligand for the
brain benzodiazepine receptor is injected intravenously, after the learned
helplessness procedure and before sacrifice.
In addition, in collaboration with Phil Skolnick, Rob is investigating the
mechanism by which inescapable tailshock reduces binding of 3h-Ro15-1788 to rat
kidney. Neither hypophysectomy nor adrenalectomy blocked this effect, suggesting
that behavioral stressors affect this peripheral -type benzodiazepine receptor by a
mechanism other than the hypothalamic-pituitary adrenal -axis.
PROPOSED COURSE
Rob will be completing the ongoing studies with chloride flux, in vivo
3h-Ro-17887 binding and 3h-Ro-4864 binding in the learned helplessness
paradigm. A new post -doctoral PRAT fellow will join our Unit in August, to
continue this progra Sandra Cottingham will use all of our behavioral and
biochemical assays to characterize the Maudsley Reactive rat, bred for
"hyperemotionality". We hope that she will also test this substrain for genetic
differences in structure or regulation of the genes for tyrosine hydroxylase and
gamma aminobutyric acid, in collaboration with Ed Ginns in our branch. Maudsley
Reactive and Non-reactive rats are currently breeding in our branch vivarium for
these studies.
490
ZOl MH 02178-04 NS
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
Our work on neural mechanisms mediating anxiety is designed to identify brain
transmitters, receptors, effector systems, and genetic factors which may be
responsible for syndromes of extreme and chronic anxiety.
PUBLICATIONS
Crawley, J. N., Glowa, J. R., Majewska, M. D., and Paul, S. M.: Anxiolytic
activity of an endogenous steroid. Brain Res. 398:382-385, 1986.
Drugan, R. C, Basile, A. S., Crawley, J. N., Paul, S. M., and Skolnick, P.:
Inescapable shock reduces [-^H] Ro5-4864 binding to "peripheral -type"
benzodiazepine receptors in the rat. Pharmacol. Biochem. Behav. 24:1673-1677,
1986.
Drugan, R. C, Basile, A. S., Crawley, J. N., Paul, S. M., and Skolnick, P.:
"Peripheral" benzodiazepine binding sites in the Maudsley Reactive Rat: Selective
decrease confined to peripheral tissue. Brain Research Bulletin 18:143-145,
1987.
Paul, S. M., Crawley, J. N., and Skolnick, P.: The neurobiology of anxiety.
American Handbook of Psychiatry (in press).
Drugan, R. C, Crawley, J. N., Paul, S. M. and Skolnick, P: Buspirone attenuate
learned helplessness behavior in rats. Drug Devel Res. 10:63-67, 1987.
Smith, C. B. and Crawley, J. N.: Anxiolytic action of CGS 9896 on mouse
exploratory behavior. Europ. J. Pharmacol. 132:259-262, 1986.
491
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02179-05 NS
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 chanctars or less. TMe must fit on ona Una between ttia txirders.)
Animal Models of Neuropsychiatric Disorders
PRINCIPAL INVESTIGATOR (Ust other prolesslonaJ personnel tielow the Principal Investigator.) (Name, title, laboratory, and institute aHillation)
PI: J.N. Crawley
Others: S.M. Paul
P. Suzdak
G. Wand
Senior Staff Fellow
Chief
Staff Fellow
Asst. Professor
Dept of Neuroscience, Johns Hopkins
University School of Medicine
NS, NIMH
NS, NIMH
NS, NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Molecular Pharmacology
INSTITUTE AND LOCATION
NIMH. Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.5
PROFESSIONAL;
0.5
0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Characterization of a benzodiazepine, Rol5-4513, which antagonizes the
biochemical actions of ethanol in the rat, was performed on the behavioral test
for intoxication. Other inverse agonists such as FG-7142 and e-CCE did not
block the intoxicating effects of ethanol, suggesting that Rol5-4513 acts
through a mechanism which is independent of any direct behavioral effects of
inverse agonists of the central benzodiazepine receptor.
Preliminary assays of neuropeptides in the hypothalamus of dwarf hamsters found
increased levels of NPY in separated males. Microinjection of NPY in rats is
known to stimulate feeding; separated male dwarf hamsters have increased body
weight.
493
PHS 6040 (Rev 1/84)
GPO 91 4-918
ZOl MH 02179-05 NS
OBJECTIVES
Development and evaluation of a new hamster model of depression. Application of
animal models to study the biochemical basis of neuropsychiatric disorders
including depression, schizophrenia, obesity and alcoholism.
METHODS
(SEE: 1985 Annual Report, Project Number ZOl MH 02179-03 NS, pp 513-515)
Breeding, pairing, separating, and neurochemical assays of Siberian dwarf
hamsters. Behavioral testing for locomotion, feeding, and alcohol -induced
intoxication. Chronic drug treatments, cannulation and microinjection into
discrete anatomical loci.
MAJOR FINDINGS
Rol5-4513, was found to both prevent and reverse the intoxication produced by
2g/kg ethyl alcohol in a dose-related fashion. Lower doses, of Rol5-4513 were
required to prevent than to reverse intoxication. Intoxication induced by
methanol and by t-butyl alcohol was also prevented Rol5-4513. The benzodiazepine
receptor antagonists, Rol5-1788 and CGS 8162, blocked the actions of Rol5-4513 on
both prevention and reversal of alcohol intoxication. Two other inverse
antagonists of the central benzodiazepine receptor failed to reverse
ethanol -induced intoxication, FG-7142 (10 mg/kg and 30 mg/kg) and B-CCE (10
mg/kg). However, combined treatment of Ro4513 and 6-CCE produced competitive
interactions. High doses of Rol5-4513 failed to reverse intoxication induced by
high doses of ethanol (4 grams/kg). These data suggest that Rol5-4513 can
effectively inhibit alcohol -induced intoxication at an unknown site which is
different than, but linked to, the site at which Rol5-4513 and other inverse
agonists bind to the central benzodiazepine receptor.
In collaboration with Dr. Gary Wand, several peptides have been assayed in
pituitary hypothalamus and cortex of paired and separate male dwarf hamsters.
Separated males were found to have significantly lower levels of ACTH in the
pituitary, and significantly higher levels of neuropeptide Y in the hypothalamus.
These preliminary data are interesting in that separated males are less active
and have higher body weights than paired males. Neuropeptide Y has been found to
be the most potent peptide which increases feeding behavior in male rats.
PROPOSED COURSE
The behavioral analysis of Rol5-4513 is being completed for publication. Further
testing of this class of drugs for antagonism of alcohol -induced intoxication
will be undertaken if such compounds become available.
Replications of assays for NPY, ACTH, and CRF are in progress in paired and
separated dwarf hamsters, to determine the importance of these neuropeptides for
the "separation syndrome".
494
ZOl MH 02179-05 NS
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
Our Unit is the only laboratory in NIMH for the development of new animal model
of neuropsychiatric disorders. We continue to investigate the biological bases
of dysfunctions including depression, schizophrenia, anxiety, obesity, and
alcoholism. Our applications goal is to help develop rational, specific drugs
for these illnesses.
PUBLICATIONS
Angel, I., Kiss, A., Stivers, J. A., Skirboll, L. R., Crawley, J. N., and Paul,
S. M.: Regulation of [^Hlmazindol binding to subhypothalamic areas:
involvement in glucoprivic feeding. Brain Res. Bull. 17:873-877, 1986.
Angel, I., Stivers, J. A., Paul, S. M. and Crawley, J. N.: Site of action of
anorectic drugs: glucoprivic- versus food deprivation-induced feeding.
Pharmacol. Biochem. Behav., in press.
495
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE PROJECT NUMBER
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1. 1986 to September 30. 1987
ZOl MH 02180-05 NS
TniB OF PROJECT (ao characters or less. TWe must m on one line betwam the borders.) El GCt TOphysi Ol OQI Cdl StudieS Of
Peptidergic and GABAergic Function in Mammalian Brain.
PRINCIPAL INVESTIGATOR (Ust other pmtessioni personnel below the Principal Investigator.) (Name. Wto. laboratory, and institute altlllation)
PI: D. Hommer Staff Psychiatrist NS, NIMH
Others: L. Skirboll Sr. Staff Fellow NS, NIMH
J.N. Crawley Sr. Staff Fellow NS, NIMH
S.M. Paul Chief NS, NIMH
COOPERATING UNITS (it any)
LAB/BRANCH
Clinical Npurnsr.ienr.e Branch
Unit nn Elpctrophy^inlngy, Sprtinn nn Mnlpriilar Pharmarnlngy
INSTITUTE AND LOCATION
NIMH, BPthP<;Ha, Maryland ?(]RQ?
TOTAL MAN-YEARS:
2.JL
PROFESSIONAL:
Li
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Using extracellular single unit recording techniques, we have examined the
effects of stress and pharmacological agents which either alleviate or mimic the
effects of stress on individual neurons in the rat substantia nigra (SN).
Specifically, learned helplessness induced by uncontrollable stressful shocks
results in a supersensitivity to gamma-amino butyric acid (GABA) agonists while
shocks which are controllable do not produce GABAergic supersensitivity. The
anxiogenic benzodiazepine (BZ) receptor ligand, beta-carboline carboxylate ethyl
ester (gCCE) increases the activity of neurons in the SN zona reticulata (ZR) but
had no effect on noradrenergic neurons in the locus coeruleus. Caffeine also
mimics many of the effects of BCCE in the SN but its actions are not reversed by
the specific BZ antagonist Rol5-1788 as are those of gCCE. Furthermore, the
effects of caffeine could be blocked by lesions of the caudate nucleus while the
effects of bCCE were unaffected by such lesions.
We have continued our studies on the interactions between endogenously
occurring neuropeptides and classical neurotransmitters. All varieties of
CCK-like peptides which bind to brain CCK receptors also potentiate DA in those
areas where CCK and DA coexist while those CCK-like peptides which do not bind to
this, receptor are ineffectual in facilitating DA inhibition. The putative
cholecystokinin (CCK) antagonists, proglumide and benzotript, were found to weakly
block CCK in proportion to their potency at central CCK receptors.
Dynorphin (DYN) appears to modulate the response of SNZR neurons to GABA.
Finally, we have examined the effects to cholinergic agents in the SN. Nicotinic
agents appear to activate DA neurons in the SN through a central mechanism.
497
PHS 6040 (Rev. 1/84) gpo»14-»h
ZOl MH 02180-05 NS
PROJECT DESCRIPTION
OBJECTIVES
The SNZR is a region of the brain which contains a high concentration of GABA, BZ
receptors as well the opiate-like peptide, DYN. Recently several groups have
shown that microinjections of GABA agonists into the SN, but not into adjacent
mesencephalic areas, blocks electrically-induced seizures in rats. These
microinjections also block the limbic afterdischarge following kindled seizures.
This suggests that the SN may be an important region involved in the propagation
of seizure activity as well as in modulation of limbic system function.
Furthermore, since GABA, BZ's and opiates all have been implicated in the neural
response to stress, the SN represents an excellent system in which to study the
effects of stress as well as the drugs and putative transmitters which may
modulate stress and anxiety.
Aside from peptides and GABA present in the SN, it has also been reported that
the SN is an area in nicotinic receptors. In this regard, we have been examining
the cholinergic influence on the activity of nigral neurons. We have explored
the effects of nicotine on the firing of neurons in the SNZR and SN zona compacta
(ZC) neurons.
Immunohistochemical studies have shown that there is a coexistence of DA and CCK
in a subpopulation of mesencephalic neurons in the rat which project primarily to
limbic areas. We have previously reported that these DA-CCK containing cells are
excited by either systemically or iontophoretically administered CCK, we have
investigated the question of the functional significance of this coexistence
(i.e. how does the peptide CCK and the classical neurotransmitter, DA, interact
to affect the activity of neurons). We have also investigated the interaction
between GABA and peptide dynorphine (DYN) in the SNZR. These experiments provide
another avenue toward developing an understanding of the functional relationship
between neurotransmitter systems.
METHODS
(SEE: 1985 Annual Report, Project Number ZOl MH 02180-03 NS, pp 517-521)
MAJOR FINDINGS
1. Effects of stress on GABAergic sensitivity in the SNZR.
When rats are subjected to tail shock which they cannot control, this experience
produces a supersensitive response to the intravenously administered GABA
agonist, muscimol. This effect appears to related to the uncontrollable nature
of the shock. Animals which are subjected to an identical shock exposure but
over which they have some control do not develop a supersensitive response to
muscimol. Thus, uncontrollable stress leads to an increased sensitivity of SNZR
neurons the inhibitory actions of GABAergic agents. This GABAergic
supersensitivity is preent immediately after termination of the uncontrollable
shock and persist for at least 2 hours. It is not prevented by adrenalectomy.
498
ZOl MH 02180-05 NS
2. Effects of drugs which may mimic the effects of stress.
Caffeine decreases the activity of DA neurons in the ventral tegmental (VT) area
which project to limbic regions but has little effect on DA neurons in the more
lateral SN regions which project to the striatum. This caffeine-induced
inhibition can be blocked by either diazepam or haloperidol suggesting that both
the BZ receptor and DA release may play a role in mediating caffeine's actions.
Caffeine also produces an excitatory effect in the SNZR. This excitation can be
blocked by lesions of the caudate nucleus suggesting that the pro-convulsant an
anxiogenic actions of caffeine are at least in part mediated through the basa
ganglia. In a separate series of experiments we have examined the effects of a
intravenously aadministered alprazolam, a high-potency triazolo-benzodiazepine,
on the activity of A9 and AlO DA neuron. Alprazolam had a small dose-dependent
excitatory effect on the DA nurons which project to the striatum in contrast it
demonstrated a marked, dose-dependent, excitatory effect on DA neurons in AlO.
It appears that the cell bodies of the mesolimbic and mesocortical DA systems are
much more sensitive to BZ effects than is the nigrostriatal system.
3. CCK-DA interactions
As we have previously reported, ceruletide and sulfated CCK-8 (CCK-8-US) both
potentiate the effects of the DA agonist, apomorphine, on DA neurons in the
medial SN. We have also found that unsul fated CCK-8 (CCK-8-US) and CCK-4 posse a
similar ability to potentiate apomorphine induced inhibition in the SN. The rank
order potencies were as follows: ceruletide > CCK-8-S = CCK-8-US > CCK-4; CCK-3
was without effect. These potencies directly parallel the affinity of these
peptides for the brain CCK receptor. In contrast, only CCK-8-S produced
excitatory effect on DA neurons while CCK-US, CCK-4 and CCK-3 were devoid of
effect. This profile of activity corresponds to the affinities of the various
CCK-like peptides at the peripheral CCK binding site. It appears that CCK's
different CNS effects may be mediated by different CCK receptors.
4. GABA-DYN interactions.
We have found that DYN, an opiate-like peptide present in high concentrations the
SNZR modulates the effects of the neuronal inhibition produced by GABA applied
directly to nigral neurons. DYN had two distinct actions; either potentiation or
attenuation of the effects of GABA. In cells which were themselves inhibited by
DYN, the peptide potentiated GABA's actions. In those cells which were
unresponsive to DYN, simultaneous application of DYN and GABA resulted in an
attenuation of the GABA inhibition. These findings provide further evidence for
the modulatory interaction between peptides and classical neurotransmitters.
None of the effects of DYN could be blocked by mu or sigma opiate receptor
antagonists.
5. Nicotinic influences on nigral activity.
Low systemic doses of nicotine stimulated the firing of SNZC DA cells;
experiments with antagonists showed this action to be due to a central and
probably direct action of nicotine on these cells. Nicotine also excited
non-DYN cells in the SNZR, but this action was clearly of peripheral origin.
499
ZOl MH 02180-05 NS
These results are in accord with autoradiographic data showing that nicotinic
binding sites in the SN are largely restricted to the SNZC.
PROJECTED COURSE
We plan to continue our investigations of the effects of stress in the SN and
expand our efforts to include examinations of other neurotransmitters such as
DYN and enkephalins as well as 6ABA. We also plan to more fully characterize
the phenomenon of increased sensitivity to GABAergic agents through the use of
iontophoretic techniques and an examination of the role of stress related
hormones such as the corticosteroids.
Studies of the interactions between classical transmitters and peptides will be
extended along several lines. First, the modulation of the GABA response by
DYN will be compared to other peptide like substance P and the enkephalins.
Secondly, the hypothalamus will be explored as a site of multiple transmitter
interaction. In immunohistochemical studies (see ZOl MH 00179-04 NS), we have
been exploring the transmitter systems which innervate the paraventricular
nucleus. These transmitters can be altered by adrenalectomy or systemic
administration of a phenyl -n-methyl -transferase inhibitor. We plan to explore
the functional correlate of these findings. That is, look at the electro-
physiologic response of paraventricular nucleus neurons to neuropeptide
adrenaline and adrenalectomy.
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND TO THE PROGRAM OF THE INSTITUTE
Studies of the interaction between transmitters (CCK-DA and GABA-DYN) may
provide a prototype for modulatory function between peptide and classical
transmitters. Furthermore, electrophysiological studies of BZ and GABA
complement ongoing biochemical and behavioral studies in these areas. The SN
appears to be an important brain region in the modulation of seizures and some
aspects of anxiety. Electrophysiological techniques are particularly well
suited to further our understanding of how the SN performs this function. In
addition, the hypothalamus is the site of control of many vegetative and
neuroendocrine functions which control response to stress. Thus, understanding
how stress affects the nigral and hypothalamic systems may be of potential
value in developing pharmacological approaches to stress related diseases, both
medical and psychiatric.
PUBLICATIONS
Clarke, P., Hommer, D., and Skirboll, L.: Stimulation of cholinergic projection
to substantia nigra increase neuronal activity. Brain Res, (in press).
Clarke, P. B. S., Pert, A., Hommer, D. W., and Skirboll, L. R.: Electro-
physiological actions of nicotine on substantia nigra single units. Br. J.
Pharmacol. 85:827-835, 1985.
Hommer, D., Stoner, G., and Skirboll, L.: CCK-DA coexistence: electro-
psyciological actions corresponding to CCK-receptor subtype. J. Neurosci.
6:3039-3043, 1986.
500
ZOl MH 02180-05 NS
Skirboll, L. R., Crawley, J., and Hommer, D.: Functional studies of CCK-DA
coexistence: electrophysiology and behavior. In Hokfelt, T., Pernow, B., and
Fuxe, K. (eds). Progress in Brain Research, 681, (in press).
Stoner, G. , Skirboll, L., and Hommer, D. : Differential effects of an anxiogenic
e-carboline on single unit activity in the locus coeruleus and substantia nigra
of rat brain. Neuropharmacology, (in press).
501
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02182-05 NS
^ci°oherT° 1986 to September 30, 1987
TITLE OF PROJECT (80 charactprs or Igss. Tide must tit,on one line between the borpers.)
Toward the Visuanzation of Opiate Receptors in Living Humans
PRINCIPAL INVESTIGATOR (List other professionel personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: C. B. Pert Guest Researcher NS, NIMH
Others:
N. L. Ostrowski
K. C. Rice
Senior Staff Fellow
Pharmacologist
LCM, NIMH
LC, NIDDK
COOPERATING UNITS (if any)
Laboratory of Chemistry, NIDDK
LAB/BRANCH ., . „ ,
Clinical Neuroscience Branch
SECTION
section on Brain Biochemistry
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3.5
PROFESSIONAL:
2.5
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The distribution of positron emitting substances in brain can be followed by
positron emission tomography (PET). We are developing ^sp.iabeied high affinity
opiate drugs to be injected into living humans for the visualization of opiate
receptor patterns in vivo. It will be interesting to determine whether opiate
receptor distribution patterns in cortex change as a function of attention and
emotional states. Meanwhile, we have carefully documented the receptor-binding
properties and kinetics of ^H-cyclofoxy in rat brain after in vivo injection.
503
PHS 6040 (Rev 1IBA)
ZOl MH 02182-05 NS
Project Description:
Objectives:
To demonstrate gradients of opiate receptor density in the cortex of living
humans. To examine whether differences in these gradients exist as a function
of emotional state or attentional processes.
Methods Employed:
PET Scan--using newly developed ^^F-labeled opiate analogs. Autoradiography of
rat brain slices.
Major Findings:
We managed to affix a fluoride moiety to naltrexone, a potent opiate antagonist
without losing affinity for opiate receptors. This fluoro-opiate derivative is
suitable for iji vivo injections for visualizing receptors. We have visualized
stereospecific, striking images for opiate receptors in the thalamus, basal
ganglia and frontal cortex of a living baboon. Tritium labeled cyclofoxy has
been prepared and shown in rats to have the appropriate opiate distribution
pattern.
Significance to Biomedical Research and Program of the Institute:
The notion that alterations in mood are a function of oscillations in neuro-
trans mitter receptor sensitivity is perhaps the most exciting new lead in
attempting to understand the causes of mental illness. Other leads in this
institute point to the relevance of cortical participation as a critical factor
in psychiatric disease. The opiate receptor is the most well-studied of brain
receptors and appears to be associated with the pleasure of fulfilled appetite.
Proposed Course:
Our new useful probe, [3H]-3-acetylcyclofoxy, will be characterized thoroughly
in rodents to fulfill requirements for human use. Human studies, first on
normal controls, then psychiatric patients, should enable a rigorous test of
theories of emotions which emphasize neuropeptide receptors.
Publications:
1. Ostrowksi, N.L., Burke, T., Rice, K.C., Pert, A. and Pert, C.B. The pattern
of [^Hjcyclofoxy retention in rat brain after J_n vivo injection corresponds
to the j_n vitro opiate receptor distribution. Brain Res. , 402: 275-286,
1986.
2. Ostrowski, N.L., Hill, J.M., Pert, C.B. and Pert, A. Autoradiographic
visualization of sex difference in the pattern and density of opiate
receptors. Brain Res., 421, 1-13, 1987.
504
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02183-05 NS
PERIOD COVERED
October 1. 1986 to September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the txrders.)
Is Schizophrenia an Autoimmune Neuropeptide Receptor Disease?
PRINCIPAL INVESTIGATOR (List other protessional personnel txlow the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: C. B. Pert Guest Researcher NS, NIMH
Others: J. G. Knight Guest Researcher NS, NIMH
P. Laing Visiting Associate NS, NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Brain Biochemistry
INSTITUTE AND LOCATION
NIMH, AHAMHA, NTH, RPthesda, Maryland ?QM?
TOTAL MAN-YEARS:
AJL
PROFESSIONAL:
4.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects [xl (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standaixl unreduced type. Do not exceed the space provided.)
The notion that schizophrenia has an important autoimmune component has been
around for several decades, but has not previously been subjected to analysis by
the most sensitive, modern techniques. We have hypothesized that early
(perinatal) tolerance to viruses, which use the receptor molecule to gain
cellular entry, causes this autoimmunity. We have developed a simple sensitive
assay for detecting antibodies directed against human brain found in sera of
schizophrenic patients and controls. We are now subjecting human brain
membrane receptors to either native or denaturing solubilization conditions and
separation by polyacrylamide gel electrophresis (PAGE) . After visualization by
antiserum or CSF from patients and controls, we hope to identify the specific
brain antigens against which schizophrenics have generated antibodies.
505
PHS 6040 (Rev. 1/84)
GPo ai4-aia
ZOl MH 02183-05 NS
Project Description:
Objectives:
To develop a simple assay for demonstrating brain-directed autoantibodies in
schizophrenic sera; to demonstrate the molecular properties of these brain
antigens and their distribution in brain tissue; and to explore the possibility
that the antigens are cell surface neuropeptide receptors which mediate the
biochemistry of emotion.
Methods Employed:
A novel filtration and centrifugation assay for detecting brain antigens in sera
and the (McLean, et al.. Brain Res. 278: 255-257, 1983) method for visualizing
antibody distribution patterns in brain.
Major Findings:
In collaboration with Dr. Weber, over twenty experiments were performed for the
purpose of optimizing the conditions of the antibody detection assay. In an
early blind experiment, six of the Clinical Center 4-East ward acute
schizophrenics' and controls' sera were examined. The two highest numbers in
the assay belonged to the two sickest patients. We utilized the sera from these
two patients vs. two controls in every experiment as we worked on optimization.
The assay appears to sensitively and repeatedly demonstrate differences between
controls and sera from other patients which were screened by Dr. DeLisi. The
new patient's serum level appeared elevated even after repeated blood sample
withdrawals over a period of one year. We know history of this area and are
proceeding caustiously.
A significant (14%) percentage of hospitalized chronic patients have antibrain
antibodies titers outside the control range.
A pilot study suggests macrophage chemotaxis is altered in schizophrenics.
Significance to Biomedical Research and Program of the Institute:
Schizophrenia is a crippling psychiatric disease which affects one percent of
the general population. A complete, convincing understanding of its etiology
would almost certainly lead to better therapeutic strategies and would place
this psychiatric illness in a more "normal" context with other diseases of the
body.
Proposed Course:
We must now collect a large number of determinations on many sera to describe
the incidence in normal and schizophrenic sera as well as correlating psychotic
symptoms with antibody titers over time within one patient. We plan to perform
appropriate controls for neuroleptic drug treatment. We plan future experiments
to test whether the incidence of antibodies directed against brain are much
higher in schizophrenics--and perhaps certain subtypes--than in normal controls.
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and that this incidence is not due to neuroleptic drug treatment. If schio-
phrenia is indeed a viral ly-triggered autoimmune disease, we will prove it and
provide replicatable methods for others.
Publications:
1. Knight, J.G., Knight, A. and Pert, C.B. Is schizophrenia a virally-
triggered anti -receptor autoimmune disease? In: Biological Perspectives
in Schizophrenia, Helmchen, H. and Henn, F.A. (Eds.), John Wiley and Sons
Ltd., New York, 1987, pp. 107-124.
507
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 02189-04 NS
TITLE OF PROJECT (BO characters or less. We must lit on one line between the lx>rders.)
Neuropeptides and their Receptors are Shared by the Brain and the Immune System
PRINCIPAL INVESTIGATOR (List other prolessional personnel tielow the Principal Investigator.) (Name, title, laboratory, and institute atflllation)
PI: C. B. Pert Guest Researcher MS, NIMH
Others:
P. Sacerdote
J. M. Hill
M. R. Ruff
Visiting Fellow
Senior Staff Fellow
Guest Researcher
NS, NIMH
NS, NIMH
NS, NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Brain Biochemistry
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 2089?
TOTAL MAN-YEARS:
4.0
PROFESSIONAL:
2.5
1.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
D (b) Human tissues S (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Neuropeptides, small signal peptides largely known for their role as transmitters
of nerve inpulses in the brain which mediate mood and emotion, have now been
shown to regulate immune system function. Our work reveals that human monocytes
have receptors and will respond chemotactical ly to numerous neuropeptides.
Neuropeptides which we have reported on include B-endorphin and other opiates,
substance P and bombesin. We have shown that a major class of psychoactive
drugs, the benzodiazepines, are also potent chemoattractants. In this case we
have directly demonstrated the presence of chemotactic receptors through ligand
binding experiments. The presence of diverse, distinct neuropeptide chemotactic
receptors on monocytes and other immune system cells suggests the existence of a
neuroendocrine link between the brain and the immune system whose purpose is to
integrate behavioral and emotional responses with immune system function.
In addition to the presence of neuropeptide receptors we have also been able to
demonstrate that human alveolar macrophages store and secrete the neuropeptide
bombesin. Neuropeptide synthesis is, therefore, a general feature of various
immune cell populations. Such results are consistent with a multi-directional
communication network via neuropeptides and their receptors. The purpose of this
network is to link the body's cellular defense and repair systems with the
nervous and endocrine systems and thereby integrate the internal mil lieu of the
whole organism. The flow of information in this network is perceived by the
human organisms emotional and/or altered states of consciousness. Ultimately,
this results in behavioral decisions at the whole organism level. Additional
work has suggested that a major cause of human cancer, small cell lung carcinoma,
may not, as previously thought, arise from lung epithelium but originates from
hemopoietic cells when the normal macrophage mediated repair of lung tissue is
deranged by continuous heavy smoking. ^^^
PHS 6040 (nov MBA)
ZOl MH 02189-04 NS
Project Description:
Objectives:
Neuropeptide effects on immune function: All considerations of health and
well-being must necessarily attempt to understand immune system function within
the context of the whole organism. For this reason we have considered the role
of a class of molecules termed neuropeptides - short chains of amino acids
primarily known for their role in nervous system function. What is becoming
abundantly clear however is that these compounds, far from acting solely within
the confines of the brain, are ubiquitous and have pleiotropic effects,
functioning as transmitters, growth hormones, and signal agents for all the body
systems. We feel that the neuropeptides are key components of a network whose
purpose is to integrate behavior and brain function with immunological and
endocrine systems.
In order to establish these points we have decided initially to examine the
effects of neuropeptides in several ongoing model systems which deal with the
immunological aspects of inflammatory responses, specifically the macrophage/
granulocyte component. Macrophages figure prominently in many inflammatory
processes, such as arthritis or gingivitis. Inflammation is known to have a
neurogenic component, and neuropeptides (such as substance P), released from
local nerves, appears to play a role in some inflammatory vascular reactions, as
well as in arthritis. We questioned whether such locally released neuropeptides
could exert some of their effects through immune cells such as the macrophage.
Our first objective was, therefore, to demonstrate a direct role for select
neuropeptides in immune function. Additionally, we wished to consider the
possibility that neuropeptides, as a general class of compounds, might have
effects on immune function. Sporadic reports of neuropeptide effects on in
vitro mast cell and lymphocyte function have been made over the last 10 years,
however, no unifying concepts from this diverse (sparse) literature have
emerged.
Small cell lung cancer as a disease of macrophages: Lung cancer is the leading
cause of cancer death in the United States and greater than 25,000 people die
each year of a subtype of lung cancer known as small cell (oat cell) lung cancer
(SCLC). We have recently proposed that SCLC arises not from lung epithelium but
rather from the macrophages present in the lungs of chronic smokers. Our
interpretation of the etiology of SCLC emphasizes lung emplysema, inflammation,
and tissue damage as a stimulus for myelopoiesis and recruitment of bone marrow
derived macrophages into diseased lung. These cells then becomes transformed
and give rise to the disease known as SCLC.
Our program is directed toward exploring similarities between SCLC cells and
macrophages with the intent to develop novel therapeutic modalities. These
studies will also focus on new aspects of inflammatory cell biology particularly
the conditions which may lead to cell transformation and progression towards
overt neoplastic disease.
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Methods Employed:
Currently, we are testing various neuropeptides in several standard assays of
marcrophage function. This primarily involves an evaluation of the ability of
the peptides to induce directed migration of macrophages and other cell types in
Boyden chamber type assays. Many of the known chemotactic agents have
additional effects on cell physiology and as active chemotactic peptides are
identified they can be evaluated for other actions.
In addition to the functional assay of chemotaxis we have developed radiorecep-
tor binding assays for one of the chemotactically active ligands we have studied
(benzodiazepines) and are developing assays for others. These studies will
permit a biochemical identification and, eventually, characterization of the
receptors for some of these compounds. To date no neuropeptide receptors have
been isolated in any system and binding studies (in some cases without any
ascribed function) are only now being attempted on immune cells. In concert
with these binding studies we are also attempting a preliminary biochemical
characterization of one of the neuropeptide receptors we have identified on
human monocytes (opiate receptor) through the method of chemical cross-linking
of a radiolabeled ligand to its receptor.
We are examining immune cells for their possible content of several neuropep-
tides. Cell extracts are resolved by HPLC fractionation and peptides identified
by radioimmunoassay. Specific anti-peptide antibodies are being used as
histochemical probes to detect neuropeptides in specific immune cells and
tissues.
Major Findings:
The opiate peptides (e.g., enkephalin, 3-endorphin) are potent chemoattractants
for human monocytes. Pharmacologic specificity can be demonstrated through the
use of various agonists and antagonists of the opiate receptor. These compounds
are exceeding potent; activity can be detected at 10"^'+ concentrations. Several
other neuropeptides have been found to have chemotactic activity for human
monocytes with similarly low active concentrations. These include the hypo-
tensive, bradykinin like peptide substance P, and the hypothalamic peptide
bombesin. Pharmacologic studies using closely related analogs of these peptides
indicate that chemotactic effects are mediated by specific neuropeptide
receptors.
The benzodiazepine (e.g., valium) class of drugs are also potent chemoattract-
ants for human monocytes. Binding studies confirm the presence of receptors
with the appropriate structure/function relationship. Benzodiazepines are among
the most widely prescribed drugs in the USA and no effects on macrophages or
immune function have previously been described. The endogenous ligand for this
receptor is a neuropeptide, only recently identified.
In addition to studies which have revealed a role for neuropeptides in monocyte
chemotaxis we have also been able to show that other cells also express highly
specific chemotactic receptors for these compounds. Thus, tumor cells, which
may also express migratory potential, will chemotax in response to selected
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neuropeptides. This response is not characteristic of all tumor cells but shows
selectivity, both with respect to cells which respond and to peptides which are
active. We have reported on the ability of neuropeptides such as bombesin,
B-endorphin, substance P, and other to promote SCLC chemotaxis. Ongoing studies
reveal that other highly metostatic tumors (e.g., breast) will also respond
chemotactically to select neuropeptides. We have also shown that human
spermatozoa will migrate chemotactically to various peptides. This new
methodology, modified from our monocyte techniques, can now be utilized by
researchers in human fertility who have to date not been able to assess sperm
motility in any sensitive, quantitative fashion.
Small cell lung cancer: The cell of origin for SCLC has been speculative for 50
years but has focused on a neuropeptide secreting cell sparsely distributed in
lung epithelium. Primarily, this is because SCLC cells synthesize a number of
neuropeptides; most consistantly bombesin. Our recognition of the importance of
macrophages in inflammatory diseases and our studies on neuropeptide synthesis
by macrophages prompted a direct test of the hypothesis that the putative
neuropeptide secreting precursor was not a lung epithelial cell but another
other cell which figured prominently in the pathology of smokers lung, the
macrophage. We were able to demonstrate four surface antigens, found only on
macrophages and their precursors, to be present on cell lines and tumors of
SCLC. These results are now confirmed and extended by other groups and we have
interpreted these results to support our suggestion of a macrophage derivation
for SCLC.
With regard to our studies on lung cancer it should be noted that no effective
therapy exists for this disease and it is rapidly lethal; average life
expectance for untreated SCLC is 5-8 v/eeks. Oral cancer, although less common
than lung cancer, is also associated with the use of tobacco products, and the
etiology we propose for lung cancer is quite relevant to this disease as well.
Our results have suggested a novel interpretation of the etiology of SCLC which
suggests a number of unexplored therapeutic strategems.
The cell of origin for SCLC has been speculative for 50 years but has focused on
a neuropeptide secreting cell sparsely distributed in lung epithelium.
Primarily, this is because SCLC cells synthesize a number of neuropeptides; most
consistantly bombesin. Our recognition of the importance of macrophages in
inflammatory diseases and our studies on neuropeptide synthesis by macrophages
prompted a direct test of the hypothesis that the putative neuropeptide
secreting precursor was not a lung epithelial cell but another cell which
figured prominently in the pathology of smokers lung, the macrophage. We were
able to demonstrate four surface antigens, found only on macrophages and their
precursors, to be present on cell lines and tumors of SCLC. These results are
now confirmed and extended by other groups and we have interpreted these results
to support our suggestion of a macrophage derivation for SCLC.
Among the growth regulating hormones which control monocyte growth and differen-
tiation are the interferons and colony stimulating factors. These and other
immune hormones can now be evaluated systematically within this new conceptual
framework for their effect on SCLC cells. The recent cloning of both of these
hormones makes this an attractive approach as ample precident documents the
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ZOl MH 02189-04 NS
ability of these agents to modify leukemia cell growth in some settings.
Various combined modalities may ultimately prove more efficacious than current
treatments; limited to chemotherapy.
Significance to Biomedical Research and Program of the Institute:
We have identified a group of very potent compounds, the neuropeptides, which
exert hormone effects on human monocytes and other cells. Our results,
primarily utilizing a chemotactic assay system are novel and indicative of a
broader role for these class of compounds in immune system function.
Neuropeptides are known to cause both mood and behavioral alterations when
acting within the brain, and to be released into the body during various
emotional and physical states. Because these same peptides have very potent
effects on macrophages, as well as other components of the immune system, we
feel that these compounds are a major class of biochemicals which subserve
information exchange between the brain and the body. The functional interaction
of the body's cells through networks of neuropeptides and their receptors would
be expected to be critical to the health of the organism as a whole and suggests
a mechanism by which emotional states can significantly alter the course and
outcome of biological illnesses previously considered to be strictly in the
somatic realm.
The physiological correlate of i_n vitro monocyte chemotaxis to these chemicals
is still obscure but it seems likely that the local release of neuropeptides may
have important effects on cell distribution and activities. Thus, to cite one
example, the peptide substance P has been implicated in the vascular erythe-
matouns reactions associated with inflammation and this peptide has \jery
recently been shown to exacerbate experimental arthritis. Monocytes and lympho-
cytes (which also have substance P receptors) are prominent, locally present,
cells which are primarily responsible for the degenerative changes which
characterize arthritic lesions. Thus, it seems likely that this neuropeptide,
by virtue of its ability to local ife and activate immune cells may have an
important causative role in this and other inflammatory processes. The recent
demonstration that depletion of substance P from the local area surrounding an
arthritic joint resulted in a substantial emelioration of the disease suggests
the feasibility and importance of a program directed toward the understanding of
neuropeptide effects on macrophage and immune function.
The ability of neuropeptides to effect monocyte and some tumor cell migration
suggests a further role for these agents in histogenesis and tissue
organization, serving to recruit and/or maintain resident macrophage and other
cell populations. Disseminated neoplastic diseases may, to some extent, develop
as a result of neuropeptide regulated cell trafficking. Thus, tumor cells,
which have detached from the primary mass, may respond to organ (site) specific
neuropeptide attractants. An understanding of this process could be relevant to
control line tumor spread and may help explain the frequent metastasis of some
tumors (e.g., SCLC, breast) to neuropeptide-rich body sites.
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Proposed Course:
Explore clinical settings in which neuropeptide macrophage mediated responses
may have significant causative or diagnostic potential. Various systemic
diseases with an underlying neuropeptidergic component may be reflected in
altered macrophage neuropeptide chemotactic responses. An initial survey is
being made of illnesses in which macrophages have a role, such as lung cancer to
detect such alterations. Other diseases or conditions in which neuropeptides
are known to play a role may also reveal alterations at the level of altered
macrophage neuropeptide responsiveness. Neuropeptide therapy may prove useful
in select illnesses by virtue of effects on macrophage or immune function. In
vitro systems, such as chemotaxis, could be used to facilitate design of new
drugs.
Establish the in vivo role for neuropeptides in macrophage function. Neuropep-
tides can be stimulated to be released at various sites (e.g., electrically,
mechanically, chemically) and the accumulation of immune cells studied. These
studies would support i_n vitro observations and could suggest the context in
which physiological responses may occur. Such information could be important in
understanding certain pathological states where macrophages accumulate and in
devising ways to regulate their function.
Extended studies revealing antigenic similarities between macrophages and SCLC
cells to explore functional similarities. This work will be directed at
developing strategems for indutumoring cell differentiation and growth cessation
with the aim of developing new therapeutic stategems. These studies will focus
on possible lymphokine and monokine regulation of tumor growth/differentiation.
We will also attempt to define the conditions which may result in transformation
of inflammatory macrophages into cancer.
Biochemical studies aimed at characterizing neuropeptide receptors on monocytes
and other immune cells. We will focus on receptor identification through
binding and cross-linking studies with the aim of purificaiton and raising
antibodies. These studies will make it possible to examine the mechanisms of
receptor function leading to cellular activation. Anti-receptor antibodies with
agonist on antagonist activity could be used experimentally and possibly
therapeutical ly.
Publications:
1. Wiedermann, C.J., Goldman, M.E., Serti , K., Plutchok, J.J., Kaliner, M.A.,
Johnston-Early, A., Cohen, M.H., Ruff, M.R. and Pert, C.B. Bombesin in
human and guinea pig alveolar macrophages. J. Immunol., 137: 3928-3932,
1986.
2. Wiedermann, C.J., Sertl , K. and Pert, C.B. Substance P receptors in rat
spleen: characterization and autoradiographic distribution. Blood, 68:
1398-1401, 1986.
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3. Wiedermann, C.J., Goldman, M.E. and Pert, C.B. Immunoreactive gastrin-
releasing peptide in heat inactivated feta calf serum. Cell and Tissue
Kinetics, 19: 467-470, 1986.
4. Gnessi, L., Fabbri , A., Silvestroni, L., Moretti , C, Fraioli, P., Pert,
C.B. and Isidori, A. Evidence for the presence of specific receptors for
N-formyl chemotactic peptides on human spermatozoa. J. Clin. Endocrinol.
Metab. , 63: 841-846, 1986.
5. Pert, C.B. The wisdom of the receptors: neuropeptides, the emotions, and
bodymind. Advances, 3: 8-16, 1986.
6. Wiedermann, C.J., Sertl , K. and Pert, C.B. High-affinity substance P
binding sites in rat esophagus plexus submucosus. Am. J. Physiol., 253:
R167-R171, 1987. ^
7. Hill, J.M., Lesniak, M.A., Rojeski, M., Pert, C.B. and Roth, J. Receptors
for insulin receptor peptides in the CNS: studies of localization in rat
brain. In: Insulin Structure and Function, Raizoda, M. (Ed.), Plenum
Press, New York, 1987, pp. 261-267.
8. Farrar, W.L., Hill, J.M., Ruff, M.R. and Pert, C.B. Visualization and
characterization of interleukin 1 receptor in brain. J. Immunol., 139:
459-463, 1987.
9. Farrar, W.L., Hill, J.M., Ruff, M.R. and Pert, C.B. Visualization of cyto-
kine and virus receptors common to the immune and central nervous systems.
Lymphokine Res., 6: 29-34, 1987.
10. Wiedermann, C.J., Sertl., K., and Pert, C.B. Neuropeptides and the immune
system: substance P receptors in bronchus-associated lymphoid tissue of
rat. Annals of NY Acad, of Sci., in press.
11. Sacerdote, P., Ruff, M.R. and Pert, C.B. Cholecystokinin and the immune
system: receptor-mediated chemotaxis of human and rat monocytes.
Peptides, in press.
12. Wiedermann, C.J., Sertl, K. , Zipser, B., Hill, J.M. and Pert, C.B. Vaso-
active intestinal peptide receptors in rat spleen and brain: a shared
communication network. Peptides, in press.
13. Hill, J.M., Lesniak, M.A, and Pert, C.B. Co-localization of IGF-II
receptors, IL-1 receptors and Thy 1.1 in rat brain. Peptides, in press.
14. Ruff, M.R., Sacerdote, P., Wiedermann, C.J. and Pert, C.B. Neuropeptide
receptors are shared components of nervous and immune systems. In:
Neuropeptides and Stress, Tache, Y. (Ed.), Springer-Verlag, in press.
15. Wiedermann, C.J., Jelesoff, N.E., Pert, C.B. and Hill, J.M. Distribution
of epidermal growth factor receptors in rat brain. Peptides, in press.
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16. Weber, R.J., Hill, J.M. and Pert, C.B. Regional distribution of Thy 1.1
in rat brain. J. Neuroimmunol . , in. press.
17. Hill, J.M. Neuropeptides and their receptors as the biochemicals of
emotions. In: Coping with Uncertainty: Biological Behavioral and
Developmental Perspectives, Palermo, D.S. (Ed.), Lawrence Eribaum
Associates, Inc., New Jersey, in press.
18. Hill, J.M. and Pert, C.B. Neurochemical basis of emotional behavior.
In: Handbook of Neuropsychology, Balber, F. and Grafman, J. (Eds.),
Elsevier, Amsterdam, in press.
19. Hill, J.M., Lesniak, M.A., Kiess, W. and Nissley, S.P. Radioimmuno-
histochemical localization of type II IGF receptors in rat brain.
Peptides , in press.
20. Sacerdote, P., Wiedermann, C.J., Wahl , L.M., Pert, C.B. and Ruff, M.R.
Visualization and characterization of cholecystokinin receptors on a
subset of human monocytes and in rat spleen. J. Exper. Med. , in press.
516
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1. 1986 to September 30. 1987
PROJECT NUMBER
ZOl
02190-04 NS
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Distribution and Properties of Opiate and Other Brain Receptors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atfillatlon)
PI: C. B. Pert Guest Researcher NS, NIMH
Others :
M. R. Ruff
C. M. Fraser
C. J. Venter
Guest Researcher
Pharmacologist
Chief, Recept. Biochem.
NS, NIMH
LNP, NINCDS
LNP, NINCDS
COOPERATING UNITS (if any)
Section on Receptor Biochemistry, Laboratory of Neurophysiology, NINCDS
LAB/BRANCH
Clinical Neuroscience Branch
SECTION
Section on Brain Biochemist
jqL
INSTITUTE AND LOCATION
NTMH, ADAMHA, NTH, BPth
i<;H;^, Maryland ?n8Q?
TOTAL MAN-YEARS:
PROFESSIONAL:
4.0
9 R
J_J-
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
□ (a1) Minors
D (a2) Interviews
D (b) Human tissues (xl (c) Neither
SUMMARY OF WORK (LIse standard unreduced type. Do not exceed the space provided.)
Cross-linking is a relatively recent biochemical stratepy for covalently affixing
reversible ligands to their recognition molecules for subsequent electrophoretic
analysis. [^^^Il-Tyr^^-B-endorphin (prepared as originally described by Smythe
end co-workers) binds stereospecifically to rat brain membranes. While some
studies have suggested that the B-endorphin receptor is a unique "epsilon" opiate
receptor, a larger body of evidence suggest that R-endorohin has high affinity for
most if not all of the opiate receptors types and subtypes. Cross-linking opiate
receptors from different tissue sources can potentially reveal much information
about the molecular basis of apparent opiate receptor heterogeneity. Cross-link-
ing, however, only fixes 1% of the bound trace and SDS-PAGE while exquisitely
sensitive, can fail to reveal substantial inter-molecular differences. Cross-
linkina was performed with the homo bi-functional reagent Disuccinimidyl Suberate
(DSS).' The iodinated cross-linking products of Tetrahymena, leech CNS, and
rat brain membranes (both type 1 and type 2 conditions) appeared indistinguishable
on SDS-PAGE gel with major cross-linking products at 58K and 100-llOK. The
strong cross-linked bands produced by incubation in the presence of the inactive
opiate ((+)-naloxone) was completely abolished by the same (lO'^M) concentration
of its active isomer (-)-naloxone. Although we have thus far failed to dis-
tinguish betv/een opiate receptors from a mammal , an invertebrate, and a uni-
cellular organism, we continue to explore various conditions of binding, and
electrophoresis, (e.g., reduced and unreduced) to examine possible receptor
differences, both intra and inter species. Electrophoresis of proteolytic digests
of cross-linked bands will be performed as a particularly sensitive method for
distinquishing heterogeneity. Thus far, our cross-linking experiment suggest
that the recognition molecule (the opiate receptor) which binds all opiate
alkaloids and peptides is stable across evolution. As proposed, apparent physio-
loqical receptor hrtrrogenei ty is due to coupling to other membrane components.
I'HS (iCXn (Miiv 1/11-1)
GPO Bl 4-01
ZOl MH 02190-04 NS
Project Findings:
Objectives:
To map the neuroanatomical distribution of various chemically coded pathway in
brain and to understand the neuroscientific significance of "multiple"
receptors.
Methods Employed:
Newly developed vn vitro autoradiography - unfixed frozen brain tissue is melted
onto slides, incubated in appropriate radioactive ligand to label receptors,
washed sedially, dried rapidly, fixed with paraformaldehyde vapors and dipped in
radiosensitive liquid emulsion for autoradiographic visualization.
Sophisticated computer analysis of receptor binding kinetics is used to
rigorously define conditons of multiple opiate receptor binding.
For the first time we bring together rigorous kinetic analysis with autoradio-
graphic distribution of binding sites.
We are cross-linking reversible ligands covalently to their recognition molecule
for subsequent electrophoretic analysis.
Major Findings:
One opiate delta receptor appears conformational ly fixed, while the other
appears capable of assuming mu, delta and kappa conformations.
We showed that 6-endorphin labeled opiate receptor from rat, leech and
Tetrahymena have the same molecular weights of 58 and llOKd on SDS-PAGE. This
suggests that the opiate receptor is stable across evolution.
Significance to Biomedical Research and Program of the Institute:
Pinpointing neurochemical ly coded tracts will enable us to determine the
functional significance of each newly discovered pathway. The method can be
used on human brain and ultimately should give information about the
contribution of various neurochemically coded tracts to pathology.
Proposed Course:
We plan a sophisticated biochemical and immunological approach to further
defining the molecular nature of opiate receptors. The type 1 opiate receptor
complex with its advanced evolutionary accumulation in the forebrain of humans
seems particularly worthy of further study (see Project Number ZOl MH 02182-03
NS, Toward the Visualization of Opiate Receptors in Living Human). We plan to
study the brain distribution of insulin, transferrin, and their receptors to
further demonstrate the breakdown in the distinction between "neuropeptides" and
hormones.
518
ZOl MH 02190-04 NS
Publications:
1. O'Neill, J.B., Pert, C.B., Ruff, M.R,, Smith, CC, Higgins, W.J. and
Zipser, B. Identification and characterization of the opiate receptor
in the ciliated protozoan, Tetrahymena. Brain Res. , in press.
519
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1. 1986 to September 30. 1987
PROJECT NUMBER
ZOl MH 02191-02 NS
TITLE OF PROJECT (BO charactars or less. Title must tit on one line between tite Ixrders.)
Brain Receptors for the AIDS Virus and Other Neurotrophic Viruses
PRINCIPAL INVESTIGATOR (List other professional personnel tyelow ttw Principal Investigator) (Name, title, lat>oratory, and institute atflllatlon)
PI: C. B. Pert Guest Researcher NS, NIMH
Others;
J. M. Hill
E. M. Sternberg
M. R. Ruff
P. Sacerdote
Senior Staff Fellow
Guest Researcher
Guest Researcher
Visiting Fellow
NS, NIMH
NS, NIMH
NS, NIMH
NS, NIMH
COOPERATING UNITS (H any)
Clinical Neuroscience Branch
SECTION
Section on Brain Biochemis
IS try
INSTITUTE AND LOCATION
ADAMHA. NIH.
BethRsda. Maryland 20897.
TOTAL MAN-YEARS:
PROFESSIONAL:
-Li
UL
SLh.
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed ttie space provided.)
We have demonstrated that the 60
of T lymphocytes and named "T4",
the brain and immune system. Thu
molecule can be cross-linked to ^
cipitated by the Mab 0KT4 in both
the brain distribution pattern of
and rat brain and shown that it i
hippocampus which subserves cocini
suggest that the neuropsychiatric
be due to inflammatory processes
the virus.
kD protein previously characterized on a subset
is another example of shared components between
s, we have demonstrated that this cell surface
25i labeled AIDS virus envelope and immunopre-
T cells and brain. Furthermore, we have mapped
the AIDS virus receptor, T4 in monkey, human
s most enriched in areas of the cortex and the
tion and other higher functions. Our work
effects of AIDS may not, as previously thought,
but may be due to a direct neuronal infection of
We have identified, synthesized, and studied an nctapeptide "peptide T", which
appears to be the critical attachment area of the AIDS viral envelope. Peptide T
and several rationally designed peptide analogs appear to bind with high affinity
to the AIDS virus receptor," blocking viral infectivity at very low concentrations.
We expect that synthetic peptide heteropolymers employing this core pentapeptide
attachment sequence will prove valuable as an approach for a vaccine for AIDS.
This method and approach appears useful for exploring the presence of other virus
receptors in brain. For example, we have already observed that the Epstein-Barr
virus which has been known to use the complement receptor on B cells as a
receptor entry protein, may actually infect brain via the same receptor molecule
which we have recently identified in brain.
521
I __^_
PHS 6040 (Rev 1/84) '^^° •' *■•
ZOl MH 02191-02 NS
Project Description:
Objectives:
To find the cure for AIDS.
Methods Employed:
Virus receptor binding, viral infectivity, human monocyte chemotaxis, cross-
linking to and molecular separation of brain and immune cell receptors.
Major Findings:
By molecular characterization and immunoprecipitation, we have demonstrated that
the AIDS virus receptor (T4) is present in human, monkey and rat brain in an
indistinguishable form as that present on human T cells. We are isolating an
endogenous peptide ligand that binds to these receptors, which will presumably
mediate behavioral activity as well as immune function, from peptide extracts of
rat brain. Meanwhile, synthetic peptides have been deduced with computer-
assistance which bind with \/ery high affinity (lO'^^M) to AIDS virus receptors
on rat brain membranes and displace radiolabeled viral envelope protein (gpl20)
at the same low concentrations. A series of peptide analogs have been
constructed and a structure activity relationship for T4 receptors has been
documented. This structure activity relationship appears to be constant whether
human monocyte chemotaxis, AID virus infectivity, behavioral activity of rats
after intraventricular injection, displacement of radiolabeled AIDS viral
envelope, or inhibition of lymphocyte PHA mitogenesis or DR expression is
studied.
Clearly the AIDS virus receptor and its endogenous peptide ligand are yet
another example of a neuropeptide receptor and ligand subserving intercellular
communication throughout the brain and body.
Significance to Biomedical Research and Program of the Institute:
AIS is the #1 public health problem in the USA. Unexpectedly, an understanding
of neuropeptides and their receptors, a specialty of our Institute's Program in
general and my research specifically, is highly desirable to understand AIDS.
Proposed Course:
We will optimize peptide structure to prevent proteolysis and thus obtain "The
Ultimate Peptide". We will use knowledge gained from this viral disorder to
understand schizophrenia.
Publications:
1. Hill, J.M., Farrar, W.L. and Pert, C.B. Localization of the T4 antigen/
AIDS virus receptor in monkey and rat brain: prominence in cortical
regions. Psychopharmacology Bulletin, 22: 686-694, 1986.
522
ZOl MH 02191-02 NS
2. Hill, J.M., Farrar, W.L. and Pert, C.B. Autoradiographic localization of
T4 antigen, the HIV receptor, in human brain. Intl. J. Neurosci., 29:
687-693, 1986.
3. Pert, C.B., Hill, J.M., Ruff, M.R., Berman, P.M., Robey, W.G., Arthur,
L.O., Ruscetti, F.W. and Farrar, W.L. Octapeptides deduced from the
neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit
human immunodeficiency virus receptor binding and T cell infectivity.
Proc. Natl. Acad. Sci. USA, 83: 9254-9258, 1986.
4. Pert, C.B. and Ruff, M.R. Peptide T[4-8]: a pentapeptide sequence in the
AIDS virus envelope which blocks infectivity is essentially conserved
across nine isolates. Clin. Neuropharmacol . , 9(S4): 482, 1986.
5. Ruff, M.R., Martin, B.M., Ginns, E.I., Farrar, W.L., Wahl , S.M. and Pert,
C.B. CD4 receptor binding peptides that block HIV infectivity cause human
monocyte chemotaxis: relationship to vasoactive intestinal polypeptide.
FEES Lett., 211: 17-22, 1987.
6. Wetterberg, L., Alexius, B., Saaf, J., Sonnerborg, A., Britton, S. and
Pert, C. Peptide T in treatment of AIDS. Lancet, January 17, 1987, p.
159.
7. Ruff, M.R., Hallberg, P.L., Hill, J.M. and Pert, C.B. Peptide T[4-8] is
the core HIV envelope sequence required for CD4 receptor attachment.
Lancet, 2: 751, 1987.
8. Ruscetti, F.W., Farrar, W.L., Hill, J.M. and Pert, C.B. Visualization of a
differentiation antigen of human helper T lymphocytes in primtae brain.
Peptides, in press.
9. Sacerdote, P., Ruff, M.R. and Pert, C.B. Vasoactive intestinal peptide:
a ligand for the CD4 (T4)/human immunodeficiency virus receptor present on
brain and immune cells. J. Neurosci. Res., in press.
10. Hill, J.M., Ruff, M.R., Lesniak, M.A., Roth, J. and Pert, C.B. Molecular
components common to the immune system and neurons: growth factors and
their receptors. In: AIDS: Challenge to Neuroscience, Psychology and
Drug Abuse Research: Advances in Biochemical Pharmacology, Bridge, P.
(Ed.), in press.
11. Pert, C.B., Ruff, M.R., Ruscetti, F., Farrar, W.L. and Hill, J.M. HIV
receptor in brain and deduced peptides that block viral infectivity. In:
AIDS: Challenge to Neuroscience, Psychology and Drug Abuse Research:
Advances in Biochemical Pharmacology, Bridge, P. (Edi), in press.
12. Pert, C.B., Smith, C.C, Ruff, M.R. and Hill, J.M. AIDS and its dementia
as a neuropeptide disorder: role of VIP receptor blockade by human
immunodeficiency virus (HIV) envelope. Annals of Neurology, in press.
523
13,
Farrar, W.L., Ruff, M.R., Hill, J.M
IL-1 receptor in brain. In: AIDS
and Drug Abuse Research: Advances
ZOl MH 02191-02 NS
and Pert, C.B. Characterization of
Challenge to Neuroscience, Psychology
in Biochemical Pharmacology, Bridge, P.
iEd. ) , in press.
14. Sternberg, E.M., Damschroder-Wi 11 iams , P.J., Weber, R.J. and Pert, C.B.
Peptide T, VIP and their analogs induce la expression in murine macro-
phages. Proc. Natl. Acad. Sci . USA, in press.
524
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
PROJECT NUMBER
ZOl MH 02152-08 LDP
October 1. 1986 through September 30. 1987
TITLE OF PROJECT (80 characters or less. We must fit on one tine between r/ie tMrders.)
Discipline and Parental Control in Families with Affective Disorders
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: G. Kochanska Research Psychologist LDP/NIMH
Univ. of Guelph
Guelph, Ontario
LDP/NIMH
Other: L. Kuczynski
M, Radke-Yarrow
Assoc. Prof, of Psychology
Chief
COOPERATING UNITS (if any)
University of Guelph
Guelph, Ontario Canada
LAB/BRANCH
Laboratory of Developmental Psyrhnlngy
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda. Maryland 20892
TOTAL MAN-YEARS:
.82
PROFESSIONAL:
.35
,47
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects
S (a1) Minors
® (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Mothers' discipline and control practices and their children's responses to
maternal control attempts are studied in well and clinically depressed mothers.
Impaired parental skills in managing children's behavior have often been implied
in the etiology of maladaptive patterns of child development. Depressive sympto-
matology has been linked to inappropriate control practices, but specific
difficulties of depressed mothers and possible implications for their children's
development have not been Identified. Assessments of mother and child behavior
are based on observations of their interaction in a naturalistic setting (see
Annual Report MH 02144). Detailed measures of maternal control were taken,
including goals, timing and specific techniques, and the overall interactive
quality of control episodes. Child response was also measured in terms of
compliance to maternal demands and in terras of level of social competence of non-
compliance strategies. Analyses revealed that affectively ill mothers, compared
to well mothers, have specific difficulties controlling their children. In
particular, they are more likely to avoid confronting the child and less able
to negotiate a compromise after their initial intervention meets with child's
resistance. Children of well mothers, but not of depressed mothers, became more
cooperative over time. Daughters of depressed mothers appeared at particular
risk for noncompliance problems.
525
PHS 6040 (Rev. 1/84)
GPO 91 4*«ia
ZOl MH-02152-08 LDP
Project Description
The determinants, contents, and effects of parental discipline and control
practices in families with normal and clinically depressed mothers are
investigated. Effective, yet harmonious parental control of child behavior is
crucial for child development and socialization. It becomes of particular
importance in the second and third year of life when two conflicting develop-
ments occur: the child becomes able to regulate his/her own behavior and to
comply with parental demands, but also becomes more overtly resistant towards
caregivers, which is a manifestation of emerging autonomy. If the parent is
not able to maintain effective control of the child, and at the same time to
promote the child's emerging autonomy, this may be a source of future malad-
justments and dysfunctions. Maternal depression has often been associated with
general maladaptive patterns of control, such as hostility, punitiveness , and
low involvement. However, more specific processes have not been identified.
Also, the impact of maladaptive maternal strategies on the development of the
child has not been determined.
Mothers' control practices were examined in terms of their effectiveness in
obtaining child compliance, and in terms of promoting acceptable and competent
forms of ascertaining child autonomy.
Methods
The basic paradigm is described in Annual Report // MH-021A4. Control interven-
tions of 33 well, 37 unipolar depressed and 17 bipolar depressed mothers and
their children (16 to 50 months of age) were analyzed. Each control episode
occurring during 90 minutes of naturalistic interactions was coded, starting
with the mother's attempt to regulate child behavior and continuing until the
issue was resolved or dropped. Assessments included: timing of the intervention,
specific goal, and verbal, affective and physical components of maternal inter-
vention. The child's response was coded in terms of compliance or noncompliance.
In our measures of noncompliance we tried to capture the child's developing level
of competence, revealed in his/her resistance strategies. We distinguished less
competent forms: passive noncompliance and overt defiance; direct, but nonaver-
sive refusals; and most developmentally advanced, indirect and nonaversive
attempts to bargain or modify parental demands (negotiation). Each episode was
also coded for its resolution, capturing interactive qualities and final outcome.
Categories included: immediate maternal success, ultimate maternal success by
persuasion, ultimate maternal success by enforcement, non-confrontation, ultimate
maternal failure, compromise and unresolvable episodes.
Findings
Analyses revealed that maternal psychopathology affected the pattern of control
interactions between mothers and their toddlers. The affectively ill women,
more than the well women, avoided confrontation with their children when faced
with their resistance; they also had more troubles reaching a compromise with
their children. Slightly different patterns of impairments appeared for
severely ill uni- and bipolar mothers.
526
ZOl MH-02152-08 LDP
The daughters of depressed mothers seemed most at risk for noncompliance
problems. In addition to the findings related to maternal psychopathology ,
the study gives some insight into the developmental nature of child opposition
to socialization pressure. More competent forms of resistance (negotiation)
were found to increase between the second and third year of life. Less
socially skillful forms (direct defiance, passive noncompliance) decreased
over time. The level of social competence of child resistance strategies was
found to be related to the nature of influence techniques used by the mother.
Significance to Biomedical Research
Children of depressed parents are at greater risk for psychopathology and
behavioral disorders than are children of normal parents. Research on child
development has demonstrated that aberrant parental disciplinary practices are
important contributors to children's disordered social and emotional develop-
ment. How depression affects the parent's abilities to function in controlling
child behavior is largely unresearched; yet this variable may contribute
significantly in creating a pathogenic environment for young children.
Proposed Course
Three manuscripts have been submitted to journals. At present, work is focused
on more detailed analysis of mothers' verbal and physical control strategies as
related to their history of depression, as well as their current mood state. A
manuscript is being prepared. Another focus is on mothers' socialization goals
as expressed in their control interactions. Both developmental, and psycho-
pathology issues will be addressed.
Publications
Kochanska, G. , Kuczynski, L. , Radke-Yarrow, M. , and Friedman, S. : Normal and
affectively ill mothers' beliefs about their children. Am. J. Orthopsychiatry,
57(3): 345-350, 1987.
Kochanska, G. , Kuczynski, L. , Radke-Yarrow, M. , and Welsh, J.D. : Resolutions
of control episodes between well and affectively ill mothers and their young
children. J. Abnorm Child Psychol., in press.
Kuczynski, L. , Kochanska, G. , Radke-Yarrow, M. , and Girnius-Brown, 0.: A
developmental interpretation of young children's noncompliance. Dev. Psy. , in
press.
527
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02155-08 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line t>etween the borders.)
Children of Depressed and Normal Parents
PRINCIPAL INVESTIGATOR (Ust other professional personnel t>elow the Principal Investigator) (Name, title, latxratory. and institute affiliation)
PI:
C. Zahn-Waxler
Other: E. Cummings
lannotti
Rubin
S . Denham
Chief, Sec. on
Child Behavior Disorders
Professor
Research Psychologist
Professor
Associate Professor
LDP /NIMH
University of
West Virginia
Georgetown University
University of
Waterloo
George Mason Univ.
COOPERATING UNITS (it any)
University of West Virginia
University of Waterloo
Georgetown University
George Mason University
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN-YEARS:
.82
PROFESSIONAL:
.40
.42
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
S (a1) Minors
13 (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this study is to identify early in development dimensions of per-
sonality and social-emotional functioning that relate to the expression of prob-
lem behaviors in children as they reach school age. The focus is on the develop-
ment of both externalizing (aggression) and internalizing (anxiety and depres-
sion) symptoms. Patterns of continuity and discontinuity are examined separate-
ly for children from high and low risk environments. Children's emotion expres-
sions and social interactions at age two and age five were observed. A psychia-
tric assessment was obtained at age six. Significant patterns of continuity in
internalizing and externalizing problems have been identified; early problem be-
haviors observed in children's social interactions predict internalizing and ex-
ternalizing symptoms at school age. Good social skills in toddlers appear to be
protective factors. Further coding and analyses are in progress to assess child-
dren's appropriate use of affect in guiding social interactions, their strategies
for achieving goals and solving problems in interpersonal situations, and the na-
ture of their dyadic play.
529
PHS 6040 (Rev. 1/84)
GPO SI4-9lt
ZOl MH 02155-08 LDP
Project Description
The purpose of this research is to identify early in development patterns of
behavior that relate to the expression of problem behaviors in children as they
reach school age. Internalizing symptoms or problems of overcontrol (depres-
sion, anxiety, social withdrawal) and externalizing disorders or problems of
undercontrol (aggression, conduct problems) are investigated in a sample of
six-year-old children. Patterns of continuity/discontinuity are examined
separately in children from high and low risk environments. Risk is defined
in terms of parental psychopathology (maternal depression), since this factor
has been linked both with aggression and depression in children. Problem be-
haviors in six-year-olds are examined in relation to social competence and
affective coping styles. Parental variables expected to influence aggression
and depression in children are also assessed.
Methods Employed and Major Findings
Forty-eight two-year-old children were seen in three 1 1/2 hour laboratory
sessions spaced two weeks apart. Children of both normal and depressed mo-
thers (SADS-L) were studied. Each child was exposed to a range of challenging
conditions in order to evaluate social and emotional interchanges primarily
in interactions with a familiar playmate, but also with mother and with an
adult stranger. Assessments were made of the child's ability or lack of
ability to sustain social play, compete adaptively for resources, negotiate
problems, cooperate, cope with frustration without resorting to intense ag-
gression, empathize, and solve -hypothetical social problems. Maternal char-
acteristics evaluated were sensitivity, supportive presence and quality of
assistance, and techniques used to encourage cooperation and sustained social
and task-oriented involvement with others. Children's social skills in peer
interactions were assessed again at age five. Self-report data on childrearing
practices and the marital relationship were also obtained. At age six the
Childhood Assessment Schedule, a psychiatric interview, was used to obtain
psychiatric evaluations of the children. The mothers completed the Achenbach
Child Behavior Check List.
Significant patterns of continuity in internalizing and externalizing problems
were identified in preliminary data analyses and are detailed in last year's
annual report: early problem behaviors observed in children's social interac-
tions predicted problem behaviors at follow-up and good social skills in
young children functioned as protective factors. This year's work has focused
on the development of more differentiated and elaborated systems for assessing
children's social skills and deficits in their interactions with others. Three
dimensions of social competence that might be expected to predict internalizing
and externalizing problems are examined: (1) the child's appropriate or inappro-
priate use of affect in guiding social interactions, (2) the child's strategies
for achieving goals, expressing needs, and asserting rights in interpersonal
situations, and (3) the quality and type of dyadic play patterns.
530
ZOl MH 02155-08 LDP
Proposed Course
The data have been coded, and data analyses continue. Three manuscripts are in
preparation: developmental changes and patterns of individual differences in
social competence in two- and five-year-old children, social problem-solving
strategies and play patterns in five-year-old children of depressed and non-
depressed mothers, and composites of factors early in development that predict
diagnosable problem behaviors (internalizing and externalizing patterns) in six-
year-old children.
Significance to Biomedical Research and the Program of the Institute
An aim of prevention and intervention research is to identify early in develop-
ment those child and family factors that contribute to later childhood distur-
bances. Childhood depression and antisocial behavior have tended to come to
the attention of professionals when children reach school age, but they may
have much earlier origins. If early identification of behavior problems can
be made, more effective intervention procedures could be planned.
This is a final report.
Publications
Pierrehumbert, B. , lannotti, R. , Cummings, E. , & Zahn-Waxler, C. Attachement
maternal et dependence: Quelques apports de la psychologie experimentale.
Neuropsychiatrie de I'Enfance, 1986, 34(8-9), 409-420.
Friedman, S.L., Zahn-Waxler, C. , Waxier, M. , & Werthmann, M. Effects of phy-
siologic jaundice on behavioral function in low risk pre-term infants. J^.
Appl. Dev. Psychol. , 8: 53-66, 1987.
531
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02156-08 LDP
PERIOD COVERED
October 1. 1986 through September 30 ^ 1987
TITLE OF PROJECT (BO characters or less. Title must tit on one line between the tjordersj
Personality of Children Reared by Normal and Depressed Mothers: Inhibited Children
PRINCIPAL INVESTIGATOR (List other prolessional personnel tielow the Principal Investigator) (Name, title, laboratory, and institute attillatlon)
PI: F. Bridges-Cline Guest Researcher LDP/NIMH
Other: G. Kochanska Research Psychologist LDP/NIMH
COOPERATING UNITS (it any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.22
PROFESSIONAL:
.10
.12
CHECK APPROPRIATE BOX{ES)
\^ (a) Human subjects D (b) Human tissues □ (c) Neither
Q (al) Minors
S (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This Study focuses on the role of early childhood inhibition in the development
of pathological risk indicators in children of families with and without
parental depression. Patterns of child behavior in the face of the unfamiliar
(persons, places) such as behaviors expressing inhibited exploratory activity
and social withdrawal are observed at 2 to 3 years of age in semi-naturalistic
but standard settings, which represent varied contexts of unfarailiarity.
Analyses of these data revealed that five reliable dimensions of response
styles could be empirically derived from our observation coding system. These
dimensions meaningfully distinguish groups of children in our sample at this very
young age. Comparisons of these five dimensions of early behavioral inhibition
across maternal diagnostic groups (normal, major depressive and bipolar)
indicate that the young children of the major depressive mothers typically
exhibit the most inhibited forms of exploratory behavior, a strong tendency
to cling to mother in novel situations, and characteristically flat and
withdrawn displays of affect. Children of the bipolar mothers typically
exhibit the most active and independent forms of explorations of novel
environments. In a situation of an unfamiliar person, these children
score at both the inhibited and uninhibited polar extremes. However, in this
situation, these children of bipolar mothers do not demonstrate a clinging
dependency on mother and typically present positive displays of affect.
Observational data relating to mothers' behaviors that are concurrent with
child behaviors in these situations are being analyzed to examine the ways
in which mothers in these groups function to facilitate or hinder the child's
exploration and approach of unfamiliar situations.
533
PHS 6040 (Rev. 1/84) opo »14-»i»
ZOl MH 02156-08 LDP
Project Description:
Reduced motor activity, anhedonia, disinterest in activity, and social
withdrawal are behavioral characteristics that are often associated with
depression. However, the role of early behavioral forms of these
characteristics as precursors to later manifestations of depression has
not been established. One of the early forms of these characteristics
is behavioral inhibition, as revealed by the way in which the child engages
with novelty or unfamiliarity. One purpose of this study is to identify
and describe the patterns of response to unfamiliarity that are exhibited
by young children of depressed and nondepressed mothers. Another question
of interest is how mothers' handling of their children in these situations
may differ. Of predictive interest is the consequence of these early
behavioral patterns for later manifestations of disordered behavior.
Methods Employed and Major Findings:
Patterns of response to unfamiliarity are studied in children of depressed
and nondepressed mothers at 2 to 3 years of age. The childrens' behavior
has been videotaped in varied situations of unfamiliarity, such as entrance
to an unfamiliar but attractive environment, and introduction to and
interaction with an unfamiliar but friendly adult. Child behavior measures
that are coded from these videotapes include: latency measures, such as
latency to touch objects or toys in the entrance situation, proximity to
and reliance upon mother in order to interact or explore, and levels of
exploration and interaction such as scanning and looking, or retreating
from stranger in contrast to actively manipulating objects or initiating
interaction with the unfamiliar adult. In addition, the way in which the
mother functions to facilitate or hinder the child's approach and
engagement of the unfamiliar is studied.
A factor analysis was performed. The five factors emerged: (1) Exploratory
Activity, (2) Seeks Mother Support in Exploration (3) Initiating Engagement
of Stranger (4) Responder-role in interaction with stranger, and (5) Flat
and Withdrawn. A comparison of the distributions of these response style
characteristics across the maternal diagnostic groups normal, bipolar,
and major depressive also replicated the direction and nature of the earlier
preliminary findings, indicating the robustness of these conclusions.
Children of the major depressive mothers typically show more cautious,
less active, less initiating, and more mother physical-dependency styles
in both the entrance and stranger situations of unfamiliarity. And these
children typically exhibit sad and/or flat affect, in conjunction with
withdrawal from interaction with the stranger. Children of the bipolar
mothers, on the other hand, are characteristically positive and animated
in their affect displays, and, as a group, look very similar to one another
in their active and mother-independent styles of exploring new environment.
Further, although the children of the bipolar mothers do distribute
themselves at both polar extremes of the Stranger Engagement scale, they,
in contrast to the children of the major depressive mothers, show very
little clinging to mother as a style of retreating and withdrawing from
unfamiliar people.
534
ZOl MH 02156-08 LDP
Significance to Biomedical Research;
Our observational coding system focusing on the behavioral inhibition
of the 2- to 3-year-old children in our sample yielded a set of stable
and interpretable response style patterns, which meaningfully
distinguish groups of children at this very young age. Accomplishment
of these objectives — reliable assessment of the young child's
behavioral expression of reduced activity and/or social withdrawal in
the face of the unfamiliar — is a significant step toward a better
understanding of the developmental course of these characteristics and
of their link to later manifestations of depression or other
disordered behavior.
Proposed Course:
A manuscript describing the response style patterns that emerged from
these data and the differences between the children of the three
maternal diagnostic groups is being prepared. In addition, further
analyses are being conducted to examine the role of mother behavior
in the developmental course of these child behavior patterns.
Publications
None
535
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT MUMBER
ZOl MH 02164-07 LDP
TITLE OF PROJECT (80 characters or less. Title must fit on ons lirte between me tarders.)
Biological changes and psychological functioning during adolescence
PRINCIPAL INVESTIGATOR (Ust other professional persor\nel below the Principal Investigator) (Name, title, latjoratoy, and institute atfillatton)
PI: E.J. Susman Guest Researcher LDP NIMH
OTHER:
E
D.
Nottelmann
Statistician
LDP
NIMH
G
I.
Germain
Research Psychologist
LDP
NIMH
L
D.
Dorn
Guest Researcher
LDP
NIMH
G
.P.
Chrousos
Senior Investigator
DEB
NICHD
G
.G.
Cutler
Senior Investigator
DEB
NICHD
D
L.
Loriaux
Chief
DEB
NICHD
COOPERATING UNITS (It any)
Developmental Endocrinology, NICHD
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
.97
PROFESSIONAL:
.75
,22
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects
Q (a1) Minors
CX (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The interrelations of psychological functioning and endocrine and physical
growth factors in young adolescents are investigated. Participants are 56
boys and 52 girls, 9 to 14 years old, and their parents. Participants were
evaluated three times, six months apart on (a) stage of pubertal development
(Tanner criteria), and hormone levels (serum levels of gonadotropins,
gonadal steroids, adrenal androgens, and Cortisol), and (b) psychological
status (cognitive functioning, moods, problem behavior, competencies,
social supports and interactions with parents). Higher levels of adrenal
androgens and Cortisol and older chronological age predicted higher degrees
of self-reported anxiety and depressive symptoms for boys; for girls,
these symptoms were predicted by lower levels of gonadotropins. For both
sexes, adolescents who reported an absence of psychological support from
their parents also reported higher degrees of anxiety and depressive symptoms.
In a clinical situation involving a mild stress (blood drawn for hormone
assessments), adolescents with higher Cortisol levels exhibited higher
levels of observed behavioral distress on first exposure to the stressor.
This association disappeared when the situation became familiar.
537
PHS 6040 (Rev 1/84)
SPO SI4-SI*
ZOl MH 02164-07 LDP
Project Description
The interrelations of behavioral competencies and dysfunctions and endocrine
and physical growth changes in young adolescents are examined cross-sectionally
and longitudinally.
Methods and Findings:
The interrelations of psychological functioning and endocrine and physical
growth factors in young adolescents are investigated. Participants are 56
boys and 52 girls, 9 to 14 years old, and their parents. Participants were
evaluated three times six months apart on (a) stage of pubertal development
(Tanner criteria), and hormone levels (serum levels of gonadotropins, gonadal
steroids, adrenal androgens, and Cortisol), and (b) psychological status
(cognitive functioning, moods, problem behavior, competencies, social supports
and interactions with parents). (The details of measurement are reported in
previous Annual Reports.)
A general pattern of findings, for boys, is that higher levels of adrenal
androgens and Cortisol and higher age predicted more anxious and depressive
symptoms one year later; for girls, lower levels of gonadotropins predicted
anxious and depressive symptoms. For both sexes, the absence of psychological
support from parents was associated with higher degrees of anxious and
depressive symptoms.
Physiological reactivity and behavioral reactivity were examined in terms of
adolescents' responses to a potentially stressful situation (blood drawn
for hormone levels and a physical examination for pubertal staging). Cortisol
level and changes in level were measured across a 40-minute period. Behavior
was coded for complaints of pain, muscular rigidity, crying, and physical
resistance. Adolescents with higher Cortisol levels exhibited more distress
behaviors than those with lower levels during the blood-drawing procedure at
the first time of assessment. The association disappeared when the situation
became familiar, i.e., on second and third visits. Individual differences
in the pattern of change (increased, decreased, or no change) in Cortisol
level across forty minutes were similar over the one-year period.
Significance to Biomedical Research
Increases in behavior problems, psychiatric disorders, and suicides occur
during adolescence. These behaviors are undoubtedly affected by multiple
influences. One component of potential influence is the physiological changes
that occur during this developmental period. Better knowledge of the inter-
relations among hormonal changes, physical growth changes, and family and
peer interactions investigated in this study may contribute to the under-
standing of this period.
Proposed Course
Manuscripts are in press, submitted for publication, and in preparation.
Coding of observational data and longitudinal analyses will continue
over the next year.
538
ZOl MH 02164-07 LDP
Publications
Susraan, E.J. , Inof f-Gerraain, G. , Nottelmann, E.D. , Loriaux, D.L. , Cutler,
G.B., Jr., and Chrousos, G.P. Hormones, emotional dispositions and
aggressive attributes in young adolescents. Child Dev. 58: 1114-1134, 1987.
Susman, E.J., Nottelmann, E.D. , Inof f -Germain, G. , Dorn, L.D., and
Chrousos, G.P. Hormonal influences on aspects of psychological development
during adolescence. J. Adolesc. Health Care. In press.
Susman, E. J. , Nottelmann, E.D. , Dorn, L.D., Inof f-Germain, G. , and Chrousos, G.P.
Physiological and behavioral aspects of stress in adolescence. In G.P. Chrousos
(Ed.), Mechanisms of Stress. New York: Plenum Publishers. In press.
Susman, E.J. , Dorn, L.D. , and Fletcher, J.C. Reasoning about illness in ill
and healthy children and adolescents: Cognitive and emotional developmental
aspects. J. Dev. Beh. Pediatr. In press.
Trickett, P.K. and Susraan, E.J. Perceived similarities and disagreements
about child-rearing practices in abusive and nonabusive families: Inter-
generational and concurrent family processes. In D. Cicchetti and V. Carlson
(Eds.), Theoretical Perspectives and Research on the Consequences of Child
Maltreatment. New York: Cambridge University Press. In press.
539
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02169-05 LDP
TITLE OF PROJECT (SO characters or less. Title must lit art one line tietweert ffte tiorders.)
Interactions Between Siblings With a Depressed Parent
PRINCIPAL INVESTIGATOR (List Other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute altiliation)
PI: C. Zahn-Waxler Research Psychologist LDP NIMH
OTHER:
D. Hay
M. Radke-Yarrow
Research Psychologist
Chief
Univ. of London
LDP NIMH
COOPERATING UNITS (il any)
Univ. of London
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
CHECK APPROPRIATE SOX(ES)
IZl (a) Human subjects
m (al) Minors
H (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been incorporated into project ZOl MH 02370.
541
PHS 6040 (Rev. 1/84)
:po s<4-»is
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02170-05 LDP
TITLE OF PROJECT (SO characters or less. Title must fit on one //ne between the borders.)
Psychiatric Assessment of Infants and Toddlers
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: L. Cytryn Medical Officer (Psych) LDP/NIMH
Other:
T. Sherman
D. McKnew, Jr.
Research Psychologist
Medical Officer (Psych)
LDP/NIMH
LDP /NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Affective Development
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockvllle Pike, Bethesda, MD 20892
TOTAL MAN- YEARS:
1.55
PROFESSIONAL
.45
1.10
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects
S (a1) Minors
S (a2) Interviews
□ (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Oo not exceed the space provided.)
One hundred and twenty-three children, 2 to 3 years of age, were assessed using
two structured, behavioral observation systems: in a psychiatric play inter-
view in the absence of mother, (see ZOl MH 02170-03), and in interaction with
mother (see ZOl MH 02230-02). Twenty-two mothers had Bipolar Disorder, 45
Major Depressive Disorder, 11 Minor Depressive Disorder, and 45 with no history
of psychiatric disorder. All children were assigned a rating (1 to 4) of de-
gree of risk for later development of psychopathology based on their perform-
ance in each setting. This produced four groups of children: a low risk group
with low risk ratings in both assessments, two mixed-risk groups with ratings
of 1 or 2 in one setting, and 3 or 4 in the other, and a high risk group with
ratings of 3 or 4 in both assessments. From a case-by-case analysis of the
16 children in the high risk group, three major constellations of behavioral
symptoms were discernible: One was seen in a boy from a healthy home who dis-
played little affect, had a very distant type of relationship with his mother,
and would not speak with the psychiatrist in the play session. A second pat-
tern was seen in three girls from relatively poor families in which mother
suffered from major depression. This pattern consisted of a distant type of
relationship with mother, dysphoria, and anhedonia. The third pattern was
seen in the remaining group of twelve children (4 girls and 8 boys). These
children came from horaes that varied in social class, and diagnostic status
of the parents. The behavior consisted of an angry relationship with mother,
and predominantly angry mood. The children in all three groups demonstrated
dysregulation of emotions under stress, and little or no interest in play.
543
PHS 6040 (Rev. 1/84)
SPO SI4.SI*
ZOl MH 02170-05 LDP
Project Description
The goal of this project is to develop a nosology of psychopathology for
children aged two to four years of age. To this end we have scripted a
series of behavioral episodes to reveal a wide range of the young child's
behavioral repertoire as well as his modal pattern of behavioral function-
ing. Two behavioral observation systems that allow for reliable and sys-
tematic recording of the child's behavior are used. One instrument re-
quires 30 to 40 minutes of observation of the child when separated from
mother. This instrument can be used by the clinician as s/he interacts
with the child. The second is used for evaluation of the child's inter-
action with mother. A decision tree model of the clinician's synthetic
processing of his/her behavioral observations was used and a reasonable
level of concordance was found between the ratings derived via the decision
tree and the ratings provided by experienced clinicians. The details of
the two behavioral observation systems, as well as the decision tree model
of clinical synthesis were presented in reports (ZOl MH 02170-03 and ZOl
MH 02230-02).
One hundred and twenty-three children, 2 to 3 years of age, were assessed
using the two behavioral observation sysytems. Twenty-two mothers had Bi-
polar Disorder, 45 Major Depressive Disorder, 11 Minor Depressive Disorder,
and 45 had no history of psychiatric disorder. Approximately one-half of
the husbands of the mothers with affective disorder had affective disorder
as well.
Methods and Major Findings
All children were assigned a rating (1 to 4) of degree of risk for the
later development of psychopathology based on their performance in each
of the settings. This produced four groups of children: a low risk group
who received ratings of low risk for the later development of psychopath-
ology in both assessments (60 children), two mixed-risk groups composed of
children who received a rating of high risk in one setting and low risk in
the other (12 children high risk with mother, low risk with the psychia-
trist, 35 children low risk with mother, high risk with the psychia-
trist), and a high risk group of children who received ratings of high risk
in both assessments (16 children).
From case analyses of the 16 children in the high risk group, three major
constellations of behavioral symptoms are discernible. One was seen in a
boy from a healthy home who displayed little affect, had a very distant
type of relationship with his mother, and would not speak with the psychia-
trist in the play session. A second pattern was seen in three girls from
relatively poor families in which mother suffered from major depression.
It consisted of a distant type of relationship with mother, dysphoria,
and anhedonia. The third pattern was seen in twelve children (4 girls and
8 boys). These children came from homes that varied in social class, and
diagnostic status of the parents. The behavior consisted of an angry type
of relationship with mother, and predominantly angry mood. The children
in all three groups demonstrated dysregulation of emotions under stress,
and little or no interest in play.
544
ZOl MH 02170-05 LDP
Significance to Biomedical Research
The significance of this research is multifold: The development of improved
assessment instruments will help In understanding the developmental patterns
of adaptation and maladaptation in very young children and will permit more
sensitive evaluation of the children's strengths and vulnerabilities. These
assessments enable us to see whether patterns of adaptation differentiate
between children reared by normal and depressed mothers. We will be able
to evaluate how assessments from this perspective compare with later standard-
ized psychiatric assessments of the children, and see whether any of the pat-
terns of adaptation identified in these young children predict to the later
occurrence of specific psychopathologies. This prospective information not
only adds to our understanding of the developmental course of affective ill-
ness, but may provide an Informed basis for identifying children who are most
at risk.
Proposed course;
Analyses will be completed, and manuscripts will be prepared for publication
in a scientific journal.
Publications;
None
545
I PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
ZOl MH 02171-04 LDP
TITLE OF PROJECT (80 characters or less. Title must tit art one line between the txirders.)
Protective and Risk Factors in Childrearlng: Contributions of Fathers
PRINCIPAL INVESTIGATOR (Ust other professional personnel ttelow the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: W.E. Wilson Guest Researcher DRG/NIH
OTHERS: J.E. Richters Staff Fellow LDP/NIMH
COOPERATING UNITS (if any)
Division of Research Grants, NIH
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.70
PROFESSIONAL:
.25
OTHER:
.45
CHECK APPROPRIATE BOX(ES)
[El (a) Human subjects D (b) Human tissues D (c) Neither
[1 (a1) Minors
[E (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed ttie space provided.)
The husbands of depressed women may buffer their children from the stresses
of living with a depressed mother by encouraging dialogue, maintaining
stability and equilibrium within the family, and providing children with a
supportive environment. But they may also exacerbate the stresses to which
their children are exposed by not functioning effectively in these roles.
The objective of the present study is to examine the functioning of spouses
by combining measures of psychopathology and deviance in the spouses of
depressed women with detailed assessments of their functioning with and around
their wives and children. These measures will then be examined for their
contributions to predictions of children's adjustment. Analyses will be
based on interview, case history, and direct observation data from families
participating in a follow-up phase of the NIMH Childrearlng Project, which
includes children of parents with and without a history of affective disorder.
547
PHS 6040 (Rev. 1/84)
SPO SI4-SIS
ZOl MH 02171-OA LDP
Project Description
The husbands of depressed women may buffer their children from the stresses
of living with a depressed mother by encouraging dialogue, maintaining
stability and equilibrium within the family, and providing children with a
supportive environment. But they may also exacerbate the stresses to which
their children are exposed by not functioning effectively in tnese roles.
The objective of the present study is to examine the functioning of spouses
by combining measures of psychopathology and deviance in the spouses of
depressed women with detailed assessments of their functioning with and
around their wives and children. These measures will then be examined for
their contributions to predictions of children's adjustment.
Method
Data are from families participating in a follow-up phase of the NIH
Childrearing Project (ZOl MH 02144), which includes children of parents with
and without a history of affective disorder. Follow-up assessments include
detailed psychiatric interviews with mothers, fathers, and children, interviews
with fathers concerning their role in child-rearing and family functioning,
and intensive interviews with mothers concerning their marriages and family
life. In addition, fathers were videotaped while interacting with their
children and wives — separately and in combination — in a series of
naturalistic settings within our laboratory apartment.
Proposed Course
The coding and scoring of interviews is in progress. A system for assessing
the father's role in family interaction based on our observational data is
currently being developed. Following completion of data analyses in the
Spring of 1988, manuscripts will be prepared and submitted for publication.
Significance to Biomedical Research
This study will advance our understanding of the link between deviance in
the husbands of depressed women and elevated rates of risk for maladjustment
and psychopathology among their offspring.
Publications
None
548
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl Iffl 02207-04 LDP
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
The Affective Rearing Environment: A Comparison of Normal and Depressed Parents
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory and institute affiliation)
PI:
OTHER:
M. Radke Yarrow
G. Kochanska
W.E. Wilson, Jr.
L. Kuczynski
Nottelmann
Richters
Belmont
Mayfield
StilweTI
COOPERATING UNITS (if any)
Univ. of Guelph, Guelph, Ontario, Canada
Division of Research Grants, NIH
Chief
Res. Psychologist
Res. Psychologist
Assoc. Professor
Statistician
Res. Psychologist
Soc. Sci. Analyst (Clin.)
Soc. Sci. Analyst
Nurse Prar.
Res.
fPsyr.hiarrir')
LDP NIMH
LDP NIMH
U. Of Gilelph
LDP NIMH
LDP NIMH
LDP NIMH
LDP NIMH
LDP NIMH
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
1.97
PROFESSIONAL:
.95
1.02
CHECK APPROPRIATE BOX(ES)
13 (a) Human subjects
0 (al) Minors
[^ (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Affective characteristics of depressed and well mothers are investigated
in relation to the affect and behavior of their children. The moods and
emotions observed in the parent and experienced in interaction with the
parent are significant socialization influences on the child's own
affective development. The affective impairments that are characteristic
of depression, if manifested by depressed mothers in their interactions
with their children, are likely to create unpredictable, noncontingent,
and stressful daily experiences for their children. The questions of
interest concern the nature of mother's affective profile, the concordances
between mother and child's moods and emotions, the relation of affect
to other aspects of child development. When mother and child are analyzed
as pairs, there is a high degree of concordance in their affective
expression regardless of mother's diagnosis or gender of child. Depressed
mother-child dyads are most frequently concordant in negative affect.
Mutual positive affect between mother and child is significantly diminished
in families characterized by disorganization or chaotic life conditions.
The mutual positive affect at 2 to 3 years is predictive of the child's
ability three years later to relate competently to an unfamiliar peer.
549
PHS 6040 (Rev 1/84)
sPO ai4-sia
ZOl MH 02207-04 LDP
Project Description:
Assessments of affect were made by continuous coding of mother's and child's
affective expressions over three half-days in the laboratory at the time of
the first assessment, and again three years later at the follow-up assessment.
Findings:
Not unanticipated is the greater amount of time (number of minutes) coded as
negative mood and negative emotions (sad, anxious, irritable, angry) for
depressed mothers than for well mothers. However, what is particularly
noteworthy is the finding that when mother and child are analyzed as pairs,
there is a high degree of concordance in their affective expression regardless
of mother's diagnosis or gender of child. When mothers are in the highest
quartile on negative affect, 79% of their children are above the median in
negative affect; when mothers are in the lowest quartile on negative affect
only 15% of the children are above the median. Depressed mother-child dyads
are most frequently concordant in negative affect. Mutual positive affect
between mother and child is significantly diminished in families characterized
by disorganization or chaotic life conditions. The mutual positive affect
at 2 to 3 years is predictive of the child's ability three years later to
relate competently to an unfamiliar peer.
Previously reported, for part of the present sample, were differences in
frequencies of insecure attachment relationships by diagnosis of mother.
These findings are replicated in the Fall sample: most insecure attachments
in children of bipolar mothers (76%), next most frequent in unipolar mothers
(49%), and least frequent in well mothers (36%). When data on mothers'
patterns of affect are considered, preliminary findings link, high frequency
of insecure avoidant attachment with mothers who manifest extreme sadness
and anxiety (and sometimes intense affection) in interactions with their
children. Children of angry mothers and bipolar mothers are more likely
to have anxious resistant attachments. Securely attached children of
depressed mothers are being investigated further.
Significance to Biomedical Research:
The offspring of depressed parents are at risk for the development of affective
disorders, but the relative combination of genetic and environmental factors
is not well researched. This study offers strong evidence of the specific
childrearing conditions that may contribute to the development of affective
disturbance in young children. The findings are relevant to theories of
depression and to issues of prevention.
Proposed Course:
Just beginning are analyses to examine whether affect patterns of the
depressed mothers in relating to their children are aberrant in depressive
episodes but not between episodes or whether their affect patterns with
550
ZOl MH 02207-04 LDP
their children stem from more enduring personality characteristics of
these women and vary relatively little depending on episode status. The
sample is such that there are mothers who are in a depressive episode
when they are participating in the procedures and mothers who are between
episodes. Manuscripts are in preparation.
Publications;
Radke-Yarrow, M. , Richters, J., and Wilson, W.E. : Child Development
in a Network of Relationships. In Hinde, R. and Stevenson-Hinde, J. (Eds.):
Individuals In a Network of Relationships. England, Cambridge University
Press, in press.
Radke-Yarrow, M. , and Kochanska, G. : Anger in Young Children. In
Stein, N.L., Leventhal, B. , and Trabasso, T. (Eds.): Psychological and
Biological Approaches to Emotion. Hillsdale, N.J. , Lawrence Earlbaum
Press, in press.
Radke-Yarrow, M. , Belmont, B. , Nottelmann, E. , and Bottomly, L. Young
children's self-conceptions: Origins in the natural discourse of depressed
and normal mothers and their children. In Cicchetti, D. and Beeghly, M.
(Eds.), The Development of the Self During the Preschool Years. New England,
Cambridge University Press, in press
Wylie, R.C. Mothers' attributions to their children. In Honess, T.M. and
Yardley, K. M. (Eds.), Self and .Identity: Individual Change and Development.
London, Routledge & Kegan Paul, in press.
551
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02229-03 LDP
TITLE OF PROJECT (80 characters or less. Title must fit on one line betweert the tmrders.)
Vocalic Analysis of Natural Discourse in Well and Depressed Mothers
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: T. Sherman Research Psychologist LDP/NIMH
Other: Z. Breznitz
Assistant Professor
University of Haifa
Israel
COOPERATING UNITS (if any)
University of Haifa
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Affective Development
INSTITUTE AND LOCATION
National Institute of Mental Health, NIK, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN-YEARS:
.72
PROFESSIONAL:
.30
.42
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects
S (a1) Minors
S (32) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The speech behavior of 14 depressed and 18 nondepressed mothers during conver-
sation with their 3-year-old children was examined in this study. It was found
that (Annual Report #Z01 MH 02229-02 LDP) depressed mothers vocalized less
often and responded less quickly to the cessation of their children's speech
than healthy mothers. In a mildly stressful situation, (awaiting a doctor's
visit), the depressed mothers, but not the healthy mothers, significantly in-
creased their level of speech productivity. Children of the depressed mothers
spoke less than children of healthy women, particularly while sitting and eat-
ing lunch with their mothers. The observed differences in the mothers' beha-
viors were interpreted as an indication that the two groups of children are ex-
posed to very different patterns of socialization. The offspring of depressed
women are being taught both to keep social interaction to a minimum and to be
overreactive to even mild stresses. The differences in the children's behavior
may indicate that already these 3-year-old children have learned to keep their
interactions with their mother to a minimum. This manner of adaptation may
have negative effects on the child's continued social, emotional and cognitive
development!
In order to uaderstand the developmental impact of the observed aberrant pat-
terns of vocalic interaction between depressed mothers and their young child-
ren, two further studies are underway. First, the question of continuity of
experience will be addressed by analyzing the conversation patterns of these
! women and children three years after the Initial observation. Second, the ques-
] tion of alternate environmental supports for the children's development will
be examined by assessing the conversation patterns of these young children and
their older siblings. These observations are of the children both at the time
of the original assessment of mother-child interaction and again at the time
of th«> first follow-up assessment. ,^n;-:j
PHS 6040 (Rev 1/84)
SPO 9l4->1t
ZOl MH 02229-03 LDP
Project Description
The purpose of this set of studies is to examine: 1.) the consistency of the
patterning of natural discourse of well and depressed women and their young
children, and 2.) the alternate environmental supports available to young
children from their older siblings.
The procedural details of the study were reported in the original Annual
Report in this series (# ZOl MH 02229-01 LDP). Findings presented last year
(// ZOl MH 02229-02 LDP) on a sample of 18 normal and 14 depressed mothers
and their 3-year-old children were that depressed mothers vocalized less
often and responded less quickly to the cessation of their children's
speech than healthy mothers. In a mildly stressful situation, (awaiting a
doctor's visit), the depressed mothers, but not the healthy mothers,
significantly increased their level of speech productivity. Children of the
depressed mothers spoke less than children of healthy women, particularly
while sitting and eating lunch with their mothers.
The observed differences in the mothers' behaviors were interpreted as an
indication that the two groups of children are exposed to very different
patterns of socialization. The offspring of depressed women are being taught
both to keep social interaction to a minimum and to be overreactive to even
mild stresses. The differences in the children's behavior may indicate that
already these 3-year-old children have learned to keep their interactions
with their mother to a minimum. This manner of adaptation may have negative
effects on the child's continued social, emotional and cognitive development.
A question raised by these results concerns the possible long-term sequelae
for the children of depressed mothers exposed to this aberrant patterning of
communication with their mothers. Two factors are important in projecting
long-term effects: one is whether the patterns of interaction seen when the
children were 3 years of age are stable. A second is whether the children
have alternative sources of social and cognitive support.
Conversation between mother and child, and sibling and child will be examined
for 14 (8 girls, 6 boys) children of healthy mothers and fathers, 9 children
(4 girls, 5 boys) of major depressed mothers and healthy fathers, and 11
children (4 girls, 7 boys) of major depressed mothers and fathers who also
suffer from some form of affective illness. The younger children range in
age from 26 to 39 months, the siblings range in age from 5 to 8 years of age.
These families are participants in the NIMH Childrearing Study (Annual
Report # ZOl MH 02229-01 LDP).
Measures of interest will be total amount of vocalic behavior of each
member of the dyad and the time to initiate vocalization after the other
member of the dyad has ceased vocalizing.
554
ZOl MH 02229-03 LDP
Significance to Biomedical Research
Dialogue between mother and child is a basic process by which children are so-
cialized and through which children practice aspects of their social and cog-
nitive behavioral systems. This research has demonstrated that fundamental
aspects of this dyadic system in depressed mother-child dyads are divergent
from that seen in healthy mother-child dyads.
The current study is directed towards identifying if these aberrant patterns
of vocalic interaction between young children and their depressed mothers is
an enduring aspect of their interaction. If this is found to be true, then it
is expected that there will a long-term cost to the children. One potential
source for ameliorating these experiential and socialization differences may
be the children's older siblings. Thus, the conversation patterns of the sib-
lings is also examined. Of particular interest is to see if compensatory ex-
periences are provided for children of depressed women through interaction with
their siblings.
Proposed Course:
The report on the findings of the original study have been published in Child
Development.
Data collection has been completed on the current phase of the project. Data
analysis is underway. A report on this second phase of the project will be
prepared this year.
Publications;
Breznitz, Z. , and Sherman, T. : Speech patterning of natural discourse of well
and depressed mothers and their young children. Child Development, 58, 395-
400, 1987.
555
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02231-03 LDP
TITLE OF PROJECT (80 characters or less. Title must fit art one line betv/eer) the borders.) "
Biological-Behavioral Relations in Early Adolescence
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: E.D. Nottelmann
Other: E.J. Susman
G.I. Germain
L.D. Dorn
G. P. Chrousos
G.B. Cutler, Jr.
D.L. Loriaux
COOPERATING UNITS (if any)
Developmental Endocrinology, NICHD
Statistician LDP/NIMH
Guest Researcher LDP/NIMH
Research Psychologist LDP/NIMH
Guest Researcher LDP/NIMH
Senior Investigator DEB/NICHD
Senior Investigator DEB/NICHD
Chief DEB/NICHD
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.17
PROFESSIONAL:
1.05
.12
CHECK APPROPRIATE BOX(ES)
K] (a) Human subjects
^ (a1) Minors
E] (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use Standard unreduced type. Do not exceed the space provided.)
Interrelations of pubertal development and adolescent adjustment and behavior
are investigated cross-sectionally and longitudinally. Participants are 9- to
14-year— old boys and girls and their parents. The adolescents and their parents
are seen three times, six months apart. Measures of pubertal development include
serum hormone levels (gonadotropins , sex steroids, and adrenal androgens) ,
pubertal stage (Tanner criteria), and height and weight. Psychological assess-
ments of the adolescent include adolescent- and parent-report of behavior prob-
lems and competencies and behavioral observations of parents and their adolescent
in family interactions. Comparisons of ratings of pubertal stage made by medical
personnel, the adolescents, and their parents indicate that, with appropriate
instruction, adolescents and parents can provide valid assessments of the
adolescent's pubertal stage. Analyses involving pre- and postraenarchial girls
indicate that mean levels of gonadotropins, sex steroids, and adrenal androgens
are higher in postraenarchial than premenarchial girls, but that there is consider-
able overlap in the distributions. Preliminary analyses suggest that there are
similar hormone-behavior relations in pre- and postraenarchial girls. An exami-
nation of relations between hormone levels and the adolescents' use of anger and
power in family interactions indicates that estradiol and androstenedione levels
are related to irritability and assertive forms of aggression shown by girls.
For both sexes, there was evidence that controlling behaviors shown by parents
and anger shown by adolescents are related in a pattern which results in an
escalation of conflict.
557
PHS 6040 (Rev. 1/84)
GPO 91 4.010
ZOl MH 02231-03 LDP
Project Description
Interrelations of endocrine, pubertal stage, and physical growth indices of
pubertal development and adolescent adjustment and behavior are investigated
cross-sectionally and longitudinally across three times of measurement at
six-month intervals.
Methods and Findings
The longitudinal sample consists of 54 boys and 49 girls, 9 to 14 years of
age, and their parents.
Pubertal development measures include: serum hormone levels, height and
weight, and pubertal stage (according to Tanner criteria). Psychological
measures include adolescent- and parent-report of the adolescent's behavior
problems and competencies, and behavioral observations of parents and their
adolescent in family interactions also were made. Details of procedures are
given in previous years' reports.
Methodological issues were investigated: Assessments of pubertal stage (Tanner
criteria) made by adolescents, parents, and medical personnel were compared.
Adolescents and parents rated the adolescent's pubertal stage using photographs
of the five stages of puberty. Correlations between adolescent and medical
personnel ratings ranged f rom _r = .77 to .91 (_£ ^ .001). Correlations between
ratings made by parents and by medical personnel ranged from _r = .75 to .87 (_£
_< .001). For both adolescents and parents who were not accurate, underestima-
tion occurred in the later stages of puberty; overestimation, in the earlier
stages of puberty.
At all three periods of assessment, girls were asked to report on their menar-
chial status. There were significant discrepancies from one time of assessment
to another in their reports of age at menarche and description of their men-
strual cycle. For girls who were postmenarchial at Time 1, the length of the
menstrual cycle varied from 20 to 54 days. Mean levels of gonadotropins, sex
steroids, and adrenal androgens were higher in postmenarchial than premenarchial
girls, but there was considerable overlap in the distributions. There are
similar hormone-behavior relations in pre- and postmenarchial girls.
Adolescents' use of anger and power in parent-adolescent interactions was
examined in relation to hormone status. Although, in general, hormone behavior
relations are stronger and more consistent for boys than for girls, in family
interactions findings for hormone-aggression relations are stronger for girls.
Estradiol and androstenedione levels were related to irritability and assertive
forms of aggression shown by girls in the context of family interactions. For
boys, findings were sparse, but certain of the significant relations that were
obtained — those between levels of luteinizing hormone, dehydroepiandrosterone ,
and dehydroepiandrosterone sulphate and degree of aggression — replicated
previous findings for relations between hormone levels and mother-report data
of aggressive traits of the adolescent. Aggressiveness in girls appears to
emerge only in certain contexts such as family interactions. The family may be
558
ZOl MH 02231-03 LDP
a "safe" place for girls to test power. For both sexes, there was evidence
that controlling behaviors by parents and expression of anger by adolescents
are related in a pattern that results in escalation of conflict. For both
sexes adolescents who reported an absence of psychological support from
their parents also reported higher degrees of anxiety and depressive symptoms.
Significance to Biomedical Research
Increases in problems during adolescence are well documented. Findings from
this study provide systematic data on behavior change during puberty and begin
to clarify how such problems may be related to developmental status and
rate of pubertal change in early adolescence.
Proposed Course
Analyses are under way to generate hormone profiles and identify individuals
with exceptional profiles of hormone levels. These profiles will be analyzed
in relation to physical pubertal change and to competencies and dysfunctions
in psychological domains. Ongoing analyses also include predictive analyses;
in each case, developmental status and developmental change are used to
predict subsequent adjustment and behavior.
Future work includes the examination of mediators in the relations between
biological and psychological measures, e.g., social support and parental
marital relations and interactions with adolescents.
Publications;
Inoff-Gerraain, G. , Arnold, G. S. , Nottelraann, E. D. , Susman, E. J. , Cutler,
G. B. , Jr., & Chrousos, G. P. (1987). Relations between hormone levels and
observational measures of aggressive behavior of young adolescents in family
interactions. Dev. Psychol. , in press.
Nottelmann, E. D. , Susman, E. J., Blue, J. H. , Inoff-Germain, G. , Dorn, L. D. ,
Cutler, G. B. , Jr., Loriaux, D. L. , & Chrousos, G. P.: Gonadal and adrenal
correlates of adolescent adjustment. In Lerner, R. M. & Foch, T. L. (Eds.):
Biological and Psychosocial Interactions in Early Adolescence: A Life-Span
Perspective. Hillsdale, N. J.: Erlbaum, 1987, pp. 303-323.
Nottelmann, E. D. , Susman, E. J., Dorn, L. D. , Inoff-Germain, G. , Loriaux,
D. L. , Cutler, G. B. , Jr., & Chrousos, G. P. Developmental processes in
early adolescence: Relations among chronologic age, pubertal stage, height,
weight, and serum levels of gonadotropins, sex steroids, and adrenal androgens.
J. Adolesc. Health Care. 8: 246-260, 1987.
Nottelmann, E. D. , Susman, E. J., Inoff-Germain, G. , Cutler, G. B. , Jr., Loriaux,
D. L. , & Chrousos, G. P. Developmental processes in early adolescence:
Relations between adolescent adjustment problems and chronologic age, pubertal
stage, and puberty-related serum hormone levels. J. Pediatr. 110: 473-480, 1987.
Nottelmann, E. D. Competence and self-esteem during transition from childhood
to adolescence. Dev. Psychol. 23: 441-450, 1987.
559
TITLE OF PROJECT (80 characters or less. Title must lit art one line between the tx>rderz.) ~ ~ '
Development of Ability to Concentrate in Children of Depressed and Well Mothers
PRINCIPAL INVESTIGATOR (Ust other prolesslonal personnel tMlow the Principal Investigator) (Name, title, laboratory, and institute alfiliation)
PI: Z. Breznitz Research Psychologist Univ. of Haifa
Haifa, Israel
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02232-03 LDP
Other: S.L. Friedman
Health Scientist Administrator HLB, NICHD
COOPERATING UNITS (it any)
University of Haifa, Haifa, Israel
Human Learning and Behavior Branch, NICHD
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
Q (a1) Minors
Q (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Both investigators have taken other positions,
will be carried on outside the Laboratory.
Further work on the project
561
PHS 6040 (Rev. 1/84)
CPO ei4>*it
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02233-02 LDP
TITLE OF PROJECT (BO characters or less. Title must lit on one ffne between the borders.)
The Development of Guilt: Language, Emotions, and Behavior
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliatlon)
PI: C. Zahn-Waxler Chief, Sec. on LDP/NIMH
Child Behavior Disorders
Other: G. Kochanska Research Psychologist LDP/NIMH
S. Denham Associate Professor George Mason
University
COOPERATING UNITS (If any)
George Mason University
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN- YEARS:
.92
PROFESSIONAL:
.50
,42
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
B (a1) Minors
K\ (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This research focuses on the establishment of methods to investigate the devel-
opment of guilt in children. Families and children are selected for character-
istics (e.g., socioeconomic status, gender, parental depression) that, on theo-
retical grounds, might be expected to contribute to differential development of
guilt and different forms of expression.
Two- to three-year-old children are observed in structured situations in which
a problem or mishap occurs. Analyses focus on identification of individual
differences in children's reactions. Preliminary results indicate that expres-
sions of guilt and avoidance of responsibility are already present in toddler-
age children and take varied functional and dysfunctional forms. Children be-
tween 5 and 8 years of age (ZOl-MH-02155) are administered a series of pictures
of children and adults in circumstances of distress and conflict to tap their
propensities for guilt and related affects. In normal control families, child-
ren in this age range show the characteristic increases in guilt expected with
age, but children with a depressed parent do not. Levels of guilt are lowest
in children with parents of low socio-economic status. Guilt scores on this
test relate to guilt and anxiety scores in a clinical interview, but only for
children with emotionally well parents.
563
PHS 6040 (Rev. 1/84)
CPO 9 1 4-9 It
ZOl MH 02233-02 LDP
Project Description
Certain mental disorders are characterized by atypical patterns of guilt (e.g. ,
excesses of guilt in depression and deficiencies in sociopathy). Few data are
available to indicate why some individuals develop the capacity to maintain
healthy, appropriate levels of responsibility, while others experience excesses
or deficiences in guilt at levels that seriously compromise their well-being.
This research focuses on the development of methods for investigating patterns
of adaptation and maladaptation in the development of guilt in children. Fami-
lies and children are selected for characteristics (e.g., socioeconomic status,
gender, parental depression) that, on theoretical grounds, might be expected to
contribute to differential development of guilt and different forms of expres-
sion.
Methods Employed and Major Findings
In studies 1 and 2, two- to three-year-old children are observed in structured
situations in which a problem or mishap is staged, and the child's tendency to
become involved and feel responsible is assessed. In study 1 (N = 70) the focus
is on (a) delineation of individual differences in children's patterns of guilt
and (b) maternal personality characteristics and mood expressions that may pre-
dict differences in children's guilt reactions. Study 2 (N = 65) focuses, in
addition, on exploration of mother's and children's verbal communications about
emotions with an emphasis on their emotion-induction strategies and their inter-
pretations of causality (e.g. , who is responsible and why) in situations of dis-
tress.
In study 3, 90 children between the ages of 5 and 8 (ZOl-MH-02155) are seen in
a semi-structured projective test (a series of pictures of children and adults
in circumstances of distress and conflict) which taps their propensities for
guilt and related affects and behaviors. Guilt is examined as a function of
socioeconomic status, age, gender, and parental depression. In well families,
children in this age range show the characteristic increases in guilt expected
with age, but children with a depressed caregiver do not. Levels of guilt are
lowest in children with parents of low socio-economic status. Guilt scores on
the projective test are related to guilt and anxiety scores in a structured
clinical interview with these children, but guilt is unrelated to their reports
of mood problems. The correspondence between guilt scores on the two measures
is found only for children with emotionally well parents.
Boys and girls do not differ on overall measures of guilt. However, in child-
ren's interpretations of the distress stories, girls more than boys express
concern, distress, and prosocial intentions: themes about the maintenance and
termination of interpersonal relationships (separation issues) are most common
in girls. High levels of guilt and anxiety are associated with greater concern
over relationship issues in girls. In contrast, for boys, the greater the feel-
ings of guilt, the less concern they express about relationship issues. Thus,
the dynamics of guilt and its expression are related in complex ways to family
background, cultural background, stage of development and sex of child.
564
ZOl MH 02233-02 LDP
Significance to Biomedical Research and the Program of the Institute
Emotions of guilt and related expressions of low self-esteem and worthless-
ness are major features of psychopathology. Knowledge of the developmental
course, the conditions under which different forms of guilt develop, and the
circumstances under which guilt begins to become linked with other affective
and social maladaptations will contribute an understanding of these aspects
of psychopathology. Basic research on processes of development of guilt in
children begins to provide this information.
Proposed Course
Data collection has been completed. Data are in various stages of coding, ana-
lysis and manuscript preparation. Information derived from these studies will
be incorporated into an invited presentation and chapter for the Nebraska Sym-
posium on Motivation. Journal articles are also planned. This project will
be completed within the year.
Publications
Chapman, M. , Zahn-Waxler, C. , lannotti, R. , & Cooperman, G. , Empathy and res-
ponsibility in the motivation of children's helping. Dev. Psychol. , 23(1):
140-145, 1987.
Kuczynski, L. , Zahn-Waxler, C. , & Radke-Yarrow, M. , Development and content of
imitation in the second and third years of life: A socialization perspective.
Dev. Psychol., 23(2): 276-282, 1987.
565
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02234-02 LDP
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the txrders.)
Infants of Chronically Depressed and Normal Parents
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Phncipal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: K. Suter Medical Staff Fellow LDP/NIMH
Other: J. Stilwell
Research Nurse Practitioner
(Psychiatric)
LDP /NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Affective Development
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockvllle Pike, Bethesda, MD 20892
TOTAL MAN-YEARS:
2.02
PROFESSIONAL:
1.00
1.02
CHECK APPROPRIATE BOX(ES)
@ (a) Human subjects
H (a1) Minors
13 (a2) Interviews
n (b) Hunnan tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Research has shown that children of depressed parents are at risk for development
of affective illness, but little is known about the effects of parental affective
psychopathology on infants during the first eighteen months of life. This study
examines early affective regulation, attachment behavior, and patterns of mother-
infant interaction in infants of two groups of parents: (1) depressed mothers
and well fathers, and (2) both parents without psychiatric illness. Parents are
screened by structured psychiatric interview (SADS-L) and also complete question-
naire measures of marital adjustment (DAS). Infants and their mothers are ob-
served and videotaped in a set of standard situations In a homelike laboratory
setting on two occasions five months apart, beginning when the infants are aged
3 months, 8 months, or 13 months. Data collection has been completed. Data
are currently being coded and findings are not yet available. Analyses are
planned to examine both individual and group differences in an attempt to look
for possible precursors to later difficulties in this at-risk population.
567
PHS 6040 (Rev. 1/84)
SPO si4.aii
ZOl MH 02234-02 LDP
Project Description
Research has demonstrated that children of depressed parents are at risk for
development of affective illness themselves; considerable attention has been
focused on latency age and adolescent offspring at risk for depression. More
recently, this laboratory has examined toddlers and latency age children in
families with parental history of affective disorder or no psychiatric diag-
nosis and has found a number of differences in children and parenting styles
between different parental diagnostic groups (see ZOl MH 02144). The purpose
of the current project is to look for possible precursors in how early differ-
ences are manifest by studying younger infants at risk for later affective ill-
ness. Infants of two groups of parents are included: (1) depressed mothers
and well fathers, and (2) both parents without psychiatric illness. Attach-
ment behavior, early affective regulation, and patterns of mother-infant in-
teraction are examined.
Parents are screened by structured psychiatric interview (SADS-L). Infants
are seen with their mothers in a homelike laboratory setting for two sessions
five months apart beginning at one of three ages: 3 months, 8 months, or 13
months. The 13-month group includes 10 depressed mothers and 9 control mothers;
the 8-month group, 14 depressed mothers and 9 control mothers; and the 3-month
group, 6 mothers in each diagnostic group. Mother-infant interaction is ob-
served and videotaped in a naturalistic setting in a set of standard situa-
tions, including introduction to a new environment, free play, introduction
of a stranger, brief separation, feeding, and developmental testing. Situa-
tions are adapted to be appropriate for each age group. Parents also complete
a questionnaire measure of marital adjustment (Dyadic Adjustment Scale) and
are interviewed after the second visit about life events and SADS-L status
for the interval period. Data are being coded and analyzed for a number of
variables, including attachment classification, social referencing, affective
regulation, early signs of mastery motivation, and patterns of mother-infant
interaction. Comparisons will be made between diagnostic groups as well as
examined for developmental and individual differences. Findings are not yet
available.
Significance to Biomedical Research
Little is known about the effects of maternal depression on infants. This study
will provide data on this issue as well as attempt to identify possible precur-
sors to later difficulties in a population at risk for later affective disorder.
Proposed Course
Data collection has been completed. Data are currently being coded and analyzed.
Several manuscripts are planned wthin the next year.
Publications
None
568
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02297-02 LDP
TITLE OF PROJECT (BO characters or less. Title must lit on one line between ttie tiorders.)
Generosity and Sharing in Children of Normal or Affectively Disturbed Parents
PRINCIPAL INVESTIGATOR (Ust Other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute alfillatlon)
Pi!
F.R. Asclone
Other: M. Radke-Yarrow
C. Zahn- Waxier
Guest Researcher
Chief
Chief, Sec. on Child
Behavior Disorders
LDP/NIMH
LDP /NIMH
LDP/NIMH
COOPERATING UNITS (it any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
.47
PROFESSIONAL:
.05
,42
CHECK APPROPRIATE BOX(ES)
W\ (a) Human subjects
^ (a1) Minors
IZ] (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The ability to relate positively to other persons is an important aspect of
development. One indicator of this achievement is the child's capacity for
empathy and ability to help or share with another person. The purposes of
this study are to: (1) examine 5-9 year old children's generosity in a
laboratory measure of sharing and also in a naturalistic setting with their
younger siblings; and (2) explore proximal and distal correlates of generosity
and sharing, and their relation to parental diagnostic status (affectively
disturbed or normal). (3) There is also a methodological interest in comparing
the standard laboratory measure of sharing that has been the basis of the
majority of studies of sharing with measures of sharing in the context of the
child's family interactions. This study uses data collected during the first
phase of a longitudinal study of child rearing and parental affective disorders.
569
PHS 6040 (Rev 1/84)
SPO St4-9I>
ZOl MH 02297-02 LDP
Project Description
The ability to relate positively to other persons is an important aspect of
development. One indicator of this achievement is the child's capacity for
empathy and ability to help or share with another person. The purposes of this
study are to examine 5- to 9-year-old children's generosity in a laboratory
measure of sharing and also in a naturalistic setting with their younger sib-
lings and to explore proximal and distal correlates of sharing and their
relation to parental diagnostic status (affectively disturbed or normal).
Data for 88 children (60 whose mother and/or father were diagnosed as
affectively disturbed) are examined. Mother and two siblings (mean ages 2.5 and
7 years) are observed in an informal apartment setting (Project ZOl HH 02144) and
in a variety of contexts over a 2.5 hour period. The interactions are videotaped.
The older sibling at one point accompanies the experimenter to another room and
participates in a game in which he or she wins candies. The adult model who
demonstrates the game donates some of her candy winnings to children who didn't
have candy. The older sibling then returns to the apartment and has the oppor-
tunity to share the candies he or she has won with the younger sibling and also
to share use of a game provided by the experimenter. The sibling pair is alone
during the 5 minutes when sharing is assessed.
Levels of and relations among donating, candy sharing, and game sharing are
examined. Proximal factors that may be related to generosity and sharing
include the frequency and quality of sibling interactions in a period preceding
the sharing task and the mother and younger sibling's behavior when the older
sibling returns with the candy. Distal factors to be related to generosity and
sharing include scores on a "projective" test of the older sibling's affective
attributions and aspects of the child's personality (Achenbach's Child Behavior
Checklist).
Major Findings
Data from the sharing task have been analyzed with analysis of variance (ANOVA).
Results of the ANOVA yielded a significant main effect for age of older sibling.
Subsequent analyses indicated that, although the 6 year olds shared significantly
less (M=21%) than 5 year olds (M=36%), rates of sharing candy among 5-7 (M=29%),
and 8-9 year olds (Nl=39%) were not statistically different; rates of sharing
among 6-7, and 8-9 year olds were not statistically different. No sex
differences were found nor was there systematic variation in sharing according
to parental diagnostic status. The relations of sharing to child personality
variables and sharing to parental diagnostic variables are now being examined.
Significance to Biomedical Research
Empathy and response to other person's needs are important aspects of the
development of positive interpersonal interactions. Knowledge of the antecedents
and correlates of these behaviors and the possible developmental impairments
in children of affectively disturbed parents will provide a basis for understand-
ing the processes contributing to these behaviors.
570
ZOl MH 02297-02 LDP
Proposed Course
Further analytic work is projected to relate the measures of sharing to child
personality and familial variables. The work should be accomplished in the
next year.
Publications
None
571
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02361-01 LDP
TITLE OF PROJECT (60 characters or less. Title must tit on one line twfween the tnrders.)
Relation between Self- and Teacher-reports of Social-emotional Adjustment
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, latx/ratory, and institute affiliation)
PI: T. Sherman Research Psychologist LDP/NIMH
Other: D. Pellegrini Professor Catholic University
COOPERATING UNITS (if any)
Catholic University
L^B/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Affective Development
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN-YEARS:
.52
PROFESSIONAL:
.10
.42
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects D (b) Human tissues D (c) Neither
H (a1) Minors
13 (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
One hundred and twenty-three children (the entire group of older siblings
in the NIMH Childrearlng Study, Annual Report # ZOl MH 0229-01 LDP) 8-11
years of age were adrainisterd the Child Assessment Schedule (CAS), a structured
psychiatric interview. In addition, the children's teachers filled out the
Achenbach Child Behavior Checklist - School Version. The Achenbach asks the
teachers to report on the children's ability to relate to other children, the
children's behavior, and the children's academic achievement.
The question addressed by this study is the extent to which the children's re-
ports of negative affective states, poor social relationships with family mem-
bers and/or peers, and problems in school correspond to the child's level of
adaptation (academic performance, mood regulation, and social relations with
peers and adult authority) in the school setting. A secondary question is the
extent to which patterns of adaptation in the school context relate to parental
psychopathology.
573
PHS 6040 (Rev. 1/84)
SPO Bi4-ais
ZOl MH 02361-01 LDP
Project Description
One hundred and twenty-three children (the entire group of older siblings
in the NIMH Childrearing Study, Annual Report if ZOl MH 02229-01 LDP) 8-11
years of age were administered the Child Assessment Schedule (CAS), a struc-
tured psychiatric interview. In addition, the children's teachers filled out
the Achenbach Child Behavior Checklist - School Version. The Achenbach asks
the teachers to report on the children's ability to relate to other children,
the children's behavior, and the children's academic achievement.
The question addressed by this study is the extent to which the children's re-
ports on the CAS of negative affective states, poor social relationships with
family members and/or peers, and problems in school correspond to the child's
level of adaptation (academic performance, mood regulation, and social rela-
tions with peers and adult authority) in the school setting.
Significance to Biomedical Research
A fundamental issue in understanding the developmental course of affective ill-
ness is to identify significant premorbid signs and/or symptoms. The CAS in-
terview offers the child the opportunity to share with the examiner his/her
concerns and feelings about self, peers, and family members. As with adults,
children's self reports and internal states are a significant aspect of defin-
ing psychopathology , In addition, though, it is important to establish whether
these feeling states translate into impairments in life functioning. To our
knowledge this form of validation has not been performed on child psychiatric
interviews. The contribution of this study will be to indicate the relation
for school age children between their symptom picture as revealed in their
major work and social setting and their symptom picture as revealed via self
report.
Proposed Course
Data collection is underway. The sample will be completed during the following
year. When data collection is completed in the spring of 1988, analyses will
be initiated.
Publications
None
574
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02362-01 LDP
TITLE OF PROJECT (SO characters or less. 7/(/e must tH art one line between the txrders.)
Physical/Neurological Development in Children of Healthy and Depressed Mothers
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute effillation)
PI: T. Sherman Research Psychologist LDP/NIMH
COOPERATING UNITS (It any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Affective Development
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN- YEARS:
.37
PROFESSIONAL
.05
32
CHECK APPROPRIATE BOX{ES)
B (a) Human subjects
\E (a1) Minors
S (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The exciting current work on the biochemical and genetic bases of the major
affective disorders makes more salient the need to bridge the sciences and
begin to develop concepts and markers linking the physical and neurological
substrate with the complex behavioral manifestations of these disorders. A
developmental framework provides a basis for moving beyond correlational
assessment to causal modeling. This project will be a step in this process.
A rich body of data (Childrearing Study, Annual Report # ZOl MH 02144 ) on
the children's birth complications, physical development, cognitive develop-
ment and neurological development will be brought together as profiles for
individual children. These profiles will be subjected to a cluster analysis.
It is expected that some of the clusters of children will be associated with
higher family loadings for affective illness. It is also expected that parti-
cular clusters will be associated with specific social, emotional, and/or cog-
nitive outcomes, or with the development of symptoms of affective illness,
and/or behavioral disorders in the children.
575
PHS 6040 (Rev. 1/84)
CPO 9 1 4.* It
ZOl MH 02362-01 LDP
Project Description
The exciting current work on the biochemical and genetic bases of the major
affective disorders makes more salient the need to bridge the sciences and
begin to develop concepts and markers linking the physical and neurological
substrate with the complex behavioral manifestations of these disorders. A
developmental framework provides a basis for moving beyond correlational as-
sessment to causal modeling. This project will be a step in this process.
A rich body of data (Childrearing Study, Annual Report # ZOl MH 02144) on the
children's birth complications, physical development, cognitive development
and neurological development are being brought together as profiles for indi-
vidual children. These profiles will be subjected to a cluster analysis.
At a correlational level, it is expected that some of the clusters will be
associated with higher family loadings for affective illness. At a predic-
tive level of analysis, it is expected that particular clusters will be asso-
ciated with specific social, emotional, and/or cognitive outcomes in the child-
ren. It will be of particular interest to observe if particular clusters are
associated with the development of symptoms of affective illness, and/or beha-
vioral disorders in the children.
The specific data sources are:
1.) Birth and health records of each child.
2.) Height and weight assessments of the younger children (CI) at 2 years of
age.
3.) Neurological assessments (PANESS Exam) at age 5 for CI, and at age
7-9 for C2.
4.) MacCarthy Scales of intelligence for the younger child (CI) at age 5.
Significance to Biomedical Research
The current developments in the genetic and biochemical bases of major affec-
tive illness have made more salient the need to address the question of the
relation between the identified physiological differences and the complex be-
havioral symptomatology associated with these disorders. A potential level
for beginning to build such a bridge is in the physical and neurological de-
velopment of individuals who are at risk for the development of affective
illness. This study will provide physical and neurological development pro-
files for children of affectively ill and healthy parents. The children's
profiles will be examined to determine if there are groups of children who
share developmental profiles.
These developmental characteristics may prove useful for identifying groups
of children who share a common physiological substrate for affective disorder.
It will be possible, then, to assess the extent to which these children share
common social, emotional, cognitive, and/or symptom characteristics as they
mature.
576
ZOl MH 02362-01 LDP
Proposed course
The medical records of the children will be collected from family physi-
cians this year. These records will concern birth conditions and child health,
and where possible, the age of achievement of the developmental milestones.
The PANESS and the McCarthy Scales are being administered. Data analyses will
be begun as soon as the data become available, projected in the coming year.
Publications
None
577
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02363-01 LDP
TITLE OF PROJECT (80 characters or less. Title must tit or\ one line betweery the borders.)
Information-Processing Deficits in Schizophrenic Children
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, leboratory, and institute atfillatlort)
P^- T. Sherman Research Psychologist LDP /NIMH
Other: R. Asarnow
Associate Professor
UCLA School of
Medicine
COOPERATING UNITS (if any)
UCLA School of Medicine
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Affective Development
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN-YEARS:
.37
PROFESSIONAL:
.05
32
CHECK APPROPRIATE BOX(ES)
HI (a) Human subjects
S (a1) Minors
Q (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project was designed to add to the growing body of literature concerning
core cognitive impairments in schizophrenic children. Both adult and child
schizophrenics show a wide range of impairments on a variety of cognitive
tasks. However, if these studies are examined carefully, it does not appear
that we have been able to identify a specific process that is consistently
impaired in schizophrenic individuals. This suggests that the impairment is
more systemic, not simply contained within a small set of isolable information
processing functions. In this study, based on the Feature Integration Theory
of Attention by Treisman and Gelade, the ability of the schizophrenic child
to recognize the task relevance of, and engage in, serial and parallel modes
of visual search is examined. The data reveal that schizophrenic children are
as competent as MA-matched control children in their use of a parallel visual
search strategy and a serial visual search strategy, and in their recognition
of the situations under which each is the optimal strategy. Importantly, the
data also reveal that schizophrenic children have a significantly greater
start-up time than the MA-matched controls in the initiation of their search
strategies. This difference is in the range of 480 msecs. This is much
greater than would be predicted on the basis of merely a motor delay in button
pushing. These results suggest that the schizophrenic individual does not have
a specific information processing deficit, but rather a global deficit in time
to initiate the operation of any information processing strategy, be it an
automatic strategy or an attention demanding strategy. Such a deficit would
I be apparent in all tasks requiring speeded information processing, and in real-
time processing of information.
579
PHS 6040 (Rev. 1/84)
SPO si4'eit
ZOl MH 02363-01 LDP
Project Description
This project was designed to add to the growing body of literature concerning
core cognitive impairments in schizophrenic children. Both adult and child
schizophrenics show a wide range of impairments on a variety of cognitive
tasks. However, if these studies are examined carefully, it does not appear
that we have been able to identify a specific process that is consistently
impaired in schizophrenic individuals. This suggests that the impairment is
more systemic, not simply contained within a small set of isolable information
processing functions. This study, based on the Feature Integration Theory of
Attention by Treisman and Gelade, sought to examine the ability of the schizo-
phrenic child to recognize the task relevance of and engage in serial and par-
allel modes of visual search.
Eight schizophrenic boys and 16 normal boys matched in age and mental age to
the schizophrenic children participated in the study. In addition, a develop-
mental study was conducted in order to ascertain whether observed differences
between the schizophrenic children and the MA-matched controls could be inter-
preted as a more immature form of information processing or whether their
deficits represent a pattern of information processing not normally seen in
developing children. To enable this comparison, there was an adult comparison
group (n=9), a comparison group of boys approximately one year older than the
schizophrenic children (n=ll) and a comparison group of younger boys approxi-
mately three years younger than the schizophrenic children (n=ll).
Results
The data reveal that schizophrenic children are as competent as MA-matched
control children in their use of a parallel visual search strategy and a serial
visual search strategy, and in their recognition of the situations under which
each is the optimal strategy. Importantly, the data also reveal that schizo-
phrenic children have a significantly greater start-up time than the MA-matched
controls in the initiation of their search strategies. This difference is in
the range of 480 msecs. This is much greater than would be predicted on the
basis of merely a motor delay in button pushing and is twice the difference
that distinguishes the younger and older normal boys.
These results suggest that the schizophrenic individual does not have a speci-
fic information processing deficit, but rather a global deficit in time to ini-
tiate the operation of any information processing strategy be it an automatic
strategy or an attention demanding strategy. Such a deficit would be apparent
in all tasks requiring speeded information processing, and in real-time proces-
sing of information. This first suggestion is the result found in the research
literature on information processing; the second suggestion is consistent with
the clinical picture of schizophrenic individuals.
Significance to Biomedical Research
Research on schizophrenic children provides information on a sample that is
less likely than adult samples to have suffered additional impairments due to
prolonged drug therapy or institutionalization. In addition, schizophrenic
children are probably a more homogeneous group than adult schizophrenics, and
580
ZOl MH 02363-01 LDP
are probably a group with a higher genetic loading for the illness. Thus,
research on schizophrenic children offers a potentially purer sample in which
to study cognitive impairments associated with schizophrenia. We have found
that the information processing deficits in these children are comparable to
those seen in adult samples of schizophrenics. This suggests that there is a
continuity between adult forms and childhood onset forms of schizophrenia.
The current work provides a behavior model of information processing impairment
that can guide and inform further research on the brain bases of schizophrenia
as well as provide a framework for structured intervention.
Proposed Course
Data collection is completed, and data analysis is underway. A paper is being
prepared for presentation in a scientific journal.
Publications
Mundy, P., Sigman, M. , Ungerer, J., and Sherman, T. Nonverbal communication
and play correlates of language development in autistic children. J. Autism,
in press.
Asarnow, R. , Sherman, T. , and Strandburg, R. The search for the psycho-
biological substrate of childhood onset schizophrenia. J. Am. Acad. Child.
Psychiatry. 26(5), 601-614, 1986.
Mundy, P. , Sigman, M. , Ungerer, J. , and Sherman, T. Defining the social
deficits of autism: The contribution of nonverbal communication measures,
J. Child. Psychol. Psychiatry 27(5), 657-669, 1986.
Sigman, M. , Mundy, P., Sherman, T. , and Ungerer, J.: Social interactions of
autistic, mentally retarded, and normal children and their caregivers.
J. Child. Psychol. Psychiatry 27(5), 647-655, 1986.
581
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02364-01 LDP
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
A Follow-up Investigation of Offspring of Bipolar Parents
PRINCIPAL INVESTIGATOR (List ottter professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfiliation)
PI:
C . Zahn-Waxler
Other:
L. Cytryn
A. Mayfield
D. McKnew, Jr.
M. Radke-Yarrow
COOPERATIN
3 UNITS (it any)
None
Chief, Sec. on
Child Behavior Disorders
Medical Officer (Psych)
Psychology Technician
Medical Officer (Psych)
Chief
LDP /NIMH
LDP /NIMH
LDP/NIMH
LDP /NIMH
LDP /NIMH
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, NIH, 9000 Rockville Pike, Bethesda, MD 20892
TOTAL MAN-YEARS:
.55
PROFESSIONAL:
.25
.30
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
(Xl (a1) Minors
SI {a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Seven male children of severely ill manic-depressive parents (five of whom had
a unipolar depressed spouse) were studied at one and at two years of age. They
showed a range of adjustment problems when assessed in a laboratory setting
(protocol # 78-M-75). They had Insecure attachment relationships with their
caregivers and had problems in regulation of affect. Disturbances in empathy
and aggression also were evident. Psychological and psychiatric assessments
of the children were conducted four years later to determine whether problems
identified earlier were transitory or persistent and indicated possible precur-
sors of later diagnosable disturbance.
On the follow-up at five and six years of age, these children differed from a
control group on many dimensions of functioning. In a psychiatric interview,
they reported more fears, worries, symptoms of depression and distortions in
self-image. Their mothers indicated a high incidence of externalizing, as well
as internalizing symptoms in the children. Proband children received more for-
mal (DSM-III) psychiatric diagnoses than control children. Deficits in empathy
and non-assertive strategies for resolving conflict also were identified. Many
of the problems persisted over time, even for children who were in therapy, for
whom change was evident. These patterns underscore the need for early inter-
vention in families such as these in which both parents are depressed and in
which there is a familial history of affective disorder as well.
383
PHS 6040 (Rev 1/S4)
ZOl MH 02364-01 LDP
Project Description
The purpose of this research was to conduct a follow-up evaluation of a small
sample of offspring of bipolar parents (annual report # ZOl MH 02155). From a
genetic standpoint this is a high risk, sample. Five of the seven spouses of the
bipolar parents had unipolar depression and all of the seven bipolar parents had
a familial history of affective disorder. The environments of their children
also differed from those of a control sample: the former were characterized by
disorganization, unpredictability, alienation, and weak social support systems.
Problems in relationship formation, affective self-regulation, and coping with
stress were identified in the proband children when they were infants and todd-
lers. The main goal of the current research was to determine whether the early
problems reflected transitory disturbances, or instead indicated early precursors
of later diagnosable disturbance.
Methods Employed and Major Findings
Seven male children with a severely ill manic-depressive parent were seen at ages
five and six. Four of the bipolar parents were female and three were males who
had been inpatients at NIMH. Diagnoses of bipolar affective disorder were deter-
mined on the basis of SADS-L interviews, using RDC criteria. Five of the seven
spouses were diagnosed with unipolar depression and one with substance abuse and
war neurosis. There was a comparison group of 11 children with nondepressed
parents.
At age five the children were observed in a laboratory setting in procedures cho-
sen to assess the areas of difficulty identified earlier in development. The
children were observed in interaction with a playmate, under pleasurable and
mildly stressful conditions. They were given structured tests developed to
assess empathy and conflict resolution strategies. At age six, the children
returned for psychiatric and psychological evaluation. A standard psychiatric
interview, the Childhood Assessment Schedule, was given. DSM-III diagnoses were
determined, based on the child's responses to the psychiatric interview and data
obtained from the Achenbach Child Behavior Check List filled out by the mother.
Children in the proband sample reported more fears, worries, and depression than
control children on the Childhood Assessment Schedule and they also scored higher
on a subset of depression items indicative of suicidal ideation and/or proneness
to self-injury. Their mothers reported them to be high on externalizing as well
as internalizing symptoms. Psychiatric diagnoses of children indicated that di-
agnoses were more frequent and of a more serious nature in proband than in con-
trol children. Some of the proband children showed continuity over time in the
specific symptoms of disturbance while others showed changes in the types of
problems. As a group, proband children showed atypical patterns of empathy and
social problem solving.
Significance to Biomedical Research and the Program of the Institute
The literature on children with a bipolar parent has yielded mixed findings;
few and many problems have been reported in offspring. In some studies these
children are described as super competent. It is important to begin to identify
584
ZOl MH 02364-01 LDP
the origins of these differences in order more effectively to plan prevention
and intervention strategies. This research provides information about the
frequency, nature and severity of s)rmptoms in a group of children for whom there
is high genetic loading for affective disorder. The present research helps to
provide guidelines regarding some of the specific content areas and problems in
need of modification.
Proposed Course
Data collection and analysis have been completed. A manuscript has been prepared
and submitted for consideration for publication in the American Journal of Psy-
chiatry. A report based on this work was presented in a symposium on develop-
mental psychopathology at the APA meetings in May.
Publications
Bretherton, I., Fritz, J., Zahn-Waxler, C. , & Ridgeway, D. The acquisition and
development of emotion language: A functionalist perspective. Child Dev. 57:
529-548, 1986.
585
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02365-01 LDP
TITLE OF PROJECT (BO charnctars or tesi. Title must fit on one line between the boniers.)
The Psychobiological Effects of Sexual Abuse
PRINCIPAL INVESTIGATOR (Ust Other professional personnel behw f/ie Principal Investigator.) (Name, title, laboratory, and institute atlillation)
PI: F.W. Putnam Staff Psychiatrist LDP NIMH
OTHER: P.K. Trickett Guest Researcher LDP NIMH
COOPERATING UNITS (» any)
Chesapeake Institute, Kensington, Maryland; Montgomery County Child Protection
Unit; Prince Georges County Child Protection Unit; Virginia Child Protection
Services, Fairfax County, Virginia
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.37
PROFESSIONAL
.25
.12
CHECK APPROPRIATE BOX(ES)
13 (a) Human subjects D (b) Human tissues D (c) Neither
H (a1) Minors
S (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This Study focuses on the psychological and biological effects of sexual
abuse on children. Subjects will be sexually abused females (6-15 years
of age) and their parents or guardians. Controls will be age and socio-
economically matched. The study will use a multi-method approach to gather
information on the psychological and physical development of children and
their home environment. Methods include: staging physical development,
measurement of blood hormone levels, psychological tests and measures for
both children and adults. Three hypotheses will be tested: A), that sexually
abused girls will have a more difficult transition through puberty; B), that
sexual abuse may alter the hormonal levels and effect the timing of puberty;
and C), that sexually abused children will demonstrate higher levels of
dissociation than controls.
587
PHS 6040 (Rev. 1/84)
ePO (W.*!!
ZOl MH 02365-01 LDP
Project Description:
Objectives: Sexual abuse, largely unrecognized until the 1970's, is now
known to constitute a major form of child abuse in the United States.
While statistics on the incidence of childhood sexual abuse vary widely,
even the lowest incidence rates cited indicate that it is a major public
health problem. The effects of childhood sexual abuse, both immediate and
long-term, are considerable. Prominent symptoms in adults include:
depression, suicidality, substance abuse, poor self-esteem, difficulties
with concentration, multiple somatic complaints and sexual dysfunction.
In children, the data are less clear but frequently reported symptoms include:
depression, running away, learning disabilities, self-multilation, conduct
disorders and inappropriate sexual behavior.
This study is the first attempt to follow prospectively a group of sexually
abused children longitudinally through puberty. Three hypotheses are tested:
1) that puberty exacerbates the impact of sexual abuse on the psychological
development of girls; 2) that certain specific behaviors, i.e. inappropriate
sexuality and aggression that are commonly reported in sexually abused
children, are associated with elevations in levels of adrenal androgenic
and/or gonadotropic hormones; and 3) that dissociative behavior, a psycho-
physiological response to extreme trauma, is increased in sexually abused
girls compared to matched control subjects.
Method:
The study uses a convergence or cross-sequential prospective design.
Psychological development is assessed across two broad domains: 1) indicators
of competence and coping with the developmental tasks of puberty and 2) the
presence of psychiatric symptoms and behavior problems. Physical development
will be assessed using Tanner staging and a series of adrenal and gonadal
hormonal measures. Dissociative capacity will be measured using standardized
hypnosis scales and the Dissociative Experiences Scale (DES).
Significance to Biomedical Research
This study seeks to document from a developmental perspective the impact
of childhood sexual abuse. Three mechanistic hypotheses are tested.
Proposed Course:
This project recently received a large three-year grant from the W.T. Grant
Foundation and will begin operation on June 1, 1987. Data will be collected
on 50 subjects and 50 control families a year for the first two years.
Serial follow-up measurements will begin in year two and be continued on
each child for two years.
Publications;
None
588
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 02366-01 LDP
TITLE OF PROJECT (80 characters or lea. Title must tit on one line between the borders.)
The Psychophysiology of Multiple Personality Disorder
PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfillatJon)
PI: F. Putnam Staff Psychiatrist LDP/NIMH '
Other:
R. Post
D. Weinberger
T. Zahn
0. Devinsky
N. Hall
Chief
Psychiatrist
Psychiatrist
Psychiatrist
Psychiatrist
BPP/NIMH
CBDB/NIMH
LPP/NIMH
LPP/NIMH
George Washington Univ.
COOPERATING UNITS (If any)
George Washington University, Laboratory of Psychology and Psychopathology ,
Biological Psychiatry Branch, Clinical Brain Disorder Branch
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
77
PROFESSIONAL
.65
.12
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
0 (a1) Minors
0 (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This Study measures reported psychophysiological differences that may
exist among the alternate personalities of individuals suffering from
multiple personality disorder (MPD). Alternate personalities of subjects
with MPD and imaginary personalities of normal simulating control subjects
are measured on visual and auditory evoked potentials, spontaneous EEG,
cerebral blood flow, continuous catheter venous sampling and measures of
autonomic nervous system activity such as galvanic skin response, skin
temperature, pulse and Respiration.
589
PHS 6040 (Rev 1/84)
CPO B1 4-«lt
ZOl MH 02366-01 LDP
Project Description;
Objectives: This project seeks to ascertain any psychophysiological
differences that may exist among the alternate personalities of individuals
suffering from multiple personality disorder (MPD).
Method:
A variety of psychophysiological measures have been used in this study.
These include averaged evoked potentials elicited by light and sound in
collaboration with Dr. R. Coppola (Neuropsychiatry Branch) , galvanic
skin response, respiration, pulse and skin temperature (Dr. T. Zahn,
Laboratory of Psychology and Psychopathology) , cerebral blood flow, (Dr.
D. Weinberger, Clinical Brain Disorder Branch), 24-hour continuous telemetry
EEC (Dr. 0. Devinsky, Laboratory of Psychology and Psychopathology),
and circulating immune functions (Dr. N. Hall, Department of Biochemistry,
George Washington University).
Findings:
Past findings: Studies from this project indicate that MPD patients can
produce changes in visually evoked potentials and spontaneous EEC that
cannot be duplicated by simulating control subjects.
New findings: Studies of personality switching, using 24 hour EEC telemetry,
indicate that these events are independent of epileptic activity in subjects
with concurrent MPD and temporal lobe epilepsy.
Significance to Biomedical Research
The demonstration of state-specific psychopathological responses may permit
a more extensive investigation of the role of personality factors in psy-
chosomatic disorders. Multiple personality disorder subjects provide a
unique model of the interaction of psychological states and somatic symptoms.
Proposed Course
This study continues to collect data on the psychophysiological responses
of alternate personalities in MPD subjects and simulated personalities in
control subjects. The most recent focus has been on round-the-clock EEC
monitoring of MPD subjects with temporal lobe epilepsy and alternation in
the immune functions of MPD subjects.
Publications
Putnam, F.W. : The Scientific Investigation of Multiple Personality. In
Quen, J.M. (Ed.): Split Minds Split Brains. New York, New York University
Press, 1986, pp 109-125.
590
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02367-01 LDP
PERIOD COVERED ~ ~ "
October 1, 1986 through September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) " ~ ~ ■
The Clinical Phenomenolosy of Multiple Personality Disorder
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: F.W. Putnam Staff Psychiatrist LDP/NIMH
Other: R. Lowenstein Chief Dept. Psy., UCLA
^- Post Chief BPP/NIMH
COOPERATING UNITS (it any)
UCLA
Biological Psychiatry. Branch
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Child Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
47
PROFESSIONAL:
35
,12
CHECK APPROPRIATE BOX(ES)
(3 (a) Human subjects D (b) Human tissues □ (c) Neither
£] (a1) Minors
£] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The clinical syndrome of multiple personality disorder (MPD) is an
unusual dissociative disorder that previously had been poorly characterized.
Systematic data have been collected on over 200 recent cases of MPD in treatment
in the United States. Results indicate that MPD is a clinical syndrome with
a core of dissociative and depressive symptoms that occurs in individuals with
a history of childhood trauma between ages 2 and 14 years. Male and female
MPD patients differ on the expression of aggressive and self-destructive
symptoms.
591
PHS 6040 (Rev. 1/84) CO si4-*ii
ZOl MH 02367-01 LDP
Project Description
Objectives: Until the last decade, multiple personality disorder (MPD) had
been considered to be an extremely rare dissociative condition. With the
inclusion of MPD in the DSM-III (1980) there has been a dramatic increase
in the numbers of reported cases. The purpose of this project is to survey
current cases in the United States and other countries to determine the
clinical presentation, past psychiatric, medical, childhood and family
history of these individuals, the phenomenology of the patient's system of
alternate personalities, and the responses of the patient to standard
therapeutic interventions.
Method
A 386-item questionnaire, designed to collect detailed information on a
single patient, was initially distributed to clinicians reported to be
treating MPD patients. A national sampling of patients meeting DSM-III
criteria for MPD was obtained and analyzed. Subsequent samples have been
obtained on focused samples of MPD patients. Analyses of male MPD patients
and MPD patients with concurrent temporal lobe epilepsy are nearing
completion.
Findings
Major past findings: The overall patient population was found to be
predominately female (92%) with a mean age at sampling of 35.8 years. MPD
patients are poly-symptomatic op initial clinical presentation (mean number
of symptoms 18.5) with psychiatric symptoms suggestive of depression (de-
pressed mood, mood swings, insomnia, sexual dysfunction and suicidality)
and somatic symptoms of headache and GI problems. The average number of
alternate personalities was 13.3 with a standard constellation of alternate
personality attributes appearing in most patients. A history of childhood
trauma was found in 97% of cases.
New findings: Data from this study are being analyzed for variables related
to treatment response. In addition, several subsaraples of MPD patients have
been collected, including males and patients with concurrent temporal lobe
epilepsy. Data on male patients indicate that they are more likely to pre-
sent with a history of recent aggression towards others while females are more
likely to be diagnosed after episodes of self-mutilation or suicide attempts.
Significance to Biomedical Research
This project represents the first attempt to conduct a large scale sampling
of independent cases of multiple personality disorder. The data from this
study are being used to construct profiles of MPD patients to aid in diagnosis
and to identify more effective therapeutic interventions.
5^2
ZOl MH 02367-01 LDP
Publications
Putnam, F.W. , Guroff, J.J. , Silberman, E.A. , Barban, L. and Post, R.M. : The
clinical phenomenology of multiple personality disorder: Review of 100 cases.
J. Clin. Psychiatry, 47: 285-293, 1986.
Putnam, F.W. : The treatment of multiple personality: State of the art. In
Braun, B.G. (Eds.): The treatment of multiple personality disorder.
Washington, American Psychiatric Press, pp 175-198, 1986.
593
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02368-01 LDP I
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 chanctars or less. Title must fit on one line between the txirders.)
The Dissociative Experiences Scale (DES)
PRINCIPAL INVESTIGATOR (Ust other professional personnel fie/ow the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: F.W. Putnam Staff Psychiatrist LDP NIMH
COOPERATING UNITS (it any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
SECTION
Section on Childhood Behavior Disorders
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.37
PROFESSIONAL:
25
,12
CHECK APPROPRIATE BOX(ES)
[^ (a) Human subjects
Q (al) Minors
Q (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A short, self-administered questionnaire, the Dissociative Experience
Scale (DES), has been developed and tested. The DES reliably and validly
measures frequency and types of dissociative phenomena. This instrument
has been administered to a wide variety of carefully diagnosed psychiatric
groups. The DES has been included in over 50 studies in the United States
and abroad and has been translated into French, Dutch and Cambodian.
Current research using this instrument includes studies of patients with
epilepsy, eating disorders, borderline personality disorder, affective
disorders and schizophrenia in conjunction with researchers in the IRP, NIMH;
NINCDS; and the Department of Psychiatry, UCLA and the Department of
Psychology, The American University.
595
PHS 6040 (Rev. 1/84)
GPO si4-aia
ZOl MH 02368-01 LDP
Project Description
Objective: The phenomenon of dissociation is a complex psychophysiological
process that appears to contribute to the psychopathology of a range of
disorders, primarily those associated with exposure to traumatic experiences
such as postraumatic stress disorder (PTSD) , multiple personality disorder
(MPD) and related dissociative reactions. The clinical recognition and the
study of dissociative phenomena has been limited by the lack of a reliable
and valid instrument to identify and quantify these experiences. A short,
self-administered, computer-scored questionnaire was developed that yields
an overall index score and three sub-scale scores that reliably and validly
quantify dissociative experiences.
Method
Reliability testing has included both test-retest and split-half measures.
Validity testing consists of comparing both item and overall scores from a
wide range of psychiatric and normal populations. The DES was found to have
a significant correlation (_r = 0.62, _£ = <0.01) with another measure of
dissociation, hypnotizability , measured by standardized scales. Predictive
validity studies are in progress. Our findings for normal adults and
adolescents and several psychiatric subgroups have been replicated by other
investigators.
The DES was shown to have a significant positive correlation with two
standardized scales of hypnotic capacity (DES/SHSS _r = 0.62; n = 60; _£
= < 0.01); DES/HGSHS £ = 0.51; _n = 60; £ < 0.01: Stanford Hypnotic
Susceptibility Scale (SHSS) and the Harvard Group Susceptibility to
Hypnosis Scale (HGSHS). This provides additional evidence of the validity
of the DES tc measure dissociative phenomena and indicates that normal
individuals with hypnotic capacity frequently have spontaneous dissociative
experiences.
The DES was also used with a carefully diagnosed population of Alzheimer's
disease patients. It was found that patients with organic memory deficits
do not endorse items related to dissociative memory disturbances.
Significance to Biomedical Research
Dissociation is thought to play a major role in the persistence of certain
types of symptoms secondary to traumatic experiences, e.g. flashbacks,
abreactions, and intrusive thoughts, images or affects. We have developed
a reliable and valid means of quantifying dissociative experiences that has
been rapidly adopted by researchers working with a variety of pyschiatric
disorders. While not intended as a diagnostic instrument, the DES has become
a widely used screening instrument in clinical settings where trauma patients
are common.
596
ZOl MH 02368-01 LDP
Proposed Course
Current DES studies are underway with collaborators from a number of
universities and with NIH, to determine the incidence and profiles of
experiences of dissociation and depersonalization in temporal lobe epilepsy
patients and in eating disorder patients. We plan to employ the DES as a
survey instrument on a wide range of carefully diagnosed psychiatric
populations. We will continue to increase our samples of schizophrenia,
post-traumatic stress disorder, panic anxiety disorder, multiple personality,
atypical dissociative disorder, affective, obsessive-compulsive, premenstrual
tension syndrome and organic dementia patients. We are also working on an
improved form of computer scoring. A pilot study is in progress with the
french translation of the DES seeking to explore the issues of cross-cultural
expression of dissociative symptoms. Pilot data indicate that the incidence
of dissociative phenomena in French normals and psychiatric patients is
equivalent to that found in U.S. samples.
Publications
Bernstein, E.M. , Putnam, F.W. : Development, reliability and validity of
a dissociation scale. J. Nerv. Ment. Pis., 174: 727-735, 1986.
597
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02369-01 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Mutual Interpersonal Influence in Families with and Without Affective Disorder
PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfillation)
PI: G. Kochanska Research Psychologist LDP/NiMH
COOPERATING UNITS (it any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.77
PROFESSIONAL:
.35
.42
CHECK APPROPRIATE BOX(ES)
Ck (a) Human subjects
Kl (a1) Minors
E (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Processes of mutual Interpersonal influence are studied in dyads comprised
of well and depressed mothers and their 5-year-old children. This is a longi-
tudinal study, following up the sample of 87 dyads, in which control and
discipline interactions were studied when the children were 1 1/2 to 3 1/2
years old (ZOl MH 02152), Two major themes are followed: difficulties of
depressed mothers in controlling their children, which were identified in the
original study, and the development of children's competent interpersonal
strategies of influence, which in their emerging form were studied in toddler
period. In contrast to the first study, where the focus was on the mother as
the agent of control and child as a respondent, in this study the focus is on
bilateral interpersonal influence processes. Every episode of control, which
can be initiated either by the mother or by the child, is coded.
PHS 6040 (Rev. 1/84)
OPO SI4-S1B
ZOl MH 02369-01 LDP
Pro.ject Description
Processes of mutual interpersonal influence are studied in dyads comprised
of well and depresssed mothers and their 5-year-old children. This is a longi-
tudinal study, following up the sample of 87 dyads, in which control and
discipline interactions were studied when the children were 1 1/2 to 3 1/2
years old (ZOl MH 02152). Two major themes are followed: difficulties of
depressed mothers in controlling their children, which were identified in the
original study, and the development of children's competent interpersonal
strategies of influence, which in their emerging form were studied in toddler
period.
In contrast to the first study, where the focus was on the mother as the
agent of control and child as a respondent, in this study the focus is on
bilateral interpersonal influence processes. Every episode of control, which
can be initiated either by the mother or by the child, is coded.
Method
Ninety minutes of mothers' and children's videotaped interactions in a
naturalistic setting are coded. Each episode of interpersonal influence is
described regarding its timing, goal, and verbal and physical techniques of
influence used by the initiator. Respondent's reaction is described with
the use of categories such as compliance and a variety of resistance cate-
gories (negotiation, reasoning, refusal, bargaining, excuses, conditional
statements, etc.) The interactive qualities of the entire episode are also
coded. The coding system is a modified version of the system used in the
earlier study, but it reflects children's increased social competence and
influence skills. It also allows us to capture the bilateral processes of
influence within the dyad, more appropriate at this age. Despite the
modifications, the system is compatible with the one used in the earlier
study, providing a possibility for longitudinal assessment of the mother-child
dyads.
Research Questions
Two major research questions will be addressed: the relation between psycho-
pathology and the processes of interpersonal influence, and the developmental
changes between two and five years of child's age. The impairments in control
processes of depressed mothers, such as lowered ability to reach compromise
with their children and increased tendency to avoid confrontation with the
child, were found in the earlier study; the present study will provide not
only the opportunity to replicate the findings, but also to study the possible
exacerbation of the problems over time, as well as to consider control proc-
esses in the context of our much enriched knowledge of psychiatric history
of both mother and child. Expectations of developmental change in the proc-
esses of interpersonal influence is reflected in our approach to the child
as not only a respondent to maternal control, but also as an active agent,
initiating control episodes. A rich representation of sophisticated inter-
personal strategies, included in the coding system, will allow us to capture
further development of social skills, the rudiments of which were described
in the earlier study.
600
ZOl MH 02369-01 LDP
Significance of Biomedical Research
The study will provide further insight into the interactive patterns in
families with affective disorder. Day-to-day processes of interpersonal
influence may be a significant factor in the emergence of disordered patterns
of interaction, and therefore may contribute to the increased risk for the
development of psychopathology.
Proposed Course
A reliable, modified coding system is in place, and coding has been completed.
Analyses are underway.
Publications;
None
601
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02370-01 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Caregiving Patterns in Stressed Families
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation)
PI: F. Bridges-Cline Guest Researcher LDP/NIMH
Other: w,E. Wilson
M. Radke-Yarrow
D. Hay
T. Cox
Research Psychologist
Chief
Research Psychologist
Psychiatrist
DRG/NIH
LDP/NIMH
Univ. of London
Univ. of Liverpool
COOPERATING UNITS (If any)
University of London
Division of Research Grants, NIH
Univ. of Liverpool
LAB/BRANCH
Laboratory of Developmental Psyrhnlngy
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
.37
PROFESSIONAL:
.05
.32
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
B (a1) Minors
O (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Experiences in the family are presumed to play a significant role in
the socialization of the child, the development of his or her personality,
and the emergence of any pathological characteristics. Of primary interest
in this research is the functioning of different sorts of families with
respect to a central dimension of family life, the provision of care to
its members. An immediate objective of this study is the development of a
behavioral family interaction coding system that focuses on the giving and
receiving of care among family members. The goal is for the coding system
to be at the same time sufficiently broad in the conceptualization of
"caregiving" as to be applicable to family life in different cultures and
family members of different ages, and sufficiently articulated as to be capable
of differentiating families with and without varying kinds of internal
and external stressors. An additional aim of the study is the examination
of associations of parental depression and other family stressors with
varying patterns of family caregiving. This aim derives from the view that
family caregiving is a potential mediator of factors such as economic distress
or parental pathology that place children at risk for later problems. In this
regard, the coding system is being piloted, refined, and used on a sample
of disadvantaged families in Britain and a sample of families in the NIMH
study, with and without parental depression.
603
PHS 6040 (Rev 1/84)
GPO 91 4-sie
ZOl MH 02370-01 LDP
Project Description
The effects of factors that place children at risk for later problems, such
as a family's parental psychopathology or economic distress, are likely to be
mediated through changes in or dysfunctional patterns of, the provision of
care to and by family members. An immediate objective of this study is the
development of a family interaction coding system that focuses on the giving
and receiving of care among family members. The system is to be sufficiently
broad in the conceptualization of "caregiving" as to be applicable to family
life in different cultures and family members of different ages, and
sufficiently articulated as to be capable of differentiating families with and
without varying kinds of internal and external stressors. The unit of analysis
that has been selected for this coding system differs from that used in most
other observational measures of family process. In contrast to a focus on the
individual's action or trait, the emphasis is the events and states in family
life to which members of the family may or may not respond, and the family
members' perceptions of, and responsiveness to, each other's affective, be-
havioral, and cognitive states.
In addition, an aim is to examine and describe the varying patterns of
caregiving that are associated with variations in family stressors and family
background variables. Of primary interest is the factor of parental depression
and the way in which this factor becomes manifest in the expression of and
response to particular kinds of needs of family members. One question to be
asked concerns the extent to which reversals of the usual caregiver role (e.g.
an older sibling takes on the role of parent) occur in families with parental
depression, and the extent to which it is harmful or beneficial for children
to take on caregiving roles in their families.
Method
The major goal of the coding system is to describe how members of a family,
parents and children alike, express their needs for care, react to each other's
needs, and accept or rebuff whatever care is offered. The starting point for
the description of a family's provision of care is the identification of needs
that may be perceived or inferred by family members. The observation procedure
requires four judgments: 1) determining whether a particular family member is
in need of care at a particular time, and characterizing the ways in which the
need manifests itself; 2) determining which, if any, of the other family
members respond to that need, and characterizing the nature of their response;
3) characterizing the reaction of the person in need to the other family
members' responses; and 4) recording instances in which one family member
provides or ascertains the need for care for another in the absence of any
visible need.
Two studies are involved. One study is the NIMH Childrearing Study; and the
other, to be referred to as the London Study, is conducted by Dr. Cox and his
colleagues at the University of London. The two studies share some common
research goals, as well as diverge in some aspects. Each examines families
under stress, due to the affective illness of a parent (NIMH) and conditions
associated with economic distress (London study). The issues addressed by
604
ZOl MH 02370-01 LDP
each reflect concern about the extent to which very young children's
experiences in the family place them at risk or protect them from the
likelihood of later disturbance.
Significance to Biomedical Research
Our goal in this study is to better understand the processes whereby affective
illness in a parent may affect the child's experience and understanding of the
giving and receiving of care in his/her relationships with others. Clearly,
the primary social setting within which the child acquires knowledge and
experience of caregiving is the family. The development of a robust observa-
tional measure of the family's pattern of caregiving will make available
a widely applicable tool for researchers interested in studying significant
dimensions of family environment that are associated with differential develop-
mental outcomes.
Proposed Course
The coding system has been developed. Coding of the observational data from
U.S. sample has begun, and analyses will proceed as these data are available.
A year to 1 1/2 years is an estimated time for this work.
Publications:
None
605
DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE 1 ^"°^^^^ DUMBER
NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl MH 02371-01 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the txirders.)
Patterns of Alliance in Families with and Without Parental Depression
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atfillatlon)
PI: F. Bridges-Cline Guest Researcher LDP/NIMH
Other: W. Wilson Research Psychologist DRG/NIH
COOPERATING UNITS (H any)
Division of Research Grants, NIH
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
.22
PROFESSIONAL;
.10
.12
CHECK APPROPRIATE BOX(ES)
K] (a) Human subjects □ (b) Human tissues D (c) Neither
K\ (al) Minors
K\ (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
One of the ways in which parental depression may influence the development
of the child is through the kinds of affective involvement that characterize
the relationships among various members of the family. These patterns of
affective involvement within and across family subsystems (i.e. the spouse,
parent-child, and sibling subsystems) are significant contributors to the
child's development not only through their influence on the pervasive emotional
climate of the environment within which the child functions but also through
their influence on the child's developing understanding of self in relationships.
Within the family systems literature, this aspect of family member relationships
(i.e. the direction and degree of affective responsiveness) has been referred
to as "alliances"; and it has been found that the pattern and nature of alliances
are quite different in distressed and dysfunctional as compared to effectively
functioning families. The aim of this study is to examine the nature and pattern
of family alliances — the availability and strength of interfamily communication
channels and the affective quality of these communications — in families with
and without parental depression.
607
PHS 6040 (Rev. 1/84) GPO »i4-»t»
ZOl MH 02371-01 LDP
Project Description
One of the ways in which parental depression may influence the development of
the child is through the kinds of affective involvement that characterize the
relationships among various members of the family. Within the family systems
literature, this aspect of family member relationships (i.e. the direction
and degree of affective responsiveness) has been referred to as "alliances";
and it has been found that the pattern and nature of alliances are quite
different in distressed and dysfunctional as compared to effectively functioning
families. For example, distressed families tend to display low overall levels
of alliance behavior and a weakness in the marital alliance relative to other
family subsystem alliances, for example, the parent-child alliance. The nature
and patterning of alliance behavior in families wherein parental depression is
the primary stressor has not been examined. Similarly, the role of these
differential alliance patterns in child developmental outcomes has not been
investigated. The aims of this study are (1) to examine the nature and pattern
of family alliances — the availability and strength of interfamily communication
channels, and the affective quality of these communications in families with
and without parental depression, (2) to identify the nature of alliances within
and across family subsystems (spouse, parent-child, and sibling subsystems)
and to determine whether there are patterns that distinguish groups of families
with and without parental depression, and (3) to determine the association of
particular family alliance patterns with differential child developmental
outcomes.
Method
The sample is a subset of the Childrearing Study families and includes 45
families who constitute all the families from the larger study in which the two
siblings are the oldest two siblings in the family, and in most instances are
the only two children in the family. Our reason for selecting these families
for the study derives from our concern that we have available from our ob-
servations, data relating to all of the immediate family relationship partners
that are and have been available at home to the target child(ren) of interest.
Patterns of alliance are studied in families with and without parental depres-
sion when the two siblings are between the ages of 5-6 years and 8-9 years.
Videotapes of the four family members interacting during a mealtime in our
research apartment (30 minutes) are used to code interaction patterns. For
coding, verbatim transcripts of the conversations of family members are
used in conjunction with the behavioral data available from the videotapes.
A modified version of the Family Alliances Coding System (FACS) (Gilbert,
Saltar, Deskin, Karagozian, Severance, and Christensen, 1981) is used with
additional codes developed specificaly for this study. The unit of analysis
for the FACS coding system is an individual's statement to and/or about
another family member. The additions to this coding system that are being
developed for this study focus on a more global unit of analysis wherein
dialogues or exchanges among several family members on a specific topic
serve as the object of a coder's evaluation of communication patterns. The
study is not yet at a point where findings are available.
608
ZOl MH 02371-01 LDP
Significance to Biomedical Research
Our goal in this study is to understand better the processes whereby depression
in one or both parents may affect the development of the child. A critical
place to begin the examination of such processes is at the family system level.
Our analytic strategy of examining patterns of alliance that meaningfully
distinguish clusters of families allows us to speak directly to the question
of the extent to which particular patterns correspond to maternal diagnostic
classifications. It is highly likely that such family clusters will cut across
diagnostic groups; and to the extent that we see differential outcomes for
children in the same family, and children of non-diagnosed as well as diagnosed
parents develop problems of a similar nature, this kind of finding would bring
us closer to understanding and identifying some of the more significant dimen-
sions of family environment that are associated with differential developmental
outcomes.
Proposed Course
All of the 45 verbatim transcripts of the 30 minutes of family conversation
during the mealtime have been completed. The coding system and its modifi-
cations are in the phase of development and piloting. Plans for the coming
year are to complete the coding system development, to complete the coding of
the 45 transcripts and to begin analyses.
Publications
None
609
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02372-01 LDP
PERIOD COVERED
October 1, 1986 f-hrough Sp.pfpmhpr ?n, 1Q«7
TITLE OF PROJECT (80 characters or less. Title must fit on arte line between the liorders.)
Psvchlatrlr Stafns nf rhilHrpn nf TlppT-pggpH PaT■Pn^g
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute atfitiation)
PI: M. Radke-Yarrow Chief LDP/NIMH
Other:
L. Cytryn
D, McKnew
L. Kuczynski
T. Sherman
E. Nottelmann
Med. Officer (Psychiatry)
Med. Officer (Psychiatry)
Assoc. Professor
Research Psychologist
Statistician
LDP/NIMH
LDP/NIMH
Univ. of Guelph
LDP /NIMH
LDP /NIMH
COOPERATING UNITS (If any)
University of Guelph, Guelph, Ontario
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.77
PROFESSIONAL:
.95
.82
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
0 (a1) Minors
£] (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
There is very little research that provides prospective data on the develop-
mental course of offspring of depressed parents. At each of the developmental
periods at which we have seen the children (younger child at 2 to 3 years,
followed up at 5 to 6 years; older sibling 5 to 8 years, followed up at 8 to
11 years), psychiatric assessments have been made, to be related to parental
diagnostic status, family environmental variables, and developmental variables.
In analyses to date, we have focused on associations between parental diagnoses
and children's psychiatric status. At 2 to 3 years of age, assessments did
not differentiate the children by diagnosis of parents. About 10% of the
children were rated at risk in the well and in the depressed parent groups.
We analyzed the children of middle-class background, who were initially seen as
toddlers, at their first follow-up assessment. At this time they manifested
problems (DSM-III diagnoses) with different frequencies depending on parental
diagnosis, i.e., 15% of the children of well parents, 21% of the children of
depressed mothers and well fathers, and 4 7% of the children of depressed mothers
and fathers. When their siblings were seen at 8 to 11 years, the corresponding
percentages were 8%, 28%, and 53%. The same data were re-examined in relation
to unipolar and bipolar maternal depression. There was little difference in
the frequencies of problems in these two groups.
611
PHS 6040 (Rev. 1/84)
GPO ai4>9ia
ZOl MH 02372-01 LDP
Project Description:
There is very little research that provides prospective data on the develop-
mental course of offspring of depressed parents (ZOl MH 02144). In the
present study, assessments are made of children of depressed parents and a
comparison group of children of well mothers, at each of the developmental
periods at which we have seen the children (younger child at 2 to 3 years,
followed up at 5 to 6 years; older sibling 5 to 8 years, followed up at
8 to 11 years). Psychiatric evaluations are part of an assessment battery.
Child outcomes are to be related to parental diagnostic status, family
history, and family environmental variables. In analyses to date, we have
focused on association between parental diagnoses and children's psychiatric
status at successive developmental stages
Methods and Major Findings:
Children were evaluated at 2 to 3 years of age in a psychiatric play interview
and on a sample of mother-child interactions. Based on these two psychiatric
appraisals, children were assigned a score (1 to 4) on degree of presumed risk
for later development of psychopathology. These assessments did not differ-
entiate the children by diagnosis of parents: About 10% of the children
were rated at risk in the well and in the depressed parent groups. Beginning
at 5 to 6 years of age, the children were evaluated by a psychiatric interview.
Child Assessment Schedule (CAS), supplemented by mothers' reports on the
Achenbach Behavior Checklist. The children who had been seen at the toddler
level now manifested problems (DSM-III diagnoses) with different frequencies
depending on parental diagnosis. (These analyses are of children of middle-
class background.) Problems appeared in 15% of the children of well parents,
in 21% of the children of depressed mothers and well fathers, and in 47% of
the children of depressed mothers and fathers. In their siblings, who were
seen at ages 8 to 11 years, the corresponding percentages were 8%, 28%, and
53%.
The same data were reexamined at ages 5 to 8 in relation to unipolar and
bipolar maternal depression. 39% of the children of unipolar mothers and
43% of the children of bipolar mothers manifested problems. In the sample
seen at ages 8 to 11 years, 53% of the children of unipolar mothers and 36%
of the children of bipolar mothers were given diagnoses.
Proposed Course:
Further detailed analyses are underway in which are specified (a) the
kinds of problems in the children, (b) their continuity over development,
and (c) their relation to a variety of refinements of parental diagnoses and
family variables (specified in project description). A third psychiatric
evaluation will be made, beginning this coming year, which brings the children
to adolescence. The psychiatric appraisals of the children will be considered
along with child outcome data based on observed behavior of child with family
and peers, and along with psychophysiological assessments to be made in the
follow-up.
612
ZOl MH 02372-01
Significance to Biomedical Research:
Research on the concordance of psychiatric problems of depressed parents and
their offspring is mainly without information on course of development of
offspring problems or on the conditions or processes underlying the development
of offspring problems or well-being. The data from this study begin to
provide such information.
Publications:
Radke-Yarrow, M. : Parental Depression and Parent-Child Interaction. In
Patterson, G.R. (Ed.): Family Social Interaction: Content and Methodological
Issues in the Study of Aggression and Depression. Hillsdale, New Jersey,
Lawrence Erlbaum Associates, Publishers, in press.
613
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02379-01 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between ttte borders.)
Survivor Children
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: M. Radke- Yarrow Chief LDP NIMH
OTHER: T. Sherman Research Psychologist LDP NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.12
PROFESSIONAL
.35
.77
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects
(xl (a1) Minors
S (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The focus of this study is on children who by criteria of genetic and
family variables are at high risk, but who are functioning relatively
well. Criteria of high risk were both parents are depressed or mother
is depressed and father is absent, and family environment is stressful.
Fifty children, two from each of 25 families that met these criteria
of risk, were studied. The children were between 5 and 11 years.
In-depth case analyses were done on four children who were functioning
well; namely, had no diagnosis; were liked by parents, teachers and peers;
and were performing at grade level. On this basis, critical factors in
their development were identified and hypotheses regarding conditions
or processes promoting good functioning were formulated and tested on
the 50 children to determine whether the identified factors predicted
the current status of the children in the high risk sample.
The protective factors shared by all of the children who were "surviving"
were above average intelligence, had socially winning ways or charm, and a
match, since birth, between a specific characteristic of the child and a
parental need. By fulfilling a specific need of the ill parent(s), the^
"survivor" children have received the maximum support from their family's
scant psychological resources. The children's relatively good functioning
is, however, at the expense of their own developmental needs.
615
PHS 6040 (Rev. 1/84)
ZOl MH 02379-01 LDP
Pro.ject Description:
The focus of this study is on children who by criteria of genetic and family
variables are at high risk, but who are functioning relatively well. This
work develops out of a merger of research on risk research and ego resiliency.
The objective is to examine the development of children from families in
which both parents are depressed or in which mother is depressed and father
is absent, and family environment is stressful.
Methods Employed and Major Findings:
Fifty-two children, two from each of 25 families that met these criteria of
risk, were studied. The children were between 5 and 11 years. In-depth
case analyses were done on 4 children who were functioning well; namely, had
no diagnosis, were liked by parents, teachers and peers, and were performing
at grade level. On this basis, critical factors in their development were
identified and hypotheses regarding conditions or processes promoting good
functioning were formulated and tested on the 50 children to determine whether
the identified factors predicted the current status of the children in the
high risk sample.
The protective factors shared by all of the children who were "surviving"
were above average intelligence, socially winning ways or charm, and a
match, since birth, between a specific characteristic of the child and a
parental need. The group level analyses support the findings on the smaller
group. By fulfilling a specific need of the ill parent(s), the "survivor"
children have received the maximum support from their family's scant psycho-
logical resources. The children's relatively good functioning is, however,
at the expense of their own developmental needs.
Proposed Course:
Completed.
Significance to Biomedical Research:
Risk factors in the development of psychopathology have received more attention
in research than have factors that are protective. This study focuses on
protective conditions. Understanding of processes of developmental psycho-
pathology and of effective intervention depends on clarification of both
kinds of factors.
Publications:
Radke Yarrow, M. , and Sherman, T. : Hard Growing: Children Who Survive.
In Rolf, J., Hasten, A., Cicchetti, D. , Nuechterlein, K. and Weintraub, S.
(Eds.): Risk and Protective Factors in the Development of Psychopathology.
Cambridge, England, Cambridge University Press, in press.
616
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 1 ''"°'^^*^^ number
NOTICE OF INTRAMURAL RESEARCH PROJECT
ZOl MH 02380-01 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line t>etween tt>e borders.)
Stressful Life Events and Childhood Adjustment
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: J. E. Richters Research Psychologist LDP/NIMH
Other: D. Pellegrini Psychologist Catholic Univ.
M. Radke-Yarrow Chief LDP/NIMH
COOPERATING UNITS (if any)
Catholic University
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.77
PROFESSIONAL:
30
.47
CHECK APPROPRIATE BOX(ES)
El (a) Human subjects D (b) Human tissues D (c) Neither
@ (al) Minors
^ (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is designed to investigate the links between parental
psychopathology , stressful life events, and the social-emotional adjustment
of children at-risk. The analyses are based on data from families participating
in the NIMH Childrearing Project (ZOl MH 02144), which includes children of
parents with and without a history of affective disorder. Assessments of
parental psychopathology are based on psychiatric (SADS-L) interviews with
each parent. Stressful life events to which the children have been exposed
are assessed on the basis of intensive, semi-structured interviews with
mothers. Evaluations of children's functioning are based on psychiatric
interviews, direct observations of their behavior in our laboratory apartment,
and independent reports from their parents and teachers.
In an initial set of analyses, characteristics of stressful events (e.g.
nature, severity, timing, frequency, saturation) are coded without coders'
knowledge of children's responses to the events. Events and characteristics
of events that are caused, exacerbated, or buffered by parental functioning
are distinguished. Associations between these subsets of stressful events
and their characteristics to predictions of offspring adjustment will be
examined.
617
PHS 6040 (Rev. 1/84) gpo ■14.»h
ZOl MH 02380-01 LDP
Project Description
The objective of this research is to investigate the links between parental
psychopathology, stressful life events, and the social-emotional adjustment
of children. A direct influence model holds that stressful life events play
a direct causal role in offspring maladjustment by overtaxing the developing
child's coping resources and skills. As a result, the child may adopt
maladaptive coping responses, and poor self-esteem. An alternative model holds
that stressful life events have little or no direct influence on children's
adjustment; that it is not stressful events per se, but characteristics
of parents that engender and/or exacerbate those events, that give rise to
maladjustment and psychopathology in offspring. In this research, data will
be examined to relate to these models.
Methods
The analyses are based on data from families participating in the NIMH
Childrearing Project (ZOl MH 021A4), which includes children of parents with
and without a history of affective disorder. Assessments of parental
psychopathology are based primarily on psychiatric (SADS-L) interviews with
each parent; ratings of maternal and paternal competence/functioning will be
derived from direct observation and interview data. Data on stressful life
events to which the children have been exposed are obtained in intensive,
semi-structured interviews with mothers. Assessments of children's functioning
are based on psychiatric interviews, direct observations of their interactions
with a same-age peer in our laboratory apartment, and independent reports
from their parents and teachers..
Stressful life events are classified by type; direct focus of event; long- and
short-terra stress-value; control ability timing relative to the child's
age; the roles played by each family member in either causing, exacerbating,
or lessening the negative impact of each event. Procedures for coding the
interviews ensure that the estimated stressf ulness of events is not contaminated
by the coders' knowledge of the child's functioning.
Proposed Course
To date, 91 mothers and their children have been observed and interviewed in
follow-up visits to our laboratory apartment. Life event interviews with
mothers have been processed to identify stressful events across these families.
A coding system has been developed for the detailed scoring of events, and has
been tested on a random sample of event summaries. Coding is in progress, and
will be followed by the formal scoring of events. Analyses will then be
conducted, and manuscripts will be prepared for publication during this year.
Significance to Biomedical Research
Although the link between stressful life events and psychopathology has been
demonstrated repeatedly over the years, the mechanisms through which they
are related, and their interrelated impact on children, are not well
understood. This research will contribute to our understanding of these links.
618
ZOl MH 02380-01 LDP
Publications
Richters, J. E. Exposure to parental psychopathology and children's adjustment:
A developmental analysis. In Hahlweg, K. , and Goldstein, M. (Eds.), Family
Interaction Research and Psychopathology. New York, Plenum, in press.
619
PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
i
ZOl MH 02381-01 LDP
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line between the tmrders.)
Functioning of Depressed Mothers Within and Between Episodes
PRINCIPAL INVESTIGATOR (List other professional personnel Oe/o* the Principal Investigator) (Name, title, latioratory, and institute affiliation)
PI: J. E. Richters Research Psychologist LDP/NIMH
Other: M. Radke-Yarrow Chief LDP/NIMH
COOPERATING UNITS (If any)
None
LAB/BRANCH
Laboratory of Developmental Psychology
INSTITUTE AND LOCATION
National Institute of Mental Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.77
PROFESSIONAL:
.30
,47
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects D (b) Human tissues □ (c) Neither
^ (a1) Minors
E (a2) Interviews
SUMMARY OF WORK (Use Standard unreduced type. Oo not exceed the space provided.)
What are the characteristic differences between depressed and non-depressed
women in their interactions with and around their children? Are maternal
child-related behavioral deficits characteristic of women with a history of
depression, or are difficulties present only when mothers are experiencing
an acute episode of depression? More broadly, are there certain patterns
of aberrant child-related behaviors that wax and wane with depressive
episodes, and others that reflect more or less enduring behavioral charac-
teristics of depressed mothers? These questions are important to the study
of depressed women as mothers and their influence on children.
The NIMH Childrearing Project is in a unique position to explore this
issue, by comparing maternal characteristics of two groups of mothers with
a history of affective disorder: those who are within and those who are
between episodes of depression at the time of their participation in our
laboratory apartment at follow-up. These groups of mothers will be compared
on symptom-related behaviors and interactive behaviors, including patterns
of 1) eliciting compliance and cooperation from their children, 2) monitoring
of and responsiveness to their children's need states, 3) displays of affect
such as anger and affection, 4) patterns of content and topography in verbal
interactions, and 5) methods of resolving conflict between offspring siblings.
Parallel analyses will be conducted using self-reported mood as a basis for
regrouping mothers; this will allow us to address more general state-trait
questions beyond those that are specific to depressive episodes, per se.
621
PHS 6040 (Rev. 1/84) 0PO»i4-eie
ZOl MH 02381-01 LDP
Project Description
What are the characteristic differences between depressed and non-depressed
women in their interactions with and around their children? Are maternal
child-related behavioral deficits characteristic of women with a history of
depression, or are difficulties present only when mothers are experiencing
an acute episode of depression? More broadly, are there certain patterns
of aberrant child-related behaviors that wax and wane with depressive episodes,
and others that reflect more or less enduring behavioral characteristics of
depressed mothers?
These questions are important to the study of depressed women as mothers and
of their influence on children. The NIMH childrearing Project (ZOl MH-02144),
is in a unique position to explore this issue, by comparing the maternal
characteristics of mothers with no history of affective disorder, with two
groups of mothers with a history of affective disorder: those who are in an
episode of depression and those who are between episodes of depression at the
time of assessments.
Methods
Analyses will be based on data drawn from families participating in the NIMH
Childrearing Project (ZOl MH-0214A), which includes children of parents with
and without a history of affective disorder. Assessments of parental psycho-
pathology are based on psychiatric (SADS-L) interviews with each parent on
the day they are observed in the laboratory apartment. To date, 85 mothers
and their children have participated in the follow-up phase of this study,
including 28 mothers with no history of affective disorder, 33 mothers with
a history of affective disorder who were not experiencing a depressive episode
at the time of assessment, and 24 mothers with a history of affective disorder
who were currently experiencing a depressive episode. Mothers and their
children were observed in a variety of situations designed to approximate
natural rearing conditions . Mothers will be compared on a selection of
symptom-related behaviors, as well as on a range of interactive behaviors
that are the focus of related studies on this project, including patterns of
1) eliciting compliance and cooperation from their children (ZOl MH-02132),
2) monitoring of and responsiveness to their children's need states (ZOl
MH-02370), 3) displays of affect such as anger and affection (ZOl MH-02207),
4) patterns of content and topography in their verbal interactions (ZOl
MH-02220), and 5) methods of resolving conflict between offspring siblings
(ZOl MH-02169).
In addition to comparing the previously defined groups of mothers on these
characteristics, we will conduct parallel sets of analyses by re-grouping all
mothers on the basis of their self-reported (using a standardized battery)
moods prior to entering the apartment. These additional analyses will allow
us to address more general state-trait questions beyond those that are specific
to depressive episodes per se.
622
ZOl MH 02381-01 LDP
Proposed Course
Coding for the maternal behaviors described above is in varying stages of
completion. Initial reports may be prepared for publication as early as
Spring 1988.
Significance to Biomedical Research
The questions raised and addressed in this research concerning the state-trait
dependence of maternal behaviors are of crucial importance to an understanding
of the links between maternal depression and offspring adjustment.
Publications
Richters, J.E. and Weintraub, S. Beyond Diathesis: Toward and understanding
of high-risk environments. In Rolf, J., Hasten, A., Cicchetti, D. ,
Nuechterlein, K. , and Weintraub, S. , (Eds.), Risk and Protective Factors in the
Development of Psychopathology. New York, Cambridge University Press, in press.
623
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00478-31 LN
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one /ine t>etween the borders.)
Neural mechanisms of cognitive memory and habit formation
PRINCIPAL INVESTIGATOR (List other prolessiortal personnel below the Principal Investigator.) (Name, title, laboratory, and institute atllliation)
PI: M. Mishkin Chief LN NIMH
Others: E.A. Murray Senior Staff Fellow LN NIMH
J. Bachevalier Visiting Scientist LN NIMH
R.C. Saunders Staff Fellow LN NIMH
D.P. Friedman Project Officer NRB NIDA
COOPERATING UNITS (if any)
National Institute on Drug Abuse
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
9.0
PROFESSIONAL:
2.5
6.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues 13 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Every sensory modality in the macaque is served by a series of cortical stations,
each of which processes the sensory signal in turn. Signals in the later
stations, located in the anterior temporo-insular cortex, can activate a circuit
that runs through the limbic system to the modulatory neurochemical systems (e.g.
cholinergic, noradrenergic, etc.) and back to the sensory cortical stations. We
have proposed that as a result of the action of this circuit on neurochemical
release in sensory cortex,' some of the neurons whose signals have just represented
the sensory stimulus become linked together in a cell assembly that serves as the
stored representation of that stimulus. Recognition, say of an object, occurs
when an assembly formed on a first presentation of the object is reactivated by
its re-presentation on a second occasion. Also, once formed, that assembly can be
linked to assemblies representing other stimuli and other events, such as a food
reward or a location, thereby investing the recognized object with meaning. The
linkage involved in object-reward association appears to be mediated mainly by a
limbo-neurochemical circuit running through the amygdala, the medial dorsal
thalamic nucleus, orbital frontal cortex, and the basal nucleus of Meynert.
Similarly, the linkage involved in object-place association seems to be mediated
mainly by a second, parallel limbo-neurochemical circuit running through the
hippocampus , the anterior thalamic nuclei, cingulate cortex, and the medial septal
and diagonal band nuclei. Each of these circuits has reciprocal connections with
one pair or the other of the assemblies described above. Thus, if these circuits
have been activated, the sight of the object on a second occasion can lead not
only to its recognition but also to recall of both the food reward and the spatial
location with which the object had been associated. Recognition and recall are
two forms of cognitive memory, both of which can be distinguished from habit
formation . The latter form of learning involves stimulus-response association
specifically, and we liave proposed that such learning depends largely on
interactions between the cerebral cor te.x,[<jnd the basal ganglia,
PHS 6040 (Rev 1/84)
CPO «l4>Slt
ZOl MH 00478-31 LN
PROJECT DESCRIPTION:
The objective of the studies in this project is to delineate the neural system
underlying memory formation in the monkey and to differentiate it from the
neural system that underlies habit formation. The methods used include
behavioral analyses of the effects of selective cerebral ablations and
disconnections combined with anatomical analyses of functional neural
pathways. The rationale and design of the studies are often based directly on
information derived from other projects in this laboratory, many of which deal
with the pathways for, and the mechanisms of, stimulus processing and
encoding. The results from these and other projects suggest that the sensory
system for each modality is composed of two hierarchically organized
corticocortical pathways, one directed ventrally to the temporal lobe limbic
system and concerned with object perception, and the other directed dorsally
to the frontal lobe motor system and concerned with spatial perception. The
ultimate goal of this project is to determine how object and spatial
perceptions in the different modalities are formed into memories, how these
different memories are associated with each other, how they evoke emotions and
motor acts, and how they lead not only to these cognitive events but also to
habit formation. Our progress in understanding each of these processes will
be described in turn.
(1) Recognition memory
Previous work has indicated that visual recognition memory (assessed by
delayed nonmatching-to-sample with trial-unique objects) is mediated by a
cortico-limbo-thalamic system composed of two largely separate circuits
arranged in parallel. One of these circuits consists of the amygdala,
amygdalofugal pathways, and the magnocellular portion of the mediodorsal
nucleus (MDmc), and the other consists of the hippocampus, fornix, and
anterior nuclear complex of the thalamus (Ant N) . The reason for believing
that these two circuits operate in parallel is that damage to the amygdalar
and hippocampal circuits at each stage in the system (i.e. medial temporal
lobe, limbo-thalamic pathways, and medial thalamus) causes a severe loss in
recognition memory, but only when the two circuits are damaged in
combination. Damage to just one of the two circuits leads to only mild
recognition deficits, suggesting that either circuit can compensate for the
other as far as recognition is concerned. Recent results indicate that the
orbital frontal and anterior cingulate cortical zones, which are related
anatomically to the amygdalar and hippocampal circuits, respectively, must
also be removed in combination to produce a severe recognition deficit.
Removal of either the orbital frontal or anterior cingulate cortex alone, or
of prefrontal tissue outside this zone, produces little impairment. Thus, the
ventromedial prefrontal region appears to constitute yet another stage in the
limbic memory system. In an attempt to determine if the different stages have
redundant memory functions, we have begun an experiment that examines the
effects on recognition memory of combined ablations of tissue at different
stages within a circuit. Preliminary data suggest that combined hippocampal
and cingulate cortical ablation leads to more severe memory deficits than
ablation of the hippocampus alone. Likewise, combined ablation of the
amygdala and orbital frontal cortex leads to a more severe deficit than
ablation of the amygdala alone. These new results suggest that the prefrontal
626
ZOl MH 00478-31 LN
cortical stage of each limbic circuit receives a secondary input from the
other circuit.
Recent anatomical evidence has indicated that the bed nucleus of the stria
terminalis (BNST) occupies an anatomical position within the amygdalar system
that is directly comparable to that occupied by the mamillary bodies within
the hippocampal system. That is, just as the hippocampal formation projects
to the Ant N both directly and indirectly via the mamillary bodies, the
amygdala projects to MDmc both directly and indirectly via the BNST. This
anatomical correspondence suggests the possibility that the BNST and mamillary
bodies serve analogous functions within the two limbic memory circuits. Since
previous work has demonstrated that mamillary body lesions alone produce only
a mild recognition memory impairment, we are now testing whether combined
damage to the mamillary bodies and BNST will produce a more severe effect.
Since fornix transection has been shown to have behavioral effects on
recognition memory similar to those of mamillary body lesions, and since this
procedure disconnects the mammillary bodies from their hippocampal inputs,
monkeys were prepared with BNST lesions plus transection of the fornix.
Preliminary results indicate that whereas monkeys with BNST lesions alone or
fornix transection alone are only mildly impaired in object recognition
memory, monkeys with the combined BNST ablation and transection of the fornix
are more severely impaired. These results are consistent with the idea that
the BNST participates in recognition memory and that its function in the
amygdalo-thalamic circuit could be comparable to that of the mamillary bodies
in the hippocampo-thalamic circuit.
Investigations of recognition memory in monkeys have largely been confined to
vision. The one exception was an experiment from this laboratory showing that
temporal lobe limbic structures are just as important for tactile as for
visual recognition. Experiments in another project (MH 02037) have
demonstrated that a cortical tactile processing pathway runs from the
postcentral somatosensory cortex to the second somatosensory area, SIX, from
SIX to the insular cortex, and, finally, from the insular cortex to the medial
temporal region. These results suggest that the insular cortex could play a
role in tactile recognition analogous to that played by the inferior temporal
cortex (area TE) in visual recognition. We are therefore examining the
effects on recognition of resecting the insular cortex. Preliminary results
indicate that monkeys with bilateral ablations of this cortex are severely
impaired in tactile but not visual recognition of objects. Thus, the results
suggest that the somatosensory system, like the visual system, interacts with
the limbic system through a series of modality-specific areas. Besides
pursuing the tactile recognition studies, we are continuing to explore
behavioral methods for evaluating recognition memory in audition so that we
can extend our behavioral investigations to this modality.
Xn addition to examining memory of stimulus quality in each sensory mode, we
are interested in studying spatial memory. To this end, we have recently
trained monkeys on spatial delayed nonmatching-to-sample in a T-maze, a task
that evaluates recency memory for place. The results indicate that monkeys
with fornix transections are severely impaired, and that monkeys with
cingulate cortical ablations are mildly impaired relative to intact controls.
In addition to indicating that the hippocampal system in monkeys is important
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for this kind of spatial memory, the experiment helps establish a firm link
between primate and rodent memory studies, in that this particular spatial
memory task, like most such tasks employed with rodents but unlike most
employed with monkeys, involves locomotor responses.
(2) Anatomy of recognition memory
Although we have demonstrated the importance of cortical inputs to the limbic
system in object recognition memory, we had not made as much progress in
demonstrating the anatomical substrates responsible for the limbic feedback to
the cortex that allows memories to be stored. A major hypothesis has been
that the basal forebrain cholinergic system plays an important role in this
process. But the relations of the basal forebrain to the medial temporal
limbic areas and to the midline thalamus had not been completely described.
Recent studies have addressed these issues.
Injections of tritiated amino acids were made into the hippocampal formation
and amygdaloid complex in order to trace their inputs to the basal forebrain,
with the following results. The hippocampal formation projects densely to the
medial (Chl), lateral, and dorsal septum, and to the Ch2 region. There were
relatively sparse projections to restricted portions of Ch4 as well.
Experiments in which the fornix was transected demonstrated that all of these
projections ran through the fornix. The caudal hippocampus projects to the
more medial septum, whereas the rostral hippocampus projects to the more
lateral septum, although there is considerable overlap in the projections from
these two regions. The regions of the basal forebrain and septum receiving
hippocampal inputs project back to the hippocampus.
The projections from the amygdala do not overlap those from the hippocampus,
but terminate instead in the Ch3 and Ch4 fields of the substantia innominata.
In fact, the aiaygdala represents one of the major inputs to Ch4, which
provides the major portion of the cholinergic input to the cerebral cortex.
The amygdalar efferents arise from the medial, medial basal, magnocellular
portion of the accessory basal, and the central nuclei. It is the more dorsal
and medial portions of this group that receive the largest number of intrinsic
amygdalar connections. The lateral nucleus, which is the site of termination
of afferents from the major sensory systems, provides almost no outputs to the
basal forebrain.
Because lesions of the medial thalamus lead to deficits in recognition memory
like those caused by medial temporal damage, and because the medial and
midline thalamic nuclei receive inputs from the hippocampal formation and
amygdaloid complex, the structures occupying this region appear to be
intimately involved in memory formation. To examine medial thalamic outputs,
we initially made large injections of tritiated amino acids that filled the
entire rostrocaudal and dorsoventral extent of the thalamic midline.
Subsequently we made smaller isotope injections into some of the individual
nuclei along the midline, including nucleus reuniens (Re), the magnocellular
division of the medial dorsal nucleus (Mdmc), and the nuclei of the anterior
group. Because of the expected sparse nature of these projections,
autoradiograms were exposed for up to a year before they were developed and
analyzed .
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The large injections revealed an extensive cortical projection system that ran
in long bands along the outer half of layer I and commonly crossed the borders
between cortical fields. These projections were seen in ventrolateral,
orbital, and subcallosal regions of prefrontal cortex, virtually the entire
extent of the temporal lobe ventral to the superior temporal gyrus, the visual
fields of the occipital and parietal lobes, the entire extent of the insula,
much of the frontoparietal operculum including gustatory and somatic fields,
and the auditory areas that border the ventral insula. In addition, cortical
limbic areas, including the cingulate gyrus, entorhinal and perirhinal areas,
the subicular fields, and the hippocampus proper, were labeled. In addition,
there were dense patches of label in the Ch4 fields of the basal forebrain.
Injections into MDmc led to dense label in layer IV of orbital cortex and some
patches of label in the overlying layer I as well. The injections into Re
produced only layer I label in subcallosal and orbital cortex.. Projections
originating in the anterior nuclei and the medially adjacent nuclei of the
midline led to label in both layers I and III in the subcallosal region.
The findings supply evidence for a widespread system of midline thalamic
projections to layer I of higher-order sensory and limbic cortex. These layer
I projections could well form part of a cortico-limbo-thalamo-cortical
feedback circuit involved in the formation of new memories.
(3) Associative memory
Our earlier work suggested that although the amygdalar and hippocampal systems
contribute equally to recognition memory, they have selective roles in
associative memory. The amygdala, but not the hippocampus, appears to be
important for the association of object qualities from different sensory
modalities. In contrast, the hippocampus, but not the amygdala, appears to be
important for the association of object quality and place. To test these
generalizations, monkeys are being trained on a visual-visual associative
memory task. Preliminary results indicate that, compared to
hippocarapectomized monkeys, those with amygdalar ablations are retarded in
relearning a preoperatively acquired set of visual-visual associations.
Interestingly, however, neither operated group appears to be impaired in
learning new sets. The critical question now is whether monkeys with combined
ablations of the amygdala and hippocampus will be impaired in learning new
sets. If so, it would suggest that monkeys with limbic lesions resemble
amnesic humans, who cannot learn new arbitrary associations no matter how much
training they receive. Alternatively, if monkeys with the combined ablation
can learn new sets, it would show that some nonlimbic mechanism can contribute
to intramodal stimulus-stimulus learning, a mechanism that might be related to
the priming observed in amnesic humans.
We have also begun to examine the role of the hippocampal system in
conditional spatial learning, where which object of two is positive depends on
their spatial location. In one such task, monkeys were required to use the
location of the objects within the room as the conditional cue guiding their
choice, whereas in another they were required to use the location of the
objects relative to themsclve; to guide their choice. In both tasks, the
monkeys were moved passively to the test site, thus eliminating "active
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movement through space" as a possible cue. Interestingly, monkeys with fornix
transection were not impaired on either task, and monkeys with ablations of
cingulate cortex were mildly and transiently impaired on the first but not the
second. These experiments lay the groundwork for an analysis of spatial
memory in normal monkeys as well as the role of limbic structures in such
memory.
(4) Habit Formation
Whereas monkeys with limbic lesions generally exhibit poor memory on
recognition and associative memory tasks they are able to learn certain types
of object discriminations at a normal rate. For example, we have found that
such monkeys can learn as rapidly as normal control animals to discriminate 20
pairs of objects presented concurrently, even with intertrial intervals
lasting 24 hours. We have applied the label "habit formation" to this and
other examples of preserved learning ability following limbic-system lesions.
Normal monkeys trained on the 24-hr concurrent discrimination test with
several successive sets of objects learn the later ones faster than the
earlier ones. To investigate the basis of this phenomenon, we are examining
whether it is affected by limbic lesions. The results show that monkeys with
combined amygdaloid and hippocampal ablations, like normal monkeys, learn the
later sets faster than the earlier ones, suggesting that the phenomenon of
learning set formation is supported by nonlimbic mechanisms.
In a separate LN project (MH 02039), we have been attempting to examine the
role of the neostriatum in 24-hr concurrent learning by disrupting the entire
nigro-striatal dopaminergic system with the selective neurotoxin MPTP.
Because preliminary results with two different regimens of MPTP administration
suggest that there is little effect, if any, at doses that do not also produce
motor impairments, we are planning to test the effects of damaging selectively
those portions of the neostriatum to which the cortical visual system projects
(see LN project MH 02033).
Another behavioral paradigm that may provide a measure of the ability to
acquire habits is delayed nonmatching-to-sample (DNMS). At very short delays,
monkeys with limbic lesions can relearn the delayed nonmatching rule to a high
level of accuracy. It is unlikely that the same temporo-neostriatal pathway
presumed to be responsible for simple visual discrimination habits could
mediate DNMS, however, because in DNMS the monkey must avoid the previously
baited sample in favor of a novel object (i.e. it must learn a
counter-reinforcement win-shift rule). One nonlimbic circuit that might
mediate such complex rule learning is a temporo-pref ronto-neostriatal circuit,
which could act both to suppress the win-stay rule that is normally mediated
automatically by the direct temporo-neostriatal pathway and to mediate in its
place the acquisition of the higher-order win-shift rule. To test this
possibility, we are investigating whether the combination of inferior
prefrontal and limbic ablations interferes with performance on DNMS even at
very short delays. If animals with such lesions were unable to relearn the
nonmatching rule, it would open up a new chapter in frontal lobe research,
namely, the role of the prefrontal cortex in the formation of complex habits.
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SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
In the process of investigating the role of various temporal lobe structures
in the visual memory of monkeys, we obtained a result that is particularly
exciting because it appears to solve the long-standing puzzle concerning the
neuropathology underlying the syndrome of global anterograde amnesia in man.
This syndrome, which is characterized by a profound inability to remember new
experiences or acquire new information, has been attributed in the clinical
literature to destruction of the hippocampus. Yet, attempts to duplicate this
syndrome in monkeys by removal of the hippocampus alone have largely failed.
What we have found in our studies is that if damage to the hippocampus is
combined with damage to the amygdala then a profound memory loss does ensue.
The discovery has not only resolved the discrepancy between clinical and
experimental findings in nonhuman primates, but has also provided new insight
into the neural substrates of memory. Specifically, it has led to the
development of a hierarchical model of recognition and associative memory
involving a cortico-limbo-neurochemical loop that may well serve as the
foundation for all cognitive processes beyond perception, including thought.
As we gain further understanding of the memory system, and how it differs from
the noncognitive system for habit formation, we will inevitably gain a better
understanding of thought and its breakdown in normal and abnormal behavior.
PROPOSED COURSE OF RESEARCH:
Having found severe object recognition losses in both vision and touch after
lesions of the limbic system, we shall continue our attempts to devise tests
of auditory recognition and visual spatial recognition, with the aim of
determining whether the limbic system is indeed critical for recognition in
all perceptual modalities. Also, further attempts will be made to
differentiate between amygdalar and hippocampal contributions to associative
memory, and we shall test whether any functional distinctions that apply to
these temporal lobe structures are carried further through the thalamic,
prefrontal, and neurochemical segments of the two limbic circuits. In
addition, we shall continue our exploration of the neural basis of habit
formation, with particular attention initially to the neostriatal and
prefrontal targets of the occipitotemporal visual system.
PUBLICATIONS:
Aggleton, J. P. A description of the amygdalo-hippocampal interconnections in
the macaque monkey. Exp. Brain Res. 64: 515-526, 1986.
Aggleton, J. P., Friedman, D.P., and Mishkin, M. A comparison between the
connections of the amygdala and hippocampus, with the basal forebrain in the
macaque. Exp. Brain Res, (in press)
Mishkin, M. and Appenzeller, T. The anatomy of memory. S c i . Ame r . 255:
80-89, 1987.
Mishkin, M. and Phillips, R.R. A cortico-limbic memory path revealed through
its disconnection. In C. Trevarthen (Ed.): Brain Circuits and Functions of
the Mind: Festschrift for Roger Wilcott Sperry. Cambridge University Press,
New York, (in press)
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Murray, E.A. and Mishkin, M. Experimental studies of memory in monkeys:
implications for understanding human memory disorders. National Forum, Spring
1987, p. 33-37.
Nelson, R.B., Friedman, D.P., O'Neill, J.B., Mishkin, M., and Routtenberg, A.
Gradients of protein kinase C substrate phosphorylation in primate visual
system peak in visual memory storage areas. Brain Res, (in press)
Phillips, R.R., Malamut, B.L. , Bachevalier, J., and Mishkin, M. Dissociation
of the effects of inferior temporal and limbic lesions on object
discrimination learning with 24-hour intertrial intervals. Behav. Brain Res,
(in press)
Saunders, R.C. and Rosene, D.L. A comparison of the efferent connections of
the amygdala and the hippocampus. I. Convergence in the entorhinal,
prorhinal and perirhinal cortex. J. Comp. Neurol, (in press)
Saunders, R.C, Rosene, D.L., and Van Hoesen, G.W. A comparison of the
efferent connections of the amygdala and the hippocampus. II. Reciprocal and
non-reciprocal connections. J. Comp. Neurol, (in press)
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DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02032-11 LN
PERIOD COVERED
October 1, 1986 through September 30,
1987
TITLE OF PROJECT (80 characters or less. Title must tit on one /me beftveen the borders.)
Neural coding of visual stimuli in the awake monkey
PRINCIPAL INVESTIGATOR (List ottyer professional personnel txlow the Principal Investigator.) (Name, title, laboratory, and institute atfiliation)
PI: B.J. Richmond Senior Surgeon LN NIMH
Others: L. Optican
M. Mishkin
Senior Staff Fellow
Chief
LSR NEI
LN NIMH
COOPERATING UNITS (it any)
Laboratory of Sensorimotor Research, NEI
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIK. Bethesda, Maryland 20892
TOTAL MAN- YEARS:
2.0
PROFESSIONAL:
1.0
1.0
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
□ (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Single neurons were recorded from the lateral geniculate nucleus and primary
visual cortex, the first two extraretinal stages of visual processing, and
inferior temporal cortex, the last visual cortical processing station, to
study the mechanisms underlying visual perception. Neurons in all three
regions showed different temporal response patterns to different visual
stimulus patterns. When these neurons were analyzed as communication channels
carrying information about visual stimuli in their responses, the response
patterns seen could only be represented as the sum of several (3-6)
simultaneous, independent patterns of activity. Three of these activity
patterns were analyzed as a temporal code, and the information contained in
them was compared to the information conveyed by the number of action
potentials, the usual measure of neuronal response; there was twice as much
information available in the temporal code. This showed that each response
can be considered the sum of activity from several independent channels, with
each channel acting as a spatial-to-temporal filter. Traditionally, it has
been thought that information about multiple stimulus parameters, such as
luminance, pattern, and duration of presentation, must be confounded in the
neuronal responses. However, based on this multiplex-filter hypothesis, a new
analysis of the primary visual cortex responses led to the discovery that
information about each of these parameters is carried separately in the
response. This strongly suggests that a consistent neural code exists.
A geometrical analysis of these data shows a potential structure for this
i code. When a 3-dimensional space is used to represent the responses, the
responses to a single pattern appear to lie in a plane regardless of luminance
or duration. For a single neuron's response, the planes for different
patterns are frequently separable. The equations for these planes describe a
neural code.
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PROJECT DESCRIPTION:
Objectives :
The ability to perceive, recognize, and discriminate visual patterns requires
the cooperative function of a sequentially connected system of cortical brain
regions extending from primary visual cortex through inferior temporal
cortex. The functionality of these regions arises from the properties of the
single neurons in them. Thus, to understand how visual perception occurs, we
must learn how information is encoded by the neurons in these successive
stages of processing. One clear consequence of such understanding would be
the ability both to predict the single neuronal signals to arbitrarily
constructed stimuli, and to decode the responses to unknown stimuli. We have
been recording single neurons in several regions of the visual pathway with
the goal of developing a quantitative model of neuronal function. Such a
model should simulate the activity of single visual system neurons in response
to any arbitrary stimulus conditions. The development of such a model would
confirm our understanding of the functionality of these neurons, and,
therefore, would be a major step toward understanding the functional role of
single neurons in the networks that underlie higher visual function such as
perception and memory.
Major findings:
We have developed an unbiased approach to identify and quantify the
message-carrying features of single neuronal spike trains. In this approach
single neurons are considered to be communication channels that convey
messages about visual stimuli. Adoption of this approach has allowed us to
identify and quantify the stimulus related information carried by single
neurons through the use of techniques from multivariate statistics. Shannon's
information theory, and signal processing.
To apply this approach, both the inputs (visual stimuli in this case) and the
outputs (neuronal responses) had to be precisely described. For inputs, we
constructed a set of 2-dimensional black and white stimuli from a set of basic
signal elements (Walsh functions) that can be used to represent any picture.
The responses were described in terms of an optimal (in the mean squared error
sense) set of statistically derived, independent patterns of activity, the
principal components. This set of 2-dimensional black and white stimuli was
then used to stimulate neurons in both inferior temporal and primary visual
cortex. For single neurons in both regions, a statistical analysis we
developed showed that three or more principal components were related to the
stimuli presented. Application of Shannon's information theory showed that
the amount of information transmitted about the stimulus was twice as great
when three principal components were used to represent the responses as when
the spike count was used to represent the responses. Thus, the
stimulus-related responses of these neurons are multidimensional, i.e.,
several parameters are required to describe them.
From this, we inferred that each neuron can be viewed as a small (3) number of
simultaneously active spatial-to-temporal filters. In this multiplex-filter
hypothesis, each filter gives rise to a temporally modulated message that
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corresponds to a different aspect of the visual pattern. The output of the
filters, represented by the principal components, are then multiplexed onto
the spike train. This multidimensional description is a significant advance
over less rich, unidimensional descriptions of neuronal responses. Neither
static receptive field models nor response strength measures can correctly
predict the temporally modulated responses of a neuron. However, a model
based on this multiplex-filter hypothesis has predicted the temporally
modulated responses of striate cortex complex cells to arbitrarily constructed
black and white patterns. Thus, knowledge of the multidimensional nature of
the responses allowed derivation of a predictive model of stimulus-related
neuronal responses.
Two issues that arise as a consequence of this hypothesis have been
investigated during the past year. First, can the responses be decoded? To
do this a neural code and its meaning must be identified. Our strategy has
been to study the responses of neurons with stimuli that varied along several
dimensions. Second, what do the spatial parts of the spatial-to-temporal
filters look like?
The experiments that led to the multiplex-filter hypothesis were carried out
using stimuli for which the pattern varied while other parameters remained
unchanged. However, the strength of a neuron's response is modulated by many
different stimulus dimensions, e.g., color, shape, and luminance. According
to a common view of neuronal function, the strength of a neuron's response
represents how closely the stimulus matches the receptive field's
characteristics, e.g., orientation or color. Thus, if response strength were
the only parameter a neuron could use to encode information, different
stimulus features would be confounded by individual neurons.
Under the assumption that information is carried in a single parameter, the
response strength, information about multiple features can be unconfounded
only by looking at the distribution of activity over a large population of
neurons, although no explicit solution to this problem has been shown. In
light of our finding that stimulus features modulate not only the number but
also the temporal distribution of spikes in a neuron's response, it is
possible that information about multiple features may not be confounded if the
multivariate nature of the response is taken into account.
To study whether the encoding of information about different stimulus features
might be separably encoded in neuronal responses, single neurons were recorded
from primary visual cortex of awake, trained rhesus monkeys. While the
monkeys fixated, a complete set of 16 one-dimensional, optimally-oriented
Walsh patterns were flashed on the receptive fields of the neurons one at a
time. Each pattern was presented at each of four luminance combinations
(luminance range: 0.004 - 5.0 f t-lamberts ) , and flashed for each of five
durations (32 - 256 ms ) . All 320 stimulus combinations were presented to
eight cells. The principal components were extracted from 320 ms segments of
the densities, and the information transmitted about each of the different
stimulus parameters was calculated.
Transmitted information measures the discriminability among inputs given an
output for a pair of input and output codes. Information is low when inputs
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are confounded among outputs. We related four stimulus input codes to each of
two response output codes. The input codes were: 1) the four luminances
alone, 2) the five durations alone, 3) the 16 patterns alone, and 4) the 320
combinations of these features. The two output codes were: 1) the
coefficients of the first three principal components (T3) and 2) the number of
spikes in the response (Ts).
all 320 pattern duration luminance
T3(bits) 1.07+.08(SE) 0.32+.04 0.20+.05 0.19+.02
TsCbits 0.46+,04 0.15+.02 0.06+.02 0.05+.01
The information encoded by three principal components, T3, was two to four
times greater than the information encoded in the spike count, Ts , for all
input codes. Ts for pattern was much greater than that for duration or
luminance, whereas T3 had approximately the same magnitude for all three
features. Strikingly, the transmitted information based on the input code of
all 320 stimulus combinations (1.07 bits) was substantially greater than that
predicted by the sum of the information from the individual codes (0.71 bits).
Thus, the codes for the three stimulus features were synergistic : more was
known about the stimulus when a code that simultaneously accounted for all
three features was used.
Similar synergy was also observed in another seven cells tested with 16
patterns and 7 luminances at one duration. This synergism implies that the
different stimulus features were not confounded by the neuron. For such
synergy to occur, there must be "a consistent, unconfounded , mapping from
stimulus features to neuronal responses. However, information theory does not
require that the neural code be interpretable in terms of these features.
To search for a description of the encoding rules, i.e., a neural code, the
responses of the 15 striate cortical neurons described above were analyzed
further. The principal components used to quantify the responses are
orthogonal patterns of temporal activity that define the axes of a
multidimensional space. Responses with different temporal waveforms have
different amounts of each principal component in them, and, therefore, they
map to different points in this principal component space.
In a plane defined by two principal components, no relation between stimulus
features and neuronal responses was apparent. However, in a space defined bv
the first three principal components, the responses elicited by an individual
Walsh pattern, irrespective of duration or luminance, appeared to lie near a
plane. Thus, we approximated the responses to each of the 16 patterns by
their best-fit planes. The average residual variance per plane was 4.6% + 4.1
(SD, n = 112) for the 7 neurons tested with 7 luminances, and 12.0% + 5.2 (n =
128) for the 8 neurons tested with 4 luminances and 5 durations. Since the
coefficients of the principal components are uncorrelated , if the responses to
a given pattern had not been well approximated by a plane, the residual
variance would have been close to 33%.
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In any one neuron's principal component space, many of the planes representing
the 16 stimulus patterns appeared easily dif ferentiable. This geometrical
structure demonstrates that the generation of neuronal responses obeys certain
rules, which form an intrinsic temporal neural code for visual features. A
response could be decoded to determine the stimulus pattern irrespective of
duration or luminance if the plane into which the response falls could be
ascertained. Information about duration and luminance would then be encoded
relative to that plane. Since three points determine a plane, such a decoding
scheme may require as few as three complementary neurons sharing related codes.
In another set of recently begun experiments, data have been collected to
determine the spatial part of the spatial-to-temporal filters for lateral
geniculate nucleus neurons. The first station of the visual system outside of
the retina is being investigated for this project because it has been
extensively studied and its properties from conventional experiments are well
characterized. These neurons have been shown over the past 30 years to have
receptive field properties that have excitatory centers with inhibitory
surrounds. Our analysis reveals that, even in this early stage of visual
processing, information is carried by more than one temporally modulated
message. Thus, there appears to be previously unsuspected richness of
information in the temporal modulation of the responses. From these data,
collected in collaboration with members of the NEI's Laboratory of
Sensorimotor Research, we have developed a technique to estimate the spatial
distribution of the filters that give rise to each of the temporally modulated
messages. These estimates seen thus far suggest that the classical
center-surround mechanism may arise from the combined influences of two
filters. The structures of the estimated filters suggest that these neurons
may encode a nonparametric estimate of the visual scene, possibly its
luminance profile, and the first derivative of the luminance profile. If this
can be confirmed, it would suggest that lateral geniculate neurons
simultaneously encode information about the luminance and luminance gradient
of the scenes that fall within their receptive fields.
Our results imply a new functional role for neurons in the visual system.
Information about stimulus features is conveyed by individual neurons through
multiple messages carried by a temporally modulated code. Consolidation of
local messages to determine global properties of images may be accomplished
through compilation of many temporally encoded messages. Processing of
information in visual areas may consist not so much in altering the
distribution of active elements as in transforming temporally modulated
messages. We suggest that the hierarchical organization of feature
abstraction supposed for the multiple visual areas is better replaced by a
process of successive stages of spatial-to-temporal filtering that change the
emphasis of the visual features but never confound or ignore information.
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
Disorders of attention, perception, and memory accompany many psychiatric and
neurological disorders. Tliis project studies how information is encoded and
transmitted. The knowledge gained will ultimately aid in the design of
strat:egies both for more effective palliative treatment of cognitive deficits
and for restitution of cognitive function.
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PROPOSED COURSE OF RESEARCH:
Discovering that the responses of visual system neurons are multidimensional
suggests a completely new conceptional framework in which to investigate
neuronal function. This led to the discovery that information about multiple
stimulus features may not be confounded by single neurons, a result with
important, even revolutionary consequences. One presumed reason for the huge
number of single neurons has been the necessity to unconfound stimulus
features. If this need not be done on the global scale previously envisioned,
what are all these neurons doing? This issue is critical.
Since we have evidence that a neural code may exist, and we have seen a .
possible structure for it, we will pursue its delineation. The properties of
the code should give clues about the functions performed by the neurons.
Also, the structures of the spatial filters seen in the lateral geniculate
nucleus have already given some new ideas about the properties of encoded
information there. Both these issues are being pursued.
New experiments are planned to make multiple, simultaneous single neuronal
recordings. The responses from these single neurons will be related to each
other through use of recent extensions to methods of signal identification.
With these methods to extend our techniques, it should be possible relatively
rapidly to develop models that describe the roles of single neurons as
components of larger networks. These studies should yield a better
understanding of the information transmission mechanisms used for cognitive
functions such as pattern discrimination and recognition.
PUBLICATIONS:
Optican, L.M. and Richmond, B.J. Temporal encoding of two-dimensional
patterns by single units in primate visual cortex: III. Information theoretic
analysis. J. Neurophysiol. 57: 162-178, 1987.
Richmond, B.J., Optican, L.M. , Podell, M. , and Spitzer, H. Temporal encoding
of two-dimensional patterns by single units in primate visual cortex: I.
Response characteristics. J. Neurophysiol. 57: 132-146, 1987.
Richmond, B.J. and Optican, L.M. Temporal encoding of two-dimensional
patterns by single units in primate visual cortex: II. Quantification of
response waveform. J. Neurophysiol. 57: 147-161, 1987.
638
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02033-10 LN
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the tiorders.)
Functional mapping of sensory and memory systems
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: R.C. Saunders Staff Fellow LN NIMH
Others: M. Mishkin
J. Bachevalier
C . Kennedy
L. Sokoloff
Chief LN NIMH
Visiting Scientist LN NIMH
Guest Researcher LCM NIMH
Chief LCM NIMH
COOPERATING UNITS (It any)
Laboratory of Cerebral Metabolism, NIMH
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland
20892
TOTAL MAN- YEARS:
0.5
PROFESSIONAL:
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
n (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use starulard unreduced type. Do not exceed the space provided.)
The cerebral areas related to vision in the rhesus monkey were identified by
comparison of metabolic activity in visually stimulated versus visually
deafferented cerebral hemispheres. The results allowed delineation of the
visual-nonvisual borders of both an occipitotemporal and an occipitoparietal
visual pathway and specification of their points of interaction with frontal,
limbic, striatal, and diencephalic structures. In addition, it was found
that, within the occipitotemporal pathway, the forebrain commissures
contribute to the visual activation of area TE only.
639
PHS 6040 (Rev. 1/84)
SPO 9I4.»I(
ZOl MH 02033-10 LN
PROJECT DESCRIPTION:
Metabolic mapping of the primate visual system
The extent of the two main cortical pathways known to be critical for higher
order visual functions, the occipitotemporal and occipitoparietal pathways,
were revealed in a comprehensive picture of the entire visual system at work,
achieved by application of the [^^C] 2-deoxyglucose method. The method was
applied while monkeys, restrained in a primate chair, either 1) passively
viewed a high-contrast geometric pattern mounted on a surrounding rotating
drum, or 2) actively performed a visual pattern discrimination task that
required a response with the hand opposite the deafferented hemisphere.
Earlier, the monkeys had received either an optic tract section combined with
forebrain commissurotomy, and thus had one hemisphere visually deafferented,
or had an optic tract section only, and thus had one hemisphere only partially
deafferented. The comparison of local cerebral glucose utilization (LCGU) in
the visually deafferented versus intact hemispheres within the same animal
made it possible to identify and delineate the areas related to vision,
whereas comparison of LCGU in the totally versus partially deafferented
hemispheres across animals allowed assessment of the contribution to vision
made by the forebrain commissures.
The 2DG metabolic mapping procedure yielded the following picture. All
cortical tissue caudal to the junction of the lunate and the intraparietal
sulci is related to vision. Nonvisual tissue first appears in the superior
parietal lobule (probably somatosensory) and at the beginning of the lateral
fissure (probably auditory). In" the parietal lobe, the upper border always
remains within the intraparietal sulcus, about halfway down the upper bank
caudally and closer to the fundus rostrally. The lower border moves from the
lateral fissure and into the intraparietal sulcus rostrally. The rostral
limit of visual tissue is located within the intraparietal sulcus, about 5mm
behind its anterior tip. In the temporal lobe, the upper border always
remains within the superior temporal sulcus, generally about halfway down the
dorsal bank caudally but within the fundus rostrally. The lower border moves
from the calcarine fissure to the hippocampal sulcus (where it continues
midway along its length) and then turns laterally to enter the
occipitotemporal sulcus and finally the fundus of the rhinal sulcus.
Subcortically in the temporal lobe, tissue related to vision occupies the
lateral and lateral basal nuclei of the amygdala, posteroventral putamen,
ventral claustrum, and the tail of the caudate nucleus. Visual tissue is also
present in the anterior part of the head of the caudate nucleus, known to
receive input from the visually related cortex of the inferior frontal
convexity, and in both the body and the posterior portion of the head of the
caudate nucleus, known to receive input from the visually related posterior
parietal and prearcuate frontal cortices.
Comparisons of LCGU along the occipitotemporal pathway in the hemispheres with
total and partial visual deaf ferentat ion revealed a significant difference
only in area TE , reflecting a contribution of the forebrain commissures to
visual activation of this region alone. The likely explanation for the
failure of commissural fibers to activate glucose metabolism in any part of
640
ZOl MH 02033-10 LN
the occipitotemporal pathway posterior to area TE is that the primary function
of the commissural input to the posterior part of the pathway is to provide
suppressive rather than excitatory influences on neural activity (see LN
project MH 02036).
Metabolic mapping of the visual memory system
The two conditions of visual stimulation used for metabolic mapping of the
visual system in the perceptual studies described above yielded virtually
identical results. We are now attempting to map the further processing of
visual information by requiring monkeys to perform a visual learning task. In
addition, instead of using the single-label 2-DG paradigm employed in the
perceptual studies, we will use the recently developed double-label technique,
which will allow us to map brain metabolism related to two different types of
learning in the same subject. The double-label 2-DG procedures entails
separate, sequential injections of 1^C-2DG and ^U-lUG in an individual
subject, with each injection immediately followed by a different experimental
condition. Thus, the metabolic activity in the brain accompanying each
experimental condition is indexed separately by the two radioactive labels.
To enable mapping of the cognitive memory system, monkeys will be trained to
perform the visual memory task, delayed nonmatching-to-sample (DNMS). This
test has been used extensively to assess the effects of cortical, limbic, and
thalamic lesions on memory (see LN Project MH 00478). For the present
investigation the task has been modified so that the monkey's memory will be
stimulated continuously throughout the experimental session. To enable
mapping of the visual habit system for comparison, the same monkeys will also
be trained on a visual discrimination task. Performance on this task has been
shown to be unaffected by the same limbic lesions that disable the cognitive
memory system. After reaching a performance criterion of 90% correct
responses in both tasks the monkeys will be prepared with a forebrain
commissurotomy combined with a unilateral amygdalectomy plus hippocampectomy .
The monkeys will be retrained so that they perform at high levels even on the
most difficult stages of the memory task. The double-label 2-DG method will
then be applied with the animals performing the discrimination task after the
first label is injected and then the memory task after the second label is
injected. We expect to be able to map the processing of visual information in
both hemispheres beyond the areas seen in the visual perceptual studies. In
the intact hemisphere (without the limbic system ablations) we expect to be
able to visualize the proposed limbo-thalamic memory system, and in the
hemisphere with the memory system shut down, we expect to be able to visualize
structures related to the 'habit' system.
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
The 2-deoxyglucose method provides a unique method of relating neural
structure and function, permitting for the first time both the visualization
and quantification of local levels of metabolic activity simultaneously
throughout the entire brain in animals studied either under normal conditions
or following experimental intervention. The results continue to provide
important insights into the role of various cerebral structures, both cortical
and subcortical, in particular behaviors. Our initial studies contributed
valuable information as to the tissue occupied by the two cortical visual
641
ZOl MH 02033-in LN
processing pathways, and we expect equally valuable information from our
investigation of the two cortico-subcort ical learning systems. The new
double-labelling methods should prove to be an especially powerful tool for
this purpose.
PROPOSED COURSE OF RESEARCH:
Besides pursuing the metabolic mapping of learning processes, we expect to
apply the double-labelling 2-deoxyglucose method to the study of a variety of
behavioral processes in the monkey, including not only perception and memory
but also attention, emotion, and volition, for the purpose of identifying the
various structures involved in these different behaviors and quantifying the
degree of their participation.
642
OtPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
ZOl MH 02035-07 ln
PERIOD COVERED
October 1
1986 through September 30, 1987
TITLE OF PROJECT (BO chmrtctnrs of less Tiilt must lit on on« lirw b*tw»«n (r>« bonian)
Anatomy of the primate visual system
PRINCIPAL INVESTIGATOR (Ust oth*r pmlmssion*! ptrsoorml Otlow ttn Principal Invtstiguor j (Ntrrn. irtie, Ittxxmiory, tnd institutt altillsilon)
PI: L.G. Ungerleider
Others: M. Mishkin
R. Desimone
D. Boussaoud
R.J. Tusa
Research Psychologist
Chief
Research Psychologist
Visiting Fellow
Assistant Professor
LN NIMH
LN NIMH
LN NIMH
LN NIMH
Johns Hopkins
Univ.
COOPERATING UNITS (it tny)
Johns Hopkins University
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland
TOTAL MAN-YEARS:
4.75
PROFESSIONAL:
2.0
2.75
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues G (c) Neither
SUMMARY OF WORK (Us» standtnS unnductd (ype Do not •zcsad (/w jp«c» prvvidtd.)
To better understand the role of visual association cortex in perception and
memory, we have been examining the functional areas that comprise this cortex in
the macaque and exploring their interconnections by the use of neuroanatomical
tracing techniques in combination with electrophysiological recording of neural
activity. Our results indicate that a multiplicity of separate visual areas lie
beyond the primary visual cortex, or striate cortex (Vl), in the stream of
information processing. These areas are organized into two divergent
corticocortical pathways, each having VI as the source of its initial input.
One, an occipitotemporal pathway, enables the visual recognition of objects, while
the other, an occipitoparietal pathway, mediates the appreciation of the spatial
relationships among objects as well as the visual guidance of movements toward
objects in space. The visual areas along the occipitotemporal pathway (Vl, V2 ,
V3, V4, and areas TEO and TE of the inferior temporal cortex) appear to be
organized primarily as a serial hierarchy, in which each area processes both color
and form information. By contrast, the areas along the occipitoparietal pathway
(vl, MT, and MT's projection zones in parietal cortex) process the direction of
stimulus motion. Because a major component of this pathway extends anteriorly
within the superior temporal sulcus, the neural mechanisms underlying visuospatial
function may be more extensive than previously thought. To establish the links of
both the occipitotemporal and occipitoparietal pathways with the motor system, we
have been exploring the projections of visual association cortex to the striatum.
|So far, we have found that TE, TEO and V4 all project to the tail of the caudate
nucleus and ventral putamen, an arrangement that contrasts with the pattern of
projections from the occipitotemporal pathway to the limbic system, which arise
from TE only. The presence of direct projections to the striatum but not to the
limbic system from V4 and TEO may explain the ability of monkeys with TE lesions
to acquire visual habits but not visual memories.
643
PHS 60*0 |n»v 1/M|
ZOl MH 02035-07 LN
PROJECT DESCRIPTION:
The long-term objective of this project is to understand the role of visual
association cortex in perception and memory. To this end, we have been
examining the multiple functional areas that comprise this cortex in the
macaque and exploring the complex circuitry of their interconnections. So
far, we have discovered that the primary visual area, striate cortex, is the
source of two divergent corticocortical pathways: one, an occipitotemporal
pathway, which enables the visual recognition of objects; the other, an
occipitoparietal pathway, which mediates the appreciation of the spatial
relationships among objects as well as the visual guidance of movements
towards objects in space. A major question to be answered in the future is
how the object and spatial information carried in these two separate pathways
are subsequently integrated anatomically to yield a unified visual percept.
METHODS EMPLOYED:
In these studies, we have used a variety of neuroanatomical tracing techniques
(e.g., amino-acid autoradiography, horseradish peroxidase histochemistry,
axonal transport of fluorescent dyes) in combination with electrophysiological
recording of neural activity in anesthetized monkeys. In addition, we have
employed histological stains that clearly distinguish, for the first time,
differences in architecture among the multiple visual areas.
MAJOR FINDINGS:
1 . An occipitotemporal pathway for object vision
We had previously found that the occipitotemporal pathway begins with the
projection from the striate cortex, or VI, to the second and third visual
areas, V2 and V3, which project in turn to area V4. These three prestriate
areas are arranged in adjacent cortical belts that nearly surround the striate
cortex, and, like the striate cortex, each belt contains a representation of
the contralateral visual field. The major output of V4 is to a widespread
region within the inferior temporal cortex. Within posterior inferior
temporal cortex, or architectonic area TEO, label was found only after V4
injections involving the representation of the central 10° of the visual
field, whereas within anterior inferior temporal cortex, or architectonic area
TE, label was found after injections of any part of V4 . Thus, central but not
peripheral field representations in V4 project to TEO, while both central and
peripheral field representations in V4 project to TE.
Physiological studies have shown that TE has no discernible visuotopic
organization. Rather, neurons in TE have very large receptive fields that
nearly always include the center of gaze and frequently cross the vertical
meridian into the ipsilateral visual field. Thus, a single neuron in TE can
"see" an object no matter where it occurs in the field, which is in keeping
with the role this area plays in object recognition. Surprisingly, nothing is
known about the properties of neurons within TEO. As a first step in studying
these properties, we have begun to map TEO elec trophysiological ly . Thus far,
we have found that the receptive fields of neurons in TEO are mainly foveal,
which is consistent with the input this aroa receives from the central field
644
ZOl MH 02033-07 LN
representation of V4. It remains to be seen whether TEO is visuotopically
organized, like the prestriate areas that precede it, or whether it is
nontopographic , like area TE. Because lesions of area TEO, unlike those of
area TE, produce impairments in pattern perception rather than in object
recognition, a physiological comparison of TEO with TE should help in
understanding the neural mechanisms of these functions.
2. An occipitoparietal pathway for spatial vision
We had previously found that although area MT receives inputs from areas that
participate in the occipitotemporal pathway (i.e. VI, V2, V3, and V4), its
outputs appear to be mainly to areas located in the parietal lobe. One
projection zone, area VIP, lies ventrally in the anterior two-thirds of the
intraparietal sulcus, while two others, areas MST and FST, are located on the
dorsal bank and floor, respectively, of the superior temporal sulcus. To
examine the role these areas play in visual function, we have recorded the
electrophysiological properties of neurons within MT's projection zones and
compared these properties with those of neurons in MT itself. Our results
indicate that, like neurons in MT, a majority of those in MST and a third in
FST are directionally selective. Compared to neurons in MT, however, neurons
in both MST and FST integrate motion information over progressively larger
portions of the visual field and respond selectively to more complex types of
visual motion. To determine additional components of this motion-analysis
system, we have injected multiple anterograde and retrograde tracers into
physiologically identified locations within MST and FST of five monkeys.
The major connections of MST and FST are with each other and with areas in the
superior temporal sulcus (STS) and the posterior parietal cortex. In the STS,
both MST and FST project to cortex on the dorsal bank of the sulcus including
portions of the superior temporal polysensory area (STP), which contains many
cells with complex directional selectively. MST has particularly heavy
connections with a region at the bottom of the dorsal bank, which may either
be part of STP or a new visual area. In contrast, FST is connected with an
adjacent area in the floor "of the sulcus. In the posterior parietal cortex,
both MST and FST have connections with area V3A in the parieto-occipital
sulcus, and with cortex in the intraparietal sulcus, including but not limited
to areas VIP and LIP; these areas are known to project in turn to the inferior
parietal lobule. Finally, MST also has connections with medial parietal
cortex, including but not limited to area PO and the cingulate gyrus.
The results suggest that the cortical pathway for motion analysis, which
begins with the projection of VI to MT, splits into at least two components.
One component includes widespread regions of the posterior parietal cortex,
whereas the other extends into the temporal lobe and includes several areas on
the dorsal bank and floor of the STS. Thus, the neural mechanisms underlying
visuospatial function may be far more extensive than previously thought.
While it is known that lesions along the parietal component of this system
cause impairment in spatial perception, eye movements, and visually guided
hand movements, the effects of lesions along the temporal component of this
system remain to be explored.
645
ZOl MH 02035-07 LN
3. Projections of visual association cortex to the striatum
We have recently begun to explore the projections of visual association cortex
to the striatum. Neurobehavioral studies in our laboratory suggest that these
projections are the ones that mediate visual habits, whereas projections from
visual cortex to the limbic system mediate visual memories. We are
particularly interested in determining the visual cortical areas that project
to the striatum, how the projection fields relate to one another, how the
cells of origin are organized, and to which structures the visual portions of
the striatum project. Our long-term goal in this study is to establish the
links of both the occipitotemporal and occipitoparietal pathways with the
motor system.
So far, we have placed multiple anterograde and retrograde tracers into the
tail and genu of the caudate nucleus or adjacent ventral putamen under
physiological control in five monkeys. After caudate injections, labeled
cells were found both in a large continuous region of cortex topographically
related to the site of injection, and in several non-continuous cortical
regions. After injections in the rostral tail, the continuously labeled
region included rostral area TE and adjacent portions of TF, TH, TG, and,
occasionally, area 35. After injections into the posterior tail and ventral
genu, the labeled region shifted posteriorly to posterior TE, TF, and TEO and
then into prestriate areas V4, MT, PC, and (sparsely) V3 and V2. As the
injection site advanced into the dorsal genu and then to the caudal body, the
labeled region shifted toward the parietal lobe, to area 7, to areas VIP and
LIP in the intraparietal sulcus, and into area 5 and adjacent area 23. The
non-continuous areas labeled by nearly all injections included the principal
sulcus/frontal eye field region, area 24, the superior temporal polysensory
area, and, more rarely, area 25. Thus, whereas certain temporal, occipital,
and parietal cortical areas project into the striatum largely according to
proximity, prefrontal, anterior cingulate, and superior temporal sulcal areas
have a wider distribution. In all cases, labeled cortical cells were found
mostly in layer 5 but also in layer 3.
The finding that areas TE, TEO, and V4 all project to the tail of the caudate
nucleus and ventral putamen contrasts with the pattern of projections from the
occipitotemporal pathway to the limbic system, which arise from TE only. The
presence of direct projections to the striatum but not to the limbic system
from areas V4 and TEO may explain the ability of monkeys with area TE lesions
to acquire visual habits but not visual memories.
Anterograde labeling following injections into the tail of the caudate nucleus
or ventral putamen was confined to the globus pallidus (GP) and substantia
nigra pars reticulata (SNr). Because of the known projections from the GP and
SNr via the thalamus to the supplementary motor and dorsolateral prefrontal
cortex, respectively, these cortical regions may represent further stations in
the neural circuit underlying the formation of visual habits. Ultimately, we
hope to delineate the entire wiring diagram of this circuit.
646
ZOl MH 02035-07 LN
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
An understanding of the basic mechanisms mediating normal visual perception
and memory is the first step in the diagnosis, alleviation, and, ultimately,
prevention of sensory, perceptual, and mnemonic disorders. To this end, we
have been exploring projections out of the striate cortex to prestriate
association areas. Our goal has been to trace the complex system of
projections stepwise to the still higher-order visual areas located within the
temporal and parietal lobes, areas critical for object vision and spatial
vision, respectively. The combined use of axonal transport techniques and
electrophysiological recording provides a powerful tool for tracing neural
connections within these central visual pathways. In addition, the recent
development of highly selective histological stains may give us the
opportunity for the first time of identifying the same higher-order visual
areas in the human brain that we have identified in the monkey.
PROPOSED COURSE OF RESEARCH:
Thus far, we have found that visual cortex in the monkey is organized into two
divergent corticocortical pathways and that the projections of both pathways
can be traced from the striate cortex through multiple prestriate association
areas to the still higher-order visual areas in the temporal and parietal
lobes. Our recent studies suggest that both the temporal and parietal lobes
also consist of multiple visual areas, and we will continue to investigate
their organization. Since there are no direct connections between temporal
and parietal cortex, a major question to be answered is how the object and
spatial information carried in these two separate pathways are subsequently
integrated anatomically. We also plan to continue our investigations of the
links of both pathways to affective memory, and motor systems by examining the
projections of the multiple visual association areas to limbic structures, the
neostriatum, and prefrontal cortex. Finally, using a combination of
histological stains and degeneration techniques on human brain material with
occipital lobe lesions, we will attempt to identify the multiple visual areas
in the human cortex that have been differentiated in the monkey.
PUBLICATIONS:
Segraves, M.A. , Goldberg, M.E., Deng, S.-Y., Bruce, C.J., Ungerleider, L.G. ,
and Mishkin, M. The role of striate cortex in the generation of eye movements
in monkeys. J. Neurosci. (in press)
Tusa, R.J. and Ungerleider, L.G. Fiber pathways of cortical areas mediating
smooth pursuit eye movements in monkeys. Annals Neurol, (in press)
647
DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT MUMBEa
ZOl MH 02036-07 LN
PERIOD COVERED
October 1, 1985 through September 30, 1987
TITLE OF PROJECT (80 chtitctvrs or lats Tiih must lit on ont Une b«tw»«n t^e txxden )
Neural representations of visual stimuli in the extrastriate cortex
PRINCIPAL INVESTIGATOR (Ust olt>tr prntatsioml p»r.
PI
R. Desimone
Others: M. Mishkin
H. Spitzer
^/ tMtow rft« Pnncipsl Investtgator ) {Ntma. frtte. latxyatofy, and institute aftiliatk>o)
Research Psychologist LN NIMH
Chief LN NIMH
Visiting Associate LN NIMH
COOPERATING UNITS {H any)
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
2.5
PROFESSIONAL:
1.25
1.25
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
00 (c) Neither
SUMMARY OF WORK (Us* slandan) unndix—l typt Do not aic9«<l tt>t spaca ptmided )
Disorders of perception, attention, and memory frequently accompany the
major mental diseases. To begin to understand the neural mechanisms of
these mental processes, we are recording the activity of neurons in the
extrastriate cortex of monkeys engaged in tasks requiring visual
discrimination, selective attention, and recognition memory. We have found
in area V4 of the occipital cortex and area TE of the inferior temporal
cortex that selective attention gates visual processing by filtering
unwanted information from the receptive fields. Even the degree to which
attended stimuli are processed in these areas depends on "how much"
attention or effort is devoted to them. Thus, the in format ion -processing
capacity of cortical neurons depends not only on hard-wired mechanisms but
on cognitive state. To identify the mechanisms by which cognitive state
modulates cortical activity, we have begun to examine both extrastriate
neuronal activity and animal behavior in an attention-demanding task
following lesions of selective portions of the prefrontal cortex, posterior
parietal cortex, limbic-system, and basal ganglia.
649
PMS 6O40 (R»v l/Ml
ZOl MH 02036-07 LN
PROJECT DESCRIPTION:
This is a long-term project to understand both the neuronal basis of
perception and memory in extrastriate cortex and the mechanisms by which these
processes are influenced by cognitive factors such as selective attention. We
focused our initial work on the basic sensory information coded by neurons in
the extrastriate areas most directly involved in object recognition. Having
identified several of the dimensions along which extrastriate neurons code
objects, we have now turned to examining the dynamic operation of this
cortical system in awake monkeys engaged in tasks requiring selective
attention and memory.
Methods employed: Our most recent studies were carried out in awake monkeys,
trained to hold their gaze steady while they performed a match-to-sample
task. In this task, the monkey held a bar while a stimulus appeared briefly
at one retinal location followed shortly by a second, briefly presented
stimulus at the same location. The monkey was rewarded for releasing the bar
immediately if the two stimuli matched and for releasing the bar after a fixed
delay if they did not match. At a second retinal location, two other,
irrelevant stimuli were also presented on each trial, each concurrently with
one of the stimuli used in the task. After a block of trials at the first
location, a cue was given, and the previously irrelevant stimuli at the second
location became the relevant (and, therefore, attended) stimuli for the
matching task. Thus, identical sensory conditions were maintained across
trials, but the locus of the animal's attention varied.
Experiments :
1 . Neural mechanisms for the analysis of form and color in area V4.
Anatomical experiments in our laboratory have shown that visual area V4 is a
central station in the pathway from the primary visual cortex to the object
recognition system of the temporal cortex. We have completed an extensive
analysis of sensory coding in V4 and have identified for the first time many
of the different stimulus features used by V4 cells to code objects, including
the color, length, width, orientation, and contrast of contours, and the
spatial frequency, phase, and overall size of sinusoidal gratings. One
full-length paper on this work has been published and two more are in
preparation. Now that we have a better understanding of sensory coding in V4,
we will concentrate our future studies on the mechanism by which coding is
controlled by attention.
2. Selective attention gates visual processing in extrastriate cortex.
Earlier in this project, we found that when a monkey attends to a stimulus
within the receptive field of a neuron in either area V4 or area TE of
extrastriate cortex, the processing of other, distracting, stimuli within the
field is blocked. Thus, unwanted stimuli appear to be filtered out of the
visual system as a result of selective attention, explaining why we perceive
and remember only a small portion of the stimuli acting on our retinas at any
given time. In subsequent experiments, we have found that extrastriate
neuronal responses to a given stimulus are larger and more tightly tuned when
the monkey is discriminating that stimulus from one that is very similar to it
than when the monkey is discriminating it from one that is very different.
650
ZOl MH 02036-07 LN
These results suggest that even the degree to which attended stimuli are
processed depends on "how much" attention or effort is devoted to them. A
short report on these findings has been published and full-length reports are
in preparation. Having established that information processing in
extrastriate cortex is controlled by cognitive state, our next step is to
understand the neuronal basis of this control.
3. The source of the attention "gate". We found previously that the neuronal
effects of spatially directed attention do not occur in either the primary
visual cortex or area V2, so that whatever structure or structures gate
extrastriate responses to attended stimuli must work at the level of V4 and
beyond. Anatomical studies in our laboratory have identified at least four
possible direct sources of modulating inputs to V4 and/or inferior temporal
cortex, namely portions of the pulvinar, posterior parietal cortex, prefrontal
cortex, and limbic system. In addition, recent studies of the effects of
unilateral dopamine depletion by Dr. Doris Doudet in LCM suggest that dopamine
in the substantia nigra may play an indirect but very important role in
attentional modulation of cortical activity. To determine the relative role
of these structures in the control of attention, we will test individually the
effects of reversible deactivation of the lateral pulvinar and portions of the
limbic system (using the GABA agonist muscimol), unilateral lesions of the
dopaminergic system in the substantia nigra (using the neurotoxin MPTP), and
removal of specific areas of the parietal and frontal cortex on the ability of
monkeys to perform our attention task with distracting stimuli. As a control
for the possibility that any lesion effects we observe are actually due to
impairments on the non-attentional components of our task, we will also
measure the monkeys' performance on a version of the task that does not
contain distracting stimuli and is, therefore, not attention-demanding. A
group of animals has now been prepared for the study and are about to undergo
behavioral testing.
4. Neural mechanisms for recognition and associative memory. Although
monkeys and man exhibit agnosia following damage to the cortex of the temporal
lobe, there is remarkably little neurophysiological evidence that memories are
actually stored in the cortex. To test this possibility, we have developed a
system for presenting many complex visual and auditory stimuli in recognition
and associative memory tasks. Our initial recordings from neurons in area TE
of the temporal lobe in monkeys performing a recognition task have been
encouraging, in that they indicate that many cells do indeed respond
differently to a visual stimulus depending on whether or not it has been
presented previously to the monkey. Since we have so far only tested for
these differences over very short time intervals, we do not yet know if these
responses could form the basis for a long-term recognition memory. This
possibility will be examined over the next year.
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
The results from our recording experiments in extrastriate cortex demonstrate
that psychological factors such as selective attention and effort directly
affect the cortical neurons that process ;Lncoming stimuli and store items in
memory. If, during the future course of this project, we can determine exacly
how these effects take place, we will be in a better position to understand,
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and, ultimately, treat the disorders of perception, attention, and memory that
frequently characterize major mental diseases such as Alzheimer's disease,
Parkinson's disease, and schizophrenia.
PROPOSED COURSE OF RESEARCH:
A major thrust of our work over the next year will be to continue to track
down the structure or structures that modulate cortical processing as a result
of selective attention. Our general strategy will be to first identify likely
sources of the modulating input by testing the effects of lesions on the
attentional capacities of monkeys. When a structure has been so identified,
we will then record from its neurons while the animal performs our selective
attention task, looking for neuronal responses related to the animal's switch
of attention. Finally, we will attempt to model the system with
computer-simulated neuronal networks.
PUBLICATIONS:
Albright, T.D. and Desimone, R. Local precision of visuotopic organization in
the middle temporal area (MT) of the macaque. Exp. Brain Res. 65: 582-592,
1987.
Desimone, R. and Schein, S.J. Visual properties of neurons in area V4 of the
macaque: Sensitivity to stimulus form. J. Neurophysiol . 57: 835-868, 1987.
652
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02037-06 LN
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line between the tx}rders.)
Functional anatomy of the somatosensory cortex of the monkey
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T.P. Pons Guest Researcher LN NIMH
Others:
M. Mishkin
D.P. Friedman
E.A. Murray
R.J. Schneider
R.C. Saunders
P.E. Garraghty
Chief
Project Officer
Senior Staff Fellow
Guest Researcher
Staff Fellow
Research Associate
LN NIMH
NRB NIDA
LN NIMH
LN NIMH
LN NIMH
Vanderbilt Univ.
COOPERATING UNITS (If any)
National Institute on Drug Abuse
Vanderbilt University
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.25
PROFESSIONAL:
0
0.25
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues Q (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
To identify a route by which tactile information could reach limbic structures in
the temporal lobe, we used axonal transport techniques to trace the connections
between somatosensory cortical fields. On the basis of the laminar patterns of
these corticocortical connections, we identified them as 'forward' or 'backward' by
analogy to similar designations in the visual system, where they have been shown to
have functional validity. The analysis indicated that a forward-projecting route
could be traced from the subdivisions of the primary somatosensory cortex to the
second somatosensory area, SII; from SII to the granular and dysgranular fields of
the insula; and from the insula directly to the amygdala and indirectly to the
hippocampus via rhinal cortex. This multisynaptic cortico-limbic pathway in the
somatosensory system is thus organized in a manner analogous to the multisynaptic
cortico-limbic pathway in the visual system. To assess the functional importance
of the pathway, we studied the somatosensory receptive fields of neurons in SII
cortex following selective ablations within the primary somatosensory cortex and
found that elimination of any given representation of the body surface in the
postcentral strip eliminated it also in SII. For example, removing the hand
representation from the postcentral strip resulted in its disappearance from SII
cortex; conversely, removing all other body representations from the post central
strip (i.e. except that of the hand) resulted in the preservation in SII of the
hand representation only. The electrophysiological data thus provide strong
support for the conclusion, based originally on the anatomical data, that tactile
information is processed sequentially along a cort icort ical pathway. The
electrophysiological experiments also revealed a surprising degree of functional
reorganization in SII cortex following the postcentral cortical ablations. After
each partial removal, the vacated representation in SII was filled in by the
expansion of the intact, neighboring representations. These last findings point to
a previously unrecognized degree of cortical plasticity in adult primates following
brain injury. 653
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spo ai4-«it
ZOl MH 02037-06 LN
PROJECT DESCRIPTION:
Previous work in our laboratory has shown that the amygdala and hippocampus
are critical for both visual and tactual memory. The route by which visual
information gains access to these structures has been well documented. In
brief, a series of cortical fields beginning in striate cortex and progressing
through prestriate cortex to inferior temporal cortex and from there to the
amygdala and hippocampus, have been shown to be critical for the learning and
memory of visual information. But while the cortical pathway for transmitting
visual information to these limbic structures is well understood, the
comparable somatosensory pathway, if one exists, is still uncertain. We are
therefore trying to determine (a) the most direct anatomical route by which
somatosensory information could reach the amygdala and hippocampus,
(b) the physiological dependency of one cortical area on another along this
route, (c) the processing of information along this pathway, and (d) its role
in somatosensory learning and memory. If such a functional pathway could be
identified, it would provide strong support for the view that there is a
common plan of organization across all the modalities for the cortical
processing, storage, and retrieval of sensory information.
Anatomical studies
Corticocortical connections. Anatomical studies have indicated that visual
information is transmitted ventrally to the temporal lobe via a series of
relays in prestriate areas. Specifically, VI projects to V2 (area OB), V2
projects to areas V3 and V4 (both fields are part of area OA) , and V4 projects
to the inferior temporal areas TE and TEO. Area TE of inferior temporal
cortex is the last cortical visual processing station in the sequence, and
this area projects in turn to the amygdala (directly) and to the hippocampal
formation (indirectly via entorhinal cortex). Also, area TE is totally
dependent upon input from the more posterior areas in the chain for visual
activation of its neurons. This pathway, which remains modality specific
throughout its neocortical . extent , has been shown to be important for the
visual recognition and identification of objects.
By contrast, a second cortical visual pathway is thought to be important for
visuospatial perception. This is a dorsally directed cortical pathway that
begins in VI, passes through prestriate visual areas, then courses through
posterior parietal cortex and then on to the cingulate cortex before finally
reaching the amygdala and hippocampus. Much less detail is known, however,
about the anatomical and physiological relationships between the areas
comprising this pathway and the behavioral consequences of interrupting this
pathway by lesions.
To determine whether the first type of pathway described above might be a
common feature of organization for learning and memory in all of the sensory
modalities, we turned to the somatosensory system since more is known about
the cortical organization of this sensory modality than of any other except
vision. Our experiments indicated that the fields comprising postcentral
somatosensory cortex (areas 3a, 3b, 1, and 2) are richly interconnected with
each other and that these connections link corresponding representations in
the different fields. In addition these fields have further connections wLth
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areas outside the postcentral cortex. On the basis of the laminar patterns of
these various connections, each was designated as a forward projection (layer
III to layer IV) or as a backward projection (layer V to layer I) by analogy
to similar designations in the visual system. From this analysis, we were
able to identify two major pathways for the flow of somatic information out of
postcentral cortex.
The first cortical pathway is directed ventrally and begins with the primary
cortical receiving area for tactual information, area 3b. Area 3b projects
forward to area 1 and less densely to area 2. The densest cortical projection
from each of areas 3b, 1 and 2, however, is to layer IV of SII cortex. SII
then projects in a forward manner to the granular and dysgranular fields of
insular cortex, and, finally, the pathway proceeds from the fields of the
insula to the amygdala and indirectly to the hippocampus through perirhinal
cortex. We have been slowly accumulating additional physiological and
behavioral evidence that this pathway in the somatosensory system may be
analogous to the ventrally directed pathway in the visual system.
The second pathway is a dorsally projecting one, again with area 3b projecting
forward to area 1, and less densely to area 2. Area 1 in turn projects
forward to area 2 and to a specialized cutaneous portion of area 5. Area 5 in
turn projects to area 7b of posterior parietal cortex. Each of the areas
receiving a forward connection projects with a backward connection upon the
cortical area that gave rise to the forward one. It is not yet known whether
this dorsally directed somatosensory pathway eventually has connections with
the limbic system, as does the dorsal visual pathway.
Electrophysiological studies
Receptive fields of SII neurons following ablation of postcentral cortex. It
had previously been assumed that the primary thalamic nucleus for tactual
information (the ventroposterior nucleus, or VP) supplied the major activating
input for both postcentral cortex and SII. However, our anatomical studies
summarized above suggested instead that SII cortex may be receiving its
somatosensory input from postcentral cortex, rather than directly from the
ventroposterior nucleus of the thalamus. To examine this possibility, we
recorded single- and multi-unit activity from the SII region in 10 hemispheres
of 6 macaques (4 Macaca mulatta and 2 Macaca fascicularis) anesthetized with a
mixture of halothane and nitrous oxide. The electrode penetrations were
placed 0.5-1.0 mm apart in a rectangular grid across the entire extent of SII,
and neuronal responses were sampled at 200 um intervals through the depth of
this cortex. The receptive fields of the neurons at the recording sites were
determined by applying tactile stimulation at different locations on the
contralateral body surface. Of the 10 hemispheres studied, 5 were intact and
5 had received lesions 6-8 weeks earlier of selected portions of the body
representations in the postcentral strip.
In the intact hemispheres, receptive fields of neurons in SII were readily
found for tactile stimulation of all contralateral body parts, with the
majority of the fields representing loci on the glabrous and hairy surfaces of
the hand. By contrast, in recording sites through SII of hemispheres in which
the postcentral hand representations had been removed, no receptive fields
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could be found for tactile stimulation of the glabrous surface of the hand,
and only a few were found that included the hand's hairy surface. Yet there
was no difficulty in recording responses in the experimental hemispheres to
stimulation of all other body parts, indicating that the near absence of a
hand representation in SII was not due simply to a general depression of the
SII cortex. The functional dependency on postcentral cortex that was
demonstrated for the SII representation of the hand held also for the SII
representations of other body parts. For example, in recording sites
distributed through the SII region in a case with a postcentral removal that
spared only the hand representation, all of the receptive fields found were
confined to the hand. Similarly, in recording sites through SII in a case
with a total removal of the postcentral strip, no neuronal activation was
observed from tactile stimulation of any body part. In short, the elimination
of any representation in the postcentral cortex eliminated it also in SII.
The results thus support the proposal derived from our anatomical studies that
SII depends on the postcentral strip for its somatic activation and thus could
well occupy an intermediate position between the postcentral cortex and the
insula in a sequential cort ico-limbic pathway for touch.
Postoperative cortical plasticity. Another, unexpected result from the
foregoing electrophysiological work was the finding that the SII region
undergoes major functional reorganization following removal of portions of
postcentral cortex. As indicated above, removing the representations of a
body part in postcentral cortex results in the failure to record somatically
driven responses in the representation of the corresponding body part in SII.
Interestingly, the SII tissue in question does not remain silent; instead,
representations of different body parts in the adjacent portions of SII expand
to occupy the partially deafferented cortical zone. For example, following a
lesion of the postcentral representation of the hand, there is a greater
probability of recording responses in SII to stimulation of the foot. In
other words, the areal extent of the foot representation increases to occupy
part of the former hand region (indeed, the reorganizational changes occur
over a distance of 4 or more millimeters of cortex), and, similarly, the rest
of the body representation in SII also expands. These findings provide
evidence for a previously unrecognized degree of cortical plasticity in adult
primates. The results thus require major revisions of current theories, which
tend to confer static properties on cortical maps.
Receptive fields of insular neurons. In another recording project, we are
mapping the somatically responsive portions of the insular cortex, which our
anatomy shows receives a dense input from SII, in an attempt to determine the
somatotopic organization and response properties of cells in this region.
Because anesthetics are known to depress higher order sensory cortical areas
such as the insula, this study must be performed in awake responding monkeys
trained to sit in a primate chair and to allow gentle tactile stimulation of
their bodies. The preliminary results indicate that receptive fields for
insular neurons are typically very large, usually bilateral, and are modality
specific. Our recordings have also revealed that there is at most only a
rough somatotopic organization within the granular insular cortex, with the
face and intraoral regions being represented rostrally, and the rest of the
body being represented more caudally. These receptive-field properties are
clearly analogous to those of neurons in visual area TE, in that they too are
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ZOl MH 02037-06 LN
modality-specific, are large and bilateral, and have poor topographic
organization. Our data are thus consistent with the notion that insular
cortex serves as a final link in a somatosensory-limbic pathway just as area
TE does in the visual-lirabic pathway.
Neurobehavioral studies
We have been using behavioral techniques to examine the effects of removing
the insula on tactile object recognition and have postoperative results from
four animals indicating that bilateral insula lesions cause a tactile
recognition deficit. This preliminary finding is consistent with the
suggestion that insular cortex acts as a final link in a parieto-insulo-limbic
pathway for somesthesis, analogous to the occipito-temporo-limbic pathway
previously described for the visual system. This preliminary result is
particularly exciting, for, if it is upheld, the project has the potential of
revealing a mode of sensory-limbic interaction that is common to memory
formation in all sensory modalities.
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
The data from these projects are providing us with the first comprehensive
view of the entire somatosensory system as well as of its connections with the
limbic memory system. Furthermore, the studies have suggested remarkable
parallels between the organization of the somatosensory and visual systems,
and imply that similar mechanisms of perception and memory may operate within
both. These projects are yielding fundamental insights into how the cerebral
cortex processes and stores sensory information by uncovering mechanisms that
may well be common to all sensory modalities. Finally, we have demonstrated a
previously unrecognized degree of post-injury cortical plasticity in the adult
macaque with the exciting discovery that SII cortex undergoes extensive
functional reorganization following damage to primary sensory cortex.
PROPOSED COURSE OF RESEARCH:
More research is required to gather further evidence for, or against, the
possibility that the ventrally directed pathway in the somatosensory system is
analogous to the ventrally directed one in the visual system. In addition, we
plan to begin research projects designed to determine if the dorsally directed
somatic pathway might have a role in tactual spatial perception, by analogy to
the role of the dorsal visual pathway in visual spatial perception.
PUBLICATIONS:
Friedman, D.P., Murray, E.A., O'Neill, J.B., and Mishkin, M. Cortical
connections of the somatosensory fields of the lateral sulcus of macaques:
evidence for a corticolimbic pathway for touch. J. Comp. Neurol. 252:
323-347, 1986.
Friedman, D.P. and Murray, E.A. Thalamic connectivity of the second
somatosensory area and neighboring somatosensory fields of the lateral sulcus
of the macaque. J. Comp. Neurol. 252: 348-373, 1986.
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ZOl MH 02037-06 LN
Garraghty, P.E., Pons, T.P., Huerta, M.F., and Kaas, J.H. Somatotopic
organization of the third somatosensory area (SIII) in cats. Somatosens. Res.
4: 333-357, 1987.
Kaas, J.H. and Pons, T.P. The somatosensory system of primates. In H.P.
Steklis (Ed.): Comparative Primate Biology, Vol. 4, Alan Liss, New York, (in
press)
Pons, T.P., Garraghty, P.E., Friedman, D.P., and Mishkin, M. Physiological
evidence for serial processing in somatosensory cortex. Science 237:
417-419, 1987.
Pons, T.P., Wall, J.T., Garraghty, P.E., Cusick, C.G., and Kaas, J.H.
Consistent features of the representation of the hand in area 3b of macaque
monkeys. Somatosens. Res. 4: 309-331, 1987.
658
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02038-05 LN
PERIOD COVERED
October 1. 1986 through September 30. 1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line tietween the tmrders.)
Ontogenetic development of cognitive memory and habit formation
PRINCIPAL INVESTIGATOR (Ust ottter professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: J. Bachevalier Visiting Scientist LN NIMH
Others: M. Mishkin
L.G. Ungerleider
D.P. Friedman
C. Hagger
P. Merjanian
Chief LN NIMH
Research Psychologist LN NIMH
Project Officer NRB NIDA
Guest Researcher LN NIMH
Guest Researcher LN NIMH
COOPERATING UNITS (if any)
National Institute on Drug Abuse
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland
TOTAL MAN-YEARS:
2.25
PROFESSIONAL:
1.0
1.25
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Cognitive memory and habit formation are two qualitatively different
learning processes based on separate neural systems, a cortico-limbic and a
cortico-nonlimbic system, respectively. To see how emotional and social
behavior develop in animals whose infantile global amnesia might persist from
infancy through adulthood, we have prepared monkeys with neonatal limbic
lesions and followed their behavioral development. Animals with neonatal
removal of cortical area TE, a higher-order visual station- linked to both
learning systems, serve as controls. The results indicate that neonatal TE
lesions leads to a transient impairment of habit formation three months later
(compared to permanent impairment seen with the same lesion in adults),
whereas both neonatal and adult limbic lesions leave habit formation intact.
Interestingly, data on both normal and operated infants suggest that
development of the nonlimbic habit system is sexually dimorphic, and that this
is due to the high testosterone levels present in male infants before and
shortly after birth. At ten months of age, the infants with limbic lesions
show impairment in memory formation, whereas the operated controls show
significant functional sparing (compared to those that received the same
lesions as adults). These findings point to greater compensatory potential
after neonatal cortical than after neonatal limbic removals, indicating that
association areas of the cortex are immature at birth, and may thus possess
greater plasticity than limbic structures. Direct evidence of neocortical
immaturity in the macaque has been provided by our neurobiological studies on
opiatergic and cholinergic receptor distribution and on metabolic activity.
Finally, early damage to the limbic memory system produces later
socio-emotional abnormalities that are similar in many respects to the
behavioral syndrome seen in autistic children.
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GPO •! 4.911
ZOl MH 02038-OS LN
PROJECT DESCRIPTION:
Findings from studies of the effects of lesions in adult monkeys suggest that
cognitive memory and habit formation are qualitatively different retention
processes based on separate neural mechanisms. The cognitive memory system,
which serves both recognition and associative memory, utilizes a
cortico-limbo-diencephalic circuit. By contrast, the habit system, which
mediates retention of stimulus-response connections, probably depends in large
part on a cortico-striatal system. Our recent studies of behavioral
development in infant monkeys have suggested that these two systems are
developmentally dissociable, in that the nonlimbic habit system appears to
mature considerably earlier than the limbic memory system. On the evidence
that the limbic memory system is essentially nonfunctional in infants, we have
prepared monkeys with neonatal removal of this system in an attempt to see how
emotional and social behavior develop in animals whose amnesia might persist
from infancy through adulthood. In addition, since the ontogenetic
development of habit formation appears to be sexually dimorphic, we have
initiated a study of the neuroendocrinological substrate of that dimorphism.
In tandem with these developmental studies of behavior, we continue to map the
distribution of opiatergic and muscarinic cholinergic receptors in the brain
of the developing rhesus monkey.
Experiment 1
To date, eight infant rhesus monkeys received damage to the limbic system
(i.e. amygdalo-hippocampal complex) and eight others, which served as control
animals, received damage to the anterior part of inferior temporal cortex
(i.e. cytoarchitectonic area TE, a higher-order station of the visual
system). These monkeys were age-matched with thirteen normal animals and have
already undergone some testing for social behavior and learning abilities. We
are currently following the behavior of these animals from birth through early
adulthood in order to assess the effects of neonatally induced amnesia on (1)
the maturation of cognitive functions and skill learning, as measured by a
variety of visual memory, problem solving, and habit formation tasks, and (2)
the development of emotional and social behaviors, as measured by interactions
with familiar vs. unfamiliar and normal vs. operated monkeys of both sexes and
various ages, and by reactions toward familiar vs. unfamiliar and emotionally
neutral vs. emotionally challenging environments and stimuli.
Maturation of skill learning and cognitive functions in early infancy. The
results so far indicate that, at three months of age, neonatal ablation of
area TE leads to a transient impairment in habit formation (compared to the
permanent impairment seen with the same lesion in adults), whereas limbic
lesions in both infants and adults leave habit formation intact.
Interestingly, data from both the normal and the operated infants suggest that
ontogenetic development of the habit system is sexually dimorphic, this system
maturing earlier in females than in males (see Experiment 2). At ten months
of age, the infants with limbic lesions are severely impaired in cognitive
memory, whereas the operated controls show significant functional sparing of
this ability (compared to the animals given TE lesions as adults). Thus, the
mild impairment seen in infants with area TE lesions in recognition memory
points to greater compensatory potential after neonatal cortical than after
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neonatal limbic removals. The results are consistent with the notion that
association areas of the cortex are less mature at birth, and may thus possess
greater plasticity, than limbic structures. Direct evidence of neocortical
immaturity in the macaque has been provided by our neurobiological studies
showing that (a) adult levels of metabolic activity in visual association
cortex are not reached until about 4 months of age (see Experiment 3), and (b)
the distribution of both opiatergic and muscarinic cholinergic receptors is
adult-like at birth in subcortical structures and allocortical areas but is
not yet fully developed in neocortical areas, particularly the association
cortex (see Experiment 4). These behavioral and neurobiological findings
suggest that the reduced recognition ability in infant monkeys and perhaps, by
extension, infantile global amnesia in this species are due more to slow
maturation of the cortical association areas than to neonatal immaturity of
the limbic system.
To investigate whether the severe impairment in cognitive memory in infants
with the limbic lesions is due, as it is in adults, to the combined damage of
the amygdala and the hippocampus and not to the damage of either limbic
structure alone, we have prepared new groups of infant monkeys who received
damage to either the amygdala or the hippocampus. These monkeys were
age-matched with normal infants and were tested in exactly the same way as the
infants with combined limbic lesions. The findings indicate that, as in
adults, damage to the hippocampus or the amygdala alone do not yield marked
impairment in the visual recognition task. In fact, whereas both amygdalar
and hippocampal lesions in adulthood yielded a mild memory loss, only the
neonatal amygdalar lesions produced a deficit equivalent in magnitude. The
neonatal hippocampal lesions by contrast left this ability intact.
Some of the first infants to receive neonatal lesions are now five years old
and are being retested on the behavioral tasks. This will allow us to
determine what changes have taken place in their cognitive and noncognitive
learning abilities since they were last tested, which was when they were about
a year of age.
Development of emotional and social behaviors in early infancy. The slowly
accumulating data of this long-term neurobehavioral study suggest that,
compared to intact infant monkeys and others with neonatal cortical lesions,
monkeys with neonatal limbic lesions show numerous socio-emotional
abnormalities such as increased locomotion, decreased manipulation of toys,
towels, and cage surfaces and attachments, increased withdrawal from social
contacts, and increased finger-sucking. All of these abnormalities are
reminiscent of those seen in young autistic children. And, indeed, our
results, combined with Kempers's recent report of neuropathology in the
amygdala, hippocampus, and cerebellum of an autistic man, support the view
that early dysfunction of the limbic system is one cause of infantile autism.
Although amygdalar damage by itself is associated with changes in emotional
and social behavior in adult monkeys, the socio-emotional abnormalities we
observed in the developing infants with combined amygdalo-hippocarapal lesions
was not attributable to amygdalar damage alone. Rather, the full-fledged
syndrome described above was fractionated by partial limbic lesions.
Specifically, the findings indicate that whereas amygdalar lesions in infants
produce the same behavioral abnormalities that they do in adults, neonatal
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hippocampal removals also induce socio-emot ional abnormal ites , a finding that
has not been reported before at any age. Together these experiments are
providing evidence that the amygdala and hippocampus are each components not
only of a limbo-thalamic system serving cognitive functions but also of a
limbo-hypothalamic system serving emotional functions. Though much more
testing over a much longer time course is necessary, it is becoming clear that
the same neonatal damage that leads to a severe cognitive memory disorder can
have extremely serious consequences for personality and social development, in
part perhaps because the cognitive memory disorder is present from infancy
onward, but also because of the direct effect of the limbic lesions on
mechanisms of emotionality.
To compare the socio-emotional abnormalities in the neonatally operated
animals with those observed in autistic patients, we will retest the animals
as they reach adulthood and analyze in detail their individual and social
behavior for characteristic signs of autism such as disinterest in initiating
play when placed in a group, lack of separation anxiety, fearful responses to
novelty and environmental complexity, and unusual food preferences.
One way to measure separation anxiety in monkeys is to record "isolation"
calls. These calls serve to reestablish contact between a mother and her
offspring, as well as between peers raised together. Cerebral ablation
studies in squirrel monkeys have demonstrated that these calls can be
eliminated by lesions of the cingulo-thalamo-1 imbic system. In collaboration
with Dr. John D. Newman from the Laboratory of Comparative Ethology (NICHD),
we have began to record "isolation" calls in normal infants and infants who
received neonatal amygdalectomy . Our preliminary findings indicate several
alterations in the production of isolation calls in infants with
amygdalectomy, possibly reflecting a lack of separation anxiety in these
animals .
Experiment 2
The cortico-nonlimbic habit system becomes functional in female infants
earlier than in males. The evidence for this sexual dimorphism comes from the
findings that female infants learn visual discrimination habits faster than
males, and neonatal ablation of area TE impairs learning of female but not of
male infants. These two functional differences, which are apparent only when
the monkeys are less than about six-months-old, indicate that area TE or its
striatal targets matures faster in females than in males. In a recent
neuroendocrinological study, we have further demonstrated that the high levels
of testosterone present in male infants before and shortly after birth are
probably responsible for this sexual dimorphism, since a significant
correlation appeared in male infants between their testosterone levels and
learning scores (the higher the level the poorer the scores), and also because
orchiectomy in male infants actually speeded their rate of habit formation to
equal that of females. We extended this experiment bv adding a group of
infant females that were ovariec tomized at birth and then received either
testosterone proprionate (TP) or dihydrotestosterone (DHT), the two active
forms of testosterone. The treatments proved to be ineffective, but perhaps
only because they were given after the period in which testosterone exerts its
organizational effect on the brain. To test this proposal, we are currently
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adding to the experiment a group of infant females that are receiving
testosterone treatments prenatally. If the learning ability of this new group
of females were to be developmentally delayed as it is in normal male infants,
it would provide the first direct experimental evidence that gonadal hormones
in the primate fetus influence the maturation of a telencephalic system
important for learning.
Experiment 3
The 2-deoxyglucose method was applied postoperatively to a series of infant
monkeys that had received optic tract section combined with forebrain
commissurotomy at 1 day, 1 week, and 1, 2, 4, and 6 months of age. In all
visual cortical areas of the intact hemisphere, LCGU was lowest in the
youngest subjects, peaked at 4 months, and then declined in the 6-month-old
subject to levels found in adults. This finding is consistent with behavioral
data indicating that adult levels of visual object recognition probably do not
develop until about this time and that neonatal removal of inferior temporal
cortical area TE produces a significant sparing of this function. But a
sex-related factor could also have affected the results, inasmuch as the peak
metabolic activity seen at 4 months of age was found in a female infant,
whereas the lower levels of metabolic activity seen both at two months and at
six months of age were found in males. It is therefore possible that the
results reflect the sexually dimorphic maturation of the cortico-nonlimbic
visual pathway described in Experiment 2. To test this possibility, and also
to complete the developmental sequence, three-month-old male and female
infants have been added to the study.
Experiment 4
Direct evidence that limbic and cortical systems differ in rate of development
has been provided by our developmental neurobehavioral studies. In previous
experiments, we found that the distribution of opiate and muscarinic receptors
in the macaque brain is adult-like at birth in limbic and striatal structures
but is not yet fully developed in neocortical areas. Thus, like many other
aspects of neocortical maturation, opiatergic and cholinergic mechanisms
continues to develop postnatally. We are continuing to follow the
developmental course of these mechanisms by examining them in infants of
different ages.
Experiment 5
Visual recognition in adult monkeys is critically dependent on a neural system
that includes both the inferior temporal cortical area TE and limbic
structures. As described in Experiment 1, however, infant monkeys given
lesions of area TE show significant sparing of visual recognition ability.
Through the use of behavioral, anatomical, and metabolic mapping techniques,
we will test two different hypotheses that could account for this sparing of
function, namely, (1) that afferents from other visual cortical areas
innervate the limbic tissue that was deafferented by the area TE lesions, and
(2) subcortico-limbic interaction substitutes for the absent cortico-limbic
interaction.
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Experiment 6
In collaboration with investigators from The Johns Hopkins School of Medicine,
we have begun an assessment of the decline of learning and memory in aged
monkeys. We found impairments in a wide variety of learning and memory tasks,
suggesting that there is widespread cerebral dysfunction in aged rhesus
monkeys, probably due to the vulnerability of multiple neural systems to
increasing age. At the same time, these learning and memory impairments vary
widely from one aged animal to another within a given task, and there is no
correlation in degree of impairment for a given aged animal across tasks,
suggesting that the distribution and density of age-related neuropathological
changes is likely to vary considerably from animal to animal. This
possibility will be investigated directly through postmortem localization of
neuritic plaques and depletion of cholinergic and other neurotransmitters.
Thus, the use of the aged nonhuman primate as a model for human aging will
provide important information regarding the relationship between age-related
cognitive changes and pathological alterations in the brain.
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
Developmental studies of the effects of early brain damage are of great
importance for the assessment and understanding of those errors of central
nervous system maturation that cause children to become autistic, dyslexic,
learning disabled, or mentally retarded. This project will provide the first
comprehensive evaluation of the social and cognitive development of monkeys
suffering from an amnesia induced by limbic lesions early in infancy as
compared to those rendered amnesic in adulthood, i.e. after memories have been
formed and consolidated in cerebral tissue outside the limbic system. In
addition, comparison of the effects of early and late cortical and subcortical
lesions will help answer whether or not compensatory mechanisms always operate
to promote recovery from early brain injury. Our preliminary results suggest
otherwise. In assessing the effects of early and selective temporal-lobe
damage on infant, juvenile, and adult behavioral patterns, this project will
help to evaluate two provocative proposals from the clinical literature: (a)
that early dysfunction of the limbo-thalamic memory system is one cause of
childhood autism, a syndrome characterized by dramatic social and emotional
disturbances not seen in adults with the same neuropathology; and (b) that the
reason a pure case of global anterograde amnesia like the one seen in adults
has never been reported in a child is that the clinical picture of an amnesic
child, being overlaid with autism, is entirely different from the clinical
picture of an amnesic adult. In addition, whereas our neurobiological studies
indicate how brain maturation normally progresses postnatally, our
neuroendocrinological studies demonstrate how the perinatal hormonal
environment may influence brain maturation and, consequently, the development
of cognitive functions. Finally, the studies in young adult monkeys together
with those in normal aged animals are providing the anatomical and chemical
basis for understanding the memory disorders in humans that accompany
cerebrovascular nnd other cerebral accidents and diseases as well as normal
aging.
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PROPOSED COURSE OF RESEARCH:
Our goal is to continue examination of the effects of neonatal limbic lesions
on social and emotional behavior as well as on cognitive memory and habit
formation at several periods throughout development from infancy to adulthood
in order to test whether such a preparation does indeed provide an animal
model of childhood autism. With the discovery of an important sexual
dimorphism in the development of the habit system, we plan to pursue studies
aimed at determining the neuroendocrinological substrate of that dimorphism.
We shall also pursue studies to determine how recognition memory measured by
preferential viewing differ from recognition memory measured by problem
solving. This will help determine which capacities of the memory system
appear late in ontogenetic development and, by implication, whether the
phenomenon of infantile amnesia might be due to the absence of a fully
functional cognitive memory system in early childhood. We shall continue our
attempts to follow the development of neurochemical receptors in infant
monkeys. In addition, new experiments have been initiated to study the neural
mechanisms by which visual object recognition can develop in the absense of
higher-order areas of the visual pathway. Finally, we will pursue our
longitudiual study on learning and memory decline in normal aging.
PUBLICATIONS:
Presty, S.K., Bachevalier, J., Walker, L.C., Struble, R.C., Price, D.L.,
Mishkin, M. , and Cork, L.C. Age differences in recognition memory of the
rhesus monkey (Macaca mulatta). Neurobiol. Aging (in press)
665
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02039-05 LN
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 cheraclen or less. Title must tit on one line between the borders.)
Pharmacology of cognitive memory and habit formation
PRINCIPAL INVESTIGATOR (List other prolessional personne/ twtoiv the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others;
T.G. Aigner
M. Mishkin
R.Q. Wan
R.M. Brown
M.R. DeLong
D. Price
Senior Staff Fellow
Chief
Visiting Fellow
Chief
Professor
Professor
LN NIMH
LN NIMH
LN NIMH
NS NIDA
Johns Hopkins Univ.
Johns Hopkins Univ.
COOPERATING UNITS (It any)
The Johns Hopkins University School of Medicine
National Institute on Drug Abuse
UkB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
3.0
PROFESSIONAL
2.0
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
n (b) Human tissues 0 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Evidence from patients with Alzheimer's disease suggest that the basal
forebrain cholinergic system plays an important role in memory. In support of
this proposal, we have found impaired visual recognition memory in monkeys
with lesions to the major nuclei of this system. We have found further that
recognition memory in normal monkeys can be improved by the cholinesterase
inhibitor physostigmine and impaired by the cholinergic muscarinic-receptor
blocker scopolamine. In addition, our results indicate that scopolamine acts
at a very early stage of memory, preventing information from entering even
into an immediate store.
Based on previous results indicating that THC may be exerting its effects
through an action on the limbic system, we administered this drug to monkeys
performing spatial reversal, a task known to be sensitive to hippocampal
damage. Doses equal to or even greater than those that impaired recognition
memory did not affect performance on this task, though performance did appear
to be affected several days after the last dose of THC, raising the
possibility of a delayed abstinence effect.
In a series of experiments on habit formation, we administered the
dopaminergic-neurotoxin MPTP to monkeys, but failed to show learning
impairments at doses that did not also impair motor function. Administration
of MPTP did make the animals more sensitive to the disruptive effects of
scopolamine, however, suggesting that the cholinergic-dopaminergic balance in
the neostriatum had been compromised.
667
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sPo ai4.«it
ZOl MH 02039-05 LN
PROJECT DESCRIPTION:
The cholinergic system is now suspected to play a critical role in mnemonic
processes both in humans and in primates. Our work during the past year has
continued to concentrate on identifying the processes and functions in which
acetylcholine influences memory in primates. We previously showed that
scopolamine, a muscarinic-cholinergic receptor blocker, impairs recognition
memory in monkeys performing a delayed nonmatching-to-sample task with
trial-unique objects. In addition, we showed that scopolamine produced
greater impairments when administered before, rather than after, the
acquisition trials, suggesting that this drug has mainly an anterograde
effect, influencing storage more than retrieval.
Experiment 1
We previously showed that recognition memory impairments could be produced in
monkeys by combined but not by separate neurotoxin-induced lesions of the
three major nuclei of the basal forebrain cholinergic system. In addition,
these animals showed an altered sensitivity to cholinergic drugs compared to
normal animals. The cells of the basal forebrain cholinergic nuclei,
particularly those constituting the nucleus basalis of Meynert , the major
source of cholinergic innervation of the cortex, form an extremely complex
shape that renders them very difficult to locate and damage by standard
stereotaxic methods. In our original studies, we used electrophysiological
recording to identify the boundaries of the anterior commissure (AC), which
then served as a reference for locating the basal forebrain nuclei. Although
we obtained significant functional impairments with this method, the lesions
were not complete and were highly variable. We are therefore planning a
repeat experiment in which we will try two new methods for producing lesions
of this system. One method will involve direct visualization of the dorsal
surface of the AC which we will then use to calculate the areas into which the
neurotoxin will be injected. The second method involves the use of magnetic
resonance imaging (MRl) to locate the AC and surrounding structures. We have
designed and built a specially modified stereotaxic frame that will permit use
with the MRI scan. The initial scans have shown even more detail than
anticipated, so that this method promises to be useful not only for this
study, but for others as well.
Experiment 2
During the past year we have continued to develop an automated testing
apparatus that is allowing us to study drug effects on memory in monkeys more
rigorously than previously possible. The apparatus consists of a color
monitor that is outfitted with a touch-sensitive glass screen, both of which
are connected to a personal computer. We have designed and written programs
that utilize the capacity of the computer to generate and to present to the
monkeys a wide variety of graphic symbols in a series of experimental
paradigms .
In our first use of the apparatus last year, we examined the effects of
scopolamine on delayed nonmatching-to-sample (DNMS) with a small set of
symbols, a test of recency memory. In this task, a single symbol (a 2S mm
668
ZOl MH 02039-05 LN
colored square) was presented first (acquisition) and the monkey was required
to touch the screen within the boundaries of the symbol. Then, after a delay
of 0, 1, 3, 10, 30, or 60 sec, the original symbol was presented with another
symbol of a different color in a choice trial (test), and the monkey was
rewarded for touching the novel symbol. The colors of the symbols and the
delay interval were randomly selected by the computer, which recorded the
position of the touch, the symbol selected, and the reaction time to touch the
screen during correct and incorrect choices for every trial. In this initial
study, we obtained forgetting curves, that is, a decrease in the number of
correct choices as the delay interval increased, during control sessions. In
addition, we found that scopolamine, in doses known to impair performance in
the Wisconsin General Testing Apparatus (WGTA) , also impaired performance in
the automated apparatus. More importantly, we found that scopolamine produced
its greatest effect in the interval between 0 and 1 sec. That is to say,
there was no effect of scopolamine at the 0 sec delay, but by 1 sec, the
number of correct choices was significantly decreased. The forgetting curve
from 1 to 60 sec paralleled that of the control sessions, suggesting no
further decrement in performance with increasing delay. These results, when
combined with our previous findings that the effects of scopolamine are mainly
anterograde, suggested that scopolamine exerts its effects at a very early
stage of memory, preventing information from entering even into an immediate
store.
Experiment 3
In our next series of studies on the effects of cholinergic drugs on memory,
we modified the DNMS procedure in the automated apparatus to more closely
resemble that used in the WGTA, where trial-unique objects are used. In this
new test, the computer randomly generated a new series of symbols in a variety
of different shapes and colors, such that the animal never saw the same symbol
more than once each session. The monkeys were able to perform this task more
accurately than they were the recency task. Although the animals did more
poorly with long delays than with short ones, they were nonetheless able to
perform at a level of nearly 90% correct choices at the 60-sec delay, a value
approximately 15% greater than at the comparable delay on the recency test.
Although scopolamine administration (10.0, 17.8, and 32.0 ug/kg) produced
dose-related impairments in DNMS with a large sample set, the impairment was
less than that observed with the small sample set, suggesting that recognition
memory is less susceptible to disruption than recency memory.
In addition to the DNMS task, each session also included a new task, called
recognition scan. In this test, the screen is divided into nine sectors
arranged in a 3 X 3 matrix. On the first trial, a symbol is randomly located
in one of the nine sectors, which the animal must then touch for reward and
for the task to continue. After a 5-sec interval in which the screen is dark,
the original symbol, now in a randomly selected new location, and a novel
symbol are projected on the screen. In this and all subsequent trials, the
animal must touch the new symbol to obtain a reward. If the animal correctly
touches the novel object on the screen, the procedure is repeated after a
time-out, this time with three symbols, the two previously seen, plus a new,
previously unseen, symbol. With each new sjnnbol addition, the position of
all of the symbols are randomly relocated to one of the nine positions on the
669
ZOl MH 02039-05 LN
screen so that the animal cannot rely on spatial cues to determine which
symbol has been newly added. A trial is continued until the animal has either
correctly touched all nine symbols or has incorrectly touched a previously
seen symbol. Thus, we can determine the average number of correct responses,
or list length, that the animal can recognize. Three different versions of
this task are used in a session. In the first, the symbols differ in shape,
but are the same color on each trial. In the second, the symbols are the same
shape, but are different colors. In the third task, the symbols change in
both dimensions. Under control conditions, the animals correctly recognized
an average of 7 out 9 symbols correctly when both shape and color were varied,
5.7 when shape was varied, and 5.1 when color was varied. On the
recognition-scan test, scopolamine produced dose-related impairment on all
three measures, although the degree of impairment again was related to set
size. That is, 10 ug/kg of scopolamine significantly reduced the mean list
length when the symbol varied only in color, a condition in which the set size
was limited to 9, but had no effect when both symbol and color were varied, a
condition in which set size was nearly unlimited. Administration of the
cholinesterase inhibitor physostigmine produced the opposite effect, i.e. it
improved performance, and, in this case, the smaller the set size, the greater
the improvement. Taken together, these results replicate our original
findings, obtained in the WGTA, that cholinergic agonists and antagonists
improve and impair recognition memory, respectively, and do so in the same
dose range in both procedures.
Experiment 4
We have continued our collaboration with the National Institute of Drug Abuse
on the cognitive effects of del ta-9-tetrahydrocannabinol (THC), the active
ingredient of marijuana. Previously, we reported that THC impaired
recognition memory, but not habit formation, suggesting a possible selective
action of the drug on limbic structures. Indeed, a number of reports by
others suggested that THC may have a deleterious effect on the hippocampus
specifically. To examine this possibility further, we next studied the
effects of orally administered" THC on spatial reversal, a task known to be
especially sensitive to hippocampal lesions in monkeys. Daily oral doses of
4, 8, or 16 mg/kg failed to affect task performance, although all animals
showed overt behavioral signs of marijuana intoxication. Of interest,
however, was an impairment in control performance that occurred approximately
7 days after the last dose of either 8 or 16 mg/kg. We had previously
reported a delayed abstinence syndrome in DNMS performance following 21 days
of 4 mg/kg of THC that was similar to that observed here. Because recognition
memory is known to depend equally on the hippocampus and the amygdala, we next
examined the effects of THC on object reversal, a task in which impairments
are observed following lesions of the amygdala in monkeys. The same four
monkeys used in the spatial reversal task were trained to displace one of two
visually different objects to a high criterion of 90% correct choices in one
session. In the task, the left-right position of the positive object on the
lateral food wells of the test tray is varied in a pseudorandom sequence.
After reaching criterion, the animal is rewarded only for displacing the
originally negative object, and so on for several reversals. Again, however,
doses of 4, 8, or 16 mg/kg of THC failed to impair i^er f ormance , and signs of
an abstinence syndrome were noted, although these were not as severe as those
670
ZOl MH 02039-05 LN
during place reversal. Apparently, either dysfunction in limbic structures is
not responsible for the recognition impairment produced by THC or the reversal
tasks were not sensitive enough to detect the dysfunction. This issue will be
examined further with newly designed tests.
In a separate study, we examined the effects of orally administered THC (2, 4,
or 8 mg/kg) on performance of the color memory task in the automated testing
apparatus. THC, like scopolamine, impaired performance of this task in a
dose-related manner. In contrast to scopolamine, however, which produced its
greatest effect between 0 and 1 seconds, THC produced its greatest impairment
at the longer delays (30 and 60 sec). Thus, although THC and scopolamine
produce equivalent impairments in short-term recognition, they do so starting
at different delays, suggesting that these two drugs have different mechanisms
of action.
Experiment 5
In the past year we have continued our study of the contributions of the
neostriatum to habit formation. We had previously administered
l-methyl-4-phenyl-l,2,3, 6-tetrahydropyridine (MPTP), a neurotoxin that
selectively destroys the nigrostriatal dopamine system when given in high
doses, to four monkeys. This drug has been shown to impair motor function
permanently in much the same way as that observed in Parkinson's disease. Tlio
monkeys were tested both for motor function as well as concurrent
discrimination learning with 24-hour intertrial intervals, a measure of their
ability to acquire habits. One pair of monkeys was given MPTP in a series of
small doses at approximately one- month intervals, such that any effects on
learning unaccompanied by motor deficits could be identified. Only when the
cumulative dose of MPTP exceeded 4.0 mg/kg (approximately twice that required
to produce motor impairment if given in higher doses or at shorter intervals),
did we observe learning impairment. At these dose levels, however, motor
impairment was also observed. When testing was continued following
termination of drug administration, both learning and motor impairments
dissipated. In the other pair of monkeys, MPTP was administered in higher
doses and at shorter intervals (0.5 mg/kg, once per week for three weeks).
Both of these monkeys developed severe movement disorders that persisted for
two to three weeks before gradually resolving. Only one of these monkeys
showed evidence of a learning impairment after this dosing regimen, although
when a fourth dose of MPTP was administered one month later, neither animal
showed a learning deficit. Indeed, both monkeys appeared to be less sensitive
also to the drug's effects on motor function.
Because of the known interaction of the cholinergic and dopaminergic systems
in the striatum, we tested the effects of scopolamine in these animals to
determine if they might be hypersensitive to blockade of the cholinergic
system after the dopaminergic system was compromised. We had previously shown
in normal monkeys that a high dose of scopolamine (32 ug/kg) slowed learning
rates only slightly. In the two monkeys given the protracted MPTP regimen,
this same dose of scopolamine was administered prior to each daily session.
During the first scopolamine series, one monkey, which normally attained
criterion performance in six days, failed to learn within the 40 day period
allowed. When drug administration was terminated, this animal was again able
671
ZOl MH 02039-05 LN
to reach criterion within six days. The second animal, which like the first
averaged approximately six days to learn under control conditions, was able to
learn the discriminations during scopolamine testing, but required 25 days to
do so. Motor function tests in these two animals showed an initial
disruption, which recovered at a faster rate than did the learning
impairment. This procedure was repeated on two subsequent series and both
monkeys appeared to be less sensitive to disruption by scopolamine than they
were initially, although both still required more than 20 days to learn the
discriminations. The two monkeys given the accelerated MPTP dosing regimen
were even more sensitive to the scopolamine-induced learning impairments. One
animal failed to reach criterion within 40 days during the three scopolamine
test series; the second animal failed on the first series, but then learned in
22 and 35 days on the next two series. Both monkeys also showed severe
impairments in motor function during scopolamine testing. Further, their
motor performance did not improve as it did in the first two animals. These
results suggest that MPTP administration in all four animals increased their
sensitivity to the disruptive effects of scopolamine, and that the combination
of treatments affected motor performance and habit formation simultaneously.
During the next few weeks, we plan to make further examination of the
dopaminergic function of these four monkeys. Although they presently show no
explicit effects of MPTP administration and, indeed, perform normally on all
tests of motor function, the results just described for scopolamine challenge
several months after the last dose of MPTP suggest that the dopaminergic
system may have been damaged, but perhaps not sufficiently to either prevent
recovery or compensation by remaining neurons. Since the dopaminergic system
is known to compensate for neuronal loss up to a point, these animals may be
more sensitive to challenge by other drugs or may even start to show deficits
as they age, since dopaminergic activity is known to decrease with advancing
age. By means of positron emission tomography (PET) and the use of
radiolabeled compounds that specifically bind to the central Dl and D2
dopamine receptor subtypes, we may be able to identify and quantify the extent
of damage to this system in our monkeys. In addition to the PET studies, we
also plan to remove samples of cerebrospinal fluid from these animals to
determine dopamine metabolite levels. These studies may su-ggest areas for
further analysis by drug challenge.
SIGNIFICANCE TO MENTAL HEALTH RESEARCH:
Our results continue to provide convincing evidence that cholinergic
mechanisms are critical for cognitive memory in monkeys. These mechanisms
appear to play a more important role in storage than in retrieval and,
further, the effect on storage occurs at a very early stage of processing.
Our studies on the effects of MPTP on habit formation are inconclusive
concerning the role of the dopaminergic system in this function, although our
finding that motor functioning recovers even while the animals remain
hypersensitive to disruption by scopolamine has important implications for
understanding the neurochemistry and plasticity of this system. Finally,
although our findings indicate that administration of high doses of THC does
not directly impair performance on certain tests of limbic function, there is
evidence that performance may be affected several days after the last
administered dose, suggesting a delayed abstinence syndrome to the drug.
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ZOl MH 02039-05 LN
PROPOSED COURSE OF RESEARCH:
We will continue to evaluate the effects of both cholinergic and
noncholinergic compounds on cognitive memory and habit formation in monkeys.
.In addition, we will continue to examine the role of the basal forebrain
cholinergic system in memory following direct damage to this system and to
examine the actions of drugs in monkeys with lesions of this system. We will
expand our research into the role of other neurotransmitters and neuropeptides
in mnemonic processes by examining the direct effects of these agents on
memory, as well as their ability to block the now well-documented impairments
produced by scopolamine. Finally, we will continue to study the effects of
high doses of THC during and after periods of chronic drug administration.
PUBLICATIONS:
Aigner, T.G. and Mishkin, M. Naloxone improves recognition memory in monkeys.
Psychopharmacol . (in press)
Aigner, T.G., Mitchell, S.J., Aggleton, J. P., DeLong, M.R., Struble, R.G.,
Price, D.L., Wenk, G.L., and Mishkin, M. Effects of scopolamine and
physostigmine on recognition memory in monkeys with ibotenic-acid lesions of
the nucleus basalis of Meynert. Psychopharmacol . 92: 292-300, 1987.
Wenk, G., Engisch, K. , McCall, L., Mitchell, S. , Aigner, T. , Struble, R. ,
Price, D., and Olton, D. [H3]Ketanserin binding increases in monkey cortex
following basal forebrain lesions with ibotenic acid. Neurochem. Int. 9: 557-
562, 1986.
673
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02040-04 LN
PERIOD COVERED
October 1, 1986 through September 30. 1987
TITLE OF PROJECT (80 characters or less, rule musf tit on one line tKtween ttie borders.)
Functional analysis of neurotransmitter systems
PRINCIPAL INVESTIGATOR (List ottter professional personnel trelow the Principal Investigator.) (Name, title, laboratory, and Institute attiliatlon)
PI:
T
P. Pons
Others:
M
Mishkin
D
P . Friedman
J
Bachevalier
L
G. Ungerleider
C
B. Pert
A
Routtenberg
Guest Researcher
Chief
Project Officer
Visiting Scientist
Research Psychologist
Chief, Sec. Brain Chemistry
Professor
LN NIMH
LN NIMH
NRB NIDA
LN NIMH
LN NIMH
NSB NIMH
Northwestern Univ.
COOPERATING UNITS (if any)
National Institute on Drug Abuse
Section on Brain Chemistry, NIMH
Northwestern University
LAB/BRANCH
Laboratory of Neuropsychology
INSTITUTE AND LOCATION
NIMH, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0
PROFESSIONAL;
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been terminated.
675
PHS 6040 (Rev. 1/84)
SPO 9I4.SII
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MK 00471-32 LPP
PERIOD COVERED
nrrnbpr 1, 1986 to Septpmbpr 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Studies of Heredity and Environment in Schizophrenia
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute ettillation)
PI:
Allan F. Mlrsky, Ph.D.
Chief
LPP, NIMH
COOPERATING UNITS (it any)
Institute for Research on Kibbutz Education, Haifa University, Israel; Hebrew
University, Israel; Qranim Teacher's College, Israel; Bar Ilan University,
Israel; University of Chicago, Illinois
UVB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL;
1.0
0.5
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
S (a1) Minors
Q (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been composed of the following studies: (1) An intensive
multi-disciplinary study of a family with MZ quadruplets (daughters) concordant
as to schizophrenia but discordant as to severity and outcome; (2) Studies of
Danish~adoptees and their biological and adoptive families; (3) A study of
children (of schizophrenic and control parents) reared in town or kibbutz in
Israel. We maintain contact with the quadruplets but have not pursued active
studies with them during the past two years. The Danish adoptees are of
continuing Interest to us and we are preparing additional reports on factors
involved in their psychiatric outcome. The Israeli children are the subject of
Intensive research efforts and we are currently conducting further behavioral
and biological studies with them.
677
PHS 6040 (Rev 1/84)
SPO SI4-«II
Description
A. Other Personnel
Loring Ingraham, Ph.D.
Shaul C. Sohlberg, Ph.D.
Sol Kugelmass, Ph.D.
Joseph Marcus, M.D.
Judith Shotten, M.A.
Eugene P. Tassone
Olive W. Qulnn, Ph.D.
Patricia Lowing, Ph.D.
Deborah Levy, Ph.D.
ZOl MH 00471-32 LPP
Staff Fellow LPP, NIMH
Clinical Psychologist Israel
Bar Ilan University
Professor of Psychology Israel
Hebrew University
Professor of Child Psychiatry Chicago,
University of Chicago Illinois
Psychiatric Social Worker Israel
Psychologist LPP/NIMH
Guest Researcher LPP, NIMH
Private practice Michigan
Director Psychophysiology New York
Lab, SUNY Stonybrook
B. Objectives
The project is composed of the following studies: (1) An intensive multi-
disciplinary study of a family with MZ quadruplets (daughters) concordant as
to schizophrenia but discordant as to severity and outcome. We are continuing
our contacts with this family to see what happens in the clinical course of
these women and to see how the course is related to earlier and to current
life experiences; (2) Studies of adoptees and their biological and adoptive
families in Denmark; (3) A study of children (of schizophrenic and control
parents) reared in town or kibbutz in Israel.
C. Major Findings
The objectives of this project are to understand how hereditary and
environmental factors interact to make for schizophrenic outcomes of varying
types and degrees.
1. The Genain Quadruplets
Our recent studies of the Genain quadruplets are summarized in the annual
reports of the previous four years. This year, we expect to see publication
of two reviews of prior studies of these women, one in a monograph on aging
and schizophrenia and the other to be Included in a special issue of the
Schizophrenia Bulletin dedicated to long-term follow-up studies in
schizophrenia. This edition of the bulletin will be edited by Drs. William T.
Carpenter and Thomas H. McGlashan. Dr. Olive Qulnn, who is a Guest Researcher
in the LPP, maintains contact with the Genalns and will be a coauthor of the
two reviews referred to above. Interest in the quadruplets continues to be
high, as judged by the steady demand for photographs of them for psychology
text books.
Some time ago. Dr. Deborah Levy visited the Genalns and made smooth
pursuit eye movement recordings of all four women In their home. The results
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of these measurements have been incorporated in the Schizophrenia Bulletin
chapter referred to above.
2. The Danish Adoptee Study — Reanalysis of the Data
Using data from Danish health records, in a now-classic study, Rosenthal,
Kety and Wender compared the frequency of schizophrenia spectrum disorders in
two groups of persons adopted in infancy or early childhood: those with a
psychotic parent (index group) and those whose biological parents had never
had psychiatric treatment (control group). Significantly more disorder was
found in the index than the control group. We have recently completed a
reanalysis of the extensive interview data collected on this group of
subjects, focusing on stressful events during childhood and adult outcome. In
a report we have prepared for publication, we present evidence that more
stressful events were found in the childhood histories of index subjects who
developed schizophrenia or schizotypal personality disorder (SPD) than in the
histories of index subjects free from these disorders, and that greater number
of stressors are associated with more severe outcomes. In matched control
adoptees, of biological parents free from psychiatric illness, we found less
psychiatric illness but an equal number of stressful events during childhood
when compared to index adoptees. These results are consistent with a
stress-diathesis model, where both genotype and environment contribute to
adult illness.
Further analyses of these data revealed that the most common stressors
among index subjects with schizophrenia or SPD were troubled family
environments. These results are consistent with the current body of
literature investigating expressed emotion as a factor in the relapse of adult
schizophrenic patients.
3. The Israel Kibbutz — High Risk Study
In 1984, the Laboratory published in the Schizophrenia Bulletin a report
of work begun in 1962 on the study of children at risk for schizophrenia in
Israel, which was designed and initiated by David Rosenthal. The study has
examined 100 children, of whom 50 had one schizophrenic parent (index
subjects), and 50 were born to two nonschizophrenic parents (control
subjects). Half of both the index and control groups were reared in towns in
traditional nuclear families, while the remaining half were reared in communal
settings on kibbutzim.
In broad outline, the results indicate that index children were
discriminable from controls in many areas of function, but kibbutz and town
children did not differ on the experimental examinations. Furthermore,
kibbutz versus town rearing had no discernible effect on the performance or
behavior of the high-risk index children. Index children were found to be
poorer in psychosocial adjustment, perform more poorly in school, manifest a
number of neurological "soft signs," and show deficits on psychological tests
requiring high levels of attention, visual integration, and visuomotor
coordination. An important negative finding was lack of differences between
index and control children on psychophysiologic measures of arousal and
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habituation in the first examination.
We have also conducted follow-up interviews with the study subjects, who
are now in their mid-twenties, at the peak of their risk period for
schizophrenic breakdown. Ninety of the surviving 99 subjects have been seen.
Results show that nine subjects fall within the "schizophrenia spectrum" (of
whom six have DSM-III schizophrenia), six from kibbutz backgrounds, and three
from towns. When all DSM disorders are considered, more than five times as
many ill subjects fall within the index (N=23) than within the control group
(N=4). Furthermore, when schizophrenia itself is excluded, the remaining
subjects with history of illness (including DSM-III Major Affective Disorder
or Dysthymic Disorder) are found predominantly in the index-kibbutz cell (16
of the total of 23 in the cell, including 9 with affective disorder). Other
significant preliminary results include persistence of attention-related
deficits in the index group, and continued poor social and work adjustment in
high-risk subjects.
During the past year we have initiated a second follow-up of these
subjects, who are now in their early thirties. At this time, the majority of
the subjects who will develop a schizophrenic disorder should have become
ill. In order to diagnose these subjects accurately, a Hebrew version of the
SADS-L with modifications allowing the accurate assessment of schizophrenia
spectrum disorders, has been developed. Social workers who are blind to the
subjects' index or control status have been trained in its use. In addition,
our laboratory has developed and validated Hebrew versions of
neuropsychological tests for use with this population. Subjects have been
contacted and are currently being interviewed. As of this writing, we
estimate that 40 of the subject cohort have been seen.
Significance to Biomedical Research and to the Program of the Institute
The issue of the mode of heritability of schizophrenia, and factors which
modify its development, may be the highest priority of the Institute. This
work contributes significantly to our knowledge in this area and ultimately,
to our capacity to treat and prevent schizophrenia and related disorders. Our
study of childhood stress and adult schizophrenia spectrum disorder suggests a
possible direction for prevention of adult psychiatric morbidity.
The studies here, which focus on schizophrenia spectrum disorders as well
as pure DSM-III schizophrenia, aid in the identification of milder syndromes
genetically linked to schizophrenia. The identification of such syndromes
would aid in the search for clear pedigrees of schizophrenic Illness by
allowing more individuals to be studied and tested for biological markers than
the current low number of biological relatives with frank schizophrenia.
Proposed Course
Genains: Drs. Quinn and Mirsky maintain contact with the Genalns and
exchange correspondence on an occasional basis. We are considering the
possibility of inviting the Genalns back to NIMH for another series of
follow-up studies to include electrophysiological and other measurements that
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ZOl MH 00471-32 LPP
were not obtained during their visit in 1981.
Denmark: We are investigating the possibility of a follow-up study of the
Danish adoptees first studied by Rosenthal. At the time of their previous
assessment, a portion of the subjects was too young to have passed though the
major risk period for schizophrenia. Such a study would allow for continuing
Investigation of the longitudinal course of schizophrenic illness as well as
potentially confirmatory evidence on the role of stress in the development and
relapse in schizophrenia.
Israel: In addition to the current follow-up study of this population, our
laboratory has also taken steps to validate previously reported findings from
this unique sample. We are planning to interview the siblings of the probands
in this study in order to increase economically the sample size and the power
of statistical tests conducted with this sample. In addition, we are planning
to reinterview the parents in order to use contemporary diagnostic tools to
confirm the earlier diagnosis of schizophrenia in index subjects' parents. In
addition, family history interviews of these families will help clarify the
role of familial schizophrenic and affective illness in the development of
psychopathology among these subjects.
As the interviews are completed, we will be collecting neuropsychological
test data as well as CT images from consenting subjects. This information
will also build on earlier studies of this population to present a fuller
picture of the psychobiological and environmental factors leading towards
adult psychiatric illness.
Expressed emotion: Our current findings in the Danish sample, taken in
conjunction with the developing literature on expressed emotion, suggest that
further exploration of the social environment of adult schizophrenic patients
may be useful in identifying modifiable environmental factors that could
reduce the incidence of relapse. We are currently developing an approach to
the study of Expressed Emotion that would shift the focus away from the
parents of schizophrenic adults living at home and place it on the social
environment of the larger number of schizophrenic patients who live outside of
their family of origin. We believe that this approach will lessen the
potential for inappropriate stigmatizing of the families of schizophrenic
patients, and lead to more effective support strategies for adult
schizophrenic patients.
Publications
Mirsky, A. P., Quinn, O.W. , DeLisi, L., Schwerdt, P. and Buchsbaum, M.: The
Genain quadruplets: A 25 year follow-up of four monozygous women discordant
for the severity of schizophrenic Illness. Cohen, G. and Miller, N.E. (Eds.):
Schizophrenia, Paranoia, and Schizophreniform Disorders in Later Life, in
press, 1987.
Mirsky, A.F., Duncan, C.C. , Silberman, E.K., Nagler, S., Kugelmass, S.,
Sohlberg, S., and Shotten, J.H.: Early neuropsychological and other
behavioral predictors of later psychotic disorder. In Shagass, C., Simpson,
681
ZOl MH 00A71-32 LPP
G., Brldger, W, , Josiassen, R. , Stoff, D. , and Weiss, K. (Eds.): Proceedings
of the IVth World Congress of Biological Psychiatry. New York: Elsevier,
1986, pp 1118-1120.
Mirsky, A.F.: The Israeli high-risk study: Reply to Kaffman. Schizophr.
Bull. 12: 158-161, 1986.
Ingraham, L.J., and Wright, T.L.: A cautionary note on the interpretation of
relationship effects in the social relations model. Soc. Psychol. Q., 49:
93-97, 1986.
Ingraham, L. J., and Wright, T.L.: A social relations model test of
Sullivan's anxiety hypothesis. J. Pers. Soc. Psychol., 52: 1212-1218, 1987.
Mirsky, A.F., and Quinn, O.W.: The genain quadruplets. Schizophr. Bull, in
press, 1987.
Mirsky, A.F.: Children at Risk for Schizophrenia. In Watt, N.F., Anthony,
E.J., Wynne, L.C., and Rolf, J.E. (Eds.): (Book Review). Br. J. Child
Psychiatry, in press, 1987.
Mirsky, A.F.: Frontiers of Infant Psychiatry. Vol. 2. In Call, J.D.,
Galenson, E. , and Tyson, R.L. (Eds.): (Book review). Contemp. Psychol., In
press, 1987.
Mirsky, A.F.: The Frontal Lobes. In Stuss, D.T., and Benson, D.F. (book
review): J. Clin. Neurophysiol. in press, 1987.
Ingraham, L. J. A medical approach to psychodlagnosis. In review of Gold,
M.S., and Pottash, A.L.C. (Eds.): Dlagnositc and Laboratory Testing in
Psychiatry. Contemp. Psychol. 32: 659-660, 1987.
Marcus, J., Hans, S.L., Nagler, S., Auerbach, J.G., Mirsky, A.F., and Aubrey,
A. : A review of the NIMH Israeli kibbutz-city study and the Jerusalem infant
development study. Schizophr. Bull. 1987, in press.
Mirsky, A.F.: The Israeli high-risk study. In Dunner, D.L. , and Gershon,
E.S. (Eds.): Relatives at Risk for Mental Disorders. New York: Raven Press,
in press.
682
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00484-27 LPP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (SO characters or his. Title must lit on or^e line between The borders.)
Psychophys-lnlogiral Responsiviry and Rphavinr in Srh-l znphreni a
PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliation)
PI:
Theodore P. Zahn , Ph.D.
Research Psychologist
LPP, NINfH
COOPERATING UNITS (If any)
Laboratory of Socio-Environmental Studies, Child Psychiatry Branch, Laboratory
of Clinical Science, Neuroscience Branch, Biological Psychiatry Branch, and
Clinical Neurogenetics Branch, NIMH; Hypertension-Endocrine Branch, NHIBI.
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAU
0.8
0.9
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
IE] (a1) Minors
n (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (tjse standard unreduced type. Do not exceed the space provided.)
The general purpose of this project is to investigate the roles of autonomic
nervous system (ANS) activity, attention, and information processing and their
Interrelationships in the pathology, etiology, and prognosis of psychiatric
disorders. A second purpose is to determine biological and psychological
processes related to ANS activity and attention. ANS activity is assessed by
peripheral measures, such as skin conductance, heart rate, and skin temperature.
Subjects are tested under conditions of rest, presentation of tones, and
performance on tasks such as reaction time and mental arithmetic.
Biological mechanisms are investigated by correlating these variables with
enzyme activity, neuropeptides, and levels of biogenic amines and their
metabolites and with brain dysfunction as revealed by CT and PET scans.
Studies are being done on unmedicated patients with diagnoses of
schizophrenia, affective disorder, obsessive compulsive disorder, anxiety-panic
disorder, and autism to test the diagnostic specificity of patterns of ANS
activity. Children of parents with bipolar affective disorder are being studied
to determine a possible ANS trait markers. In some studies blood samples are
taken during ANS recording sessions in which stressful procedures are given. In
one, the effects of success and failure to escape an aversive noise are assessed,
and in another, the effects of a dose of yohimbine is being studied. Clinical
trials of various treatments are studied in various groups.
Psychological correlates are studied via clinical background data, clinical
ratings and questionnaires, and by procedural variations. The use of confirmatory
factor analysis in data reduction and to improve quantification of ANS activity is
being explored.
683
PHS 6040 (Rev. 1/84)
GPO S14>«ll
ZOl MH 00484-27
Project Description
A. Other Personnel
Allan F. Mirsky, Ph.D.
Carmi Schooler, Ph.D.
Dennis Murphy, M.D.
David Pickar, M.D.
Thomas Uhde , M.D.
Judith Rumsey, Ph.D.
Judith Rapoport, M.D.
Frank Putnam, M.D.
John Nurnberger, M.D.
Alan Breler, M.D.
Joseph Zohar, M.D.
Chief
LPP,
NIMH
Acting Chief
LSES
, NIMH
Chief
LCS,
NIMH
Chief
SCS,
NSB, NIMH
Staff Psychiatrist
BPB,
NIMH
Staff Fellow
CHP,
NIMH
Chief
CHP,
NIMH
Staff Psyciatrist
LDP,
NIMH
Medical Officer
CNG,
NIMH
Clinical Associate
NSB,
NIMH
Visiting Associate
LCS,
NIMH
B. Objectives
The major objective of this project is the further understanding of the
role of autonomic nervous system (ANS) activity, information processing and
attention, and their interrelationships in psychiatric disorders, primarily
schizophrenia. The overall strategy involves studies of ANS and attentional
relationships to diagnosis and prognosis, studies of the effects of drugs and
other therapeutic interventions, "high risk" and personality studies in normal
volunteers, studies of the effects of various types of stress, and studies of
the measurement of ANS activity.
C. Methods Employed
The general methods of these studies include measurement of ANS activity
through skin conductance (SC) usually measured bilaterally, heart rate (HR) ,
vascular activity (skin temperature and finger pulse volume), and respiration
while subjects are resting, . exposed to a series of nonsignal tones of constant
or variable intensity, and performing tasks. Tasks include tests of attention
using reaction time techniques, tests of perceptual speed, and tasks designed
to be moderately stressful. A mini-computer system is used to run the
experiments and to collect and analyze the data. Studies in various stages of
completion are listed below.
1. Schizophrenia Studies
a. A study of newly admitted, drug-free patients used three tasks
varying in stressfulness and task demands to test the hypothesis, developed in
previous studies, that schizophrenics' ANS does not respond appropriately to
variations in stimulus significance. This study also includes several rest
periods and a series of nonsignal tones for comparative purposes.
b. In current studies, ANS recording is being carried out in two
sessions which include rest periods, a tone series, and two reaction time
tasks. In addition, several methods of assessing more precisely the nature of
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ZOl MH 00484-27
schizophrenic attention deficits using reaction time (RT) techniques are being
compared. (See ZOl MH 00484-25 LPP, 1984-85 for details.) Patients are being
tested on standard neuroleptic medication as well as drug free. The purpose
of this is to compare our results to most of the recent published non-NIH
studies which, for the most part, use medicated patients. Some patients are
also being tested in the "learned helplessness" paradigm described in section
3b below.
c. Tests of the ANS effects of drugs such as pimozide, lithium,
naloxone, GHB, verapamil, propranolol, and prazosin, and treatments such as
hemodialysis and plasmapheresis, have been carried out.
2. Studies on Nonschizophrenic Psychopathology
a. Several confirmed psychophysiological "markers" of schizophrenic
pathology have been detailed in previous annual reports and are summarized
below. In order to determine which of these are specific to schizophrenia,
patients with other types of psychopathology are being tested on the initial
part of the current standard protocol (described in l.b. above) after being
medication free for an appropriate time. These include patients with major
depressive, obsessive-compulsive, and panlc-anxlety disorders (see ZOl MH
00071 BP, 02184 NS, 00153 CHP, and 00336 LCS). In addition, a group of young
men who had a diagnosis of early infantile autism have been studied (see ZOl
MH 00178 CHP). Some patients with affective disorder and premenstrual
syndrome are being tested in the "learned helplessness" paradigm described in
section 3b below.
b. Drug effects are being evaluated in several groups. Some
panic-anxiety patients are tested on imlpramine (double-blind) and will be
compared with patients given placebo, and some we are able to test under both
treatments. We have completed a study of the psychophysiological and
attentlonal effects of a challenge with yohimbine — a noradrenergic alpha-2
antagonist — using a protocol containing rest periods, a mental arithmetic
stress, and a continuous performance task. This was given before and after
placebo and yohimbine on two separate days in a double-blind crossover
design. Fifteen patients with panic disorder, seven of whom are on alprazolam
treatment, are compared with 12 normal controls. This study is a
collaboration with Drs. Albus of NSB and Uhde of BPB.
c. Some studies are assessing state changes Independently of
pharmacological treatment. Earlier annual reports described studies of state
changes in multiple personality patients. In collaboration with CHP we are
doing follow-up studies on formerly adolescent patients with obsessive-
compulsive disorder. Some of these patients should be basically free of
symptoms, allowing us to separate out "state vs. trait" influences.
d. Nine patients with presumed early stage Alzheimer's disorder have
been compared with 6 controls of about the same age with the aim of discerning
if aspects of memory such as those Involved in habituation of physiological
responses and sequence effects in reaction time studies show deficits
685
ZOl MH 00484-27
comparable to episodic memory deficits.
e. Patients with high levels of plasma norepinephrine (NE) were
compared with patients with similar diagnoses who have low NE in collaboration
with LCS and NHLBI. This study should help clarify the role of NE in
psychophysiological activity.
f. As part of a larger LPP project, we are testing subjects with
known brain lesions from head injuries on the standard ANS and attention
protocol used with schizophrenics. The purpose is to determine what specific
brain areas may be involved in schizophrenic psychopathology.
3. Studies on Normals
a. In collaboration with CNG, we are testing the offspring of
patients with bipolar affective illness to determine if some of the putative
ANS trait markers for affective disorder reported in the literature can be
considered to be genetic markers.
b. A collaborative study with NSB is designed to compare temporary
states of "learned helplessness" and active coping on learning, mood, ANS
activity, plasma catecholamines, and Cortisol. Subjects are given the task of
learning how to turn off an aversive noise. In one condition (nonescape), the
problem is insoluble and in the other (escape), it can be solved by correctly
pushing a button. The object is to study the neurobiology of a temporary
model depressed state in humans.
c. A study on 95 normal subjects tested the hypothesis that ANS
activity mediates the relationship between platelet MAO activity and the
personality trait of sensation seeking. This study in collaboration with LSES
(see ZOl MH 00674) also uses a method of confirmatory factory analysis to
reduce psychophysiological data as described in previous annual reports.
4. Major Findings
a. Schizophrenic Studies
1. Confirmation of previous findings of high autonomic base ].evelsand
a sluggish response to the mild stress of task performance in schizophrenics
was observed and was more extreme in eight patients with significant cortical
atrophy as shown by CT scan. These and other findings have been detailed in
previous annual reports.
b. Data are still being collected. The new data continue to show that
the phenomena of visual sensory dominance and intersensory facilitation found
in normal subjects also occur in schizophrenics. A subsample of these
patients have been used as a comparison group for the autism study described
below.
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ZOl MH 00484-27
2. Studies on Nonschlzophrenic Psychopathology
a. Results for obsessives and autistlcs were presented in some detail
In previous annual reports. Both groups showed distinct patterns of ANS
activity across the various conditions which differed from their respective
control groups and from schizophrenics. Obsessives showed generally elevated
ANS activity like schizophrenics, but unlike them had normal responses to
stimuli and tasks. Autistics had very rapid breathing, but normal or only
slightly elevated ANS base levels, showing a different pattern from
schizophrenics. However, they had even more markedly attenuated ANS responses
to significant stimuli and situations than schizophrencis and a unique
laterality of SC responses. The data suggest dysregulation of ANS activity by
brainstem mechanisms and a problem in mobilizing cognitive processing
resources in autism. A paper on autism has been published.
We have retested 19 of the obsessive child group and 20 controls after a
three year period. Preliminary results show higher heart rates in the
patients at followup but only nonsignificant differences in SC variables. The
group X sex effects observed on the original testing were no longer
significant at followup. However, a comparison of baseline and followup data
revealed that controls decreased significantly more on spontaneous SC
fluctuation rate and heart rate than the patients. The state vs trait
hypothesis could not really be tested because most patients were still at
least moderately symptomatic. Neither the followup psychophysiology nor the
change from baseline were significantly related to current clinical state.
However, the number of SC orienting responses on the original test correlated
with severity of followup symptomatology, and a similar trend occurred for
heart rate. Nonsignificant trends in the same direction were present in the
other two SC arousal indicators. Thus it may be that obsessive adolescents
with a greater biological predisposition are more refractory to long term
remission than patients without this trait. Data for other groups are still
being analyzed.
b. Results for the yohimbine challenge were described more fully last
year. Briefly, yohimbine produced differential effects compared to placebo on
mood and heart rate in patients with panic disorder than in controls
irrespective of whether the patients were being treated with alprazolam.
Heart rate variability and serum Cortisol increased about the same amount in
both groups and SC activity was not greatly affected by the drug in either
group. The data from this study support the hypothesis of alpha-2
hypersensitivity in panic disorder and suggest that hyperventilation may
contribute to the genesis of panic attacks. We are awaiting analyses of
plasma catecholamine data before writing up these results.
c. The multiple personality project results were reported previously
and will be prepared for publication.
d. In preliminary analyses, the Alzheimers patients showed generally
a strong trend for hyporesponsivity in skin conductance but because of one or
two outliers in each group, these results are not significant. None of the
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ZOl MH 00484-27
patients failed to exhibit orienting response habituation, so no evidence of a
short term memory deficit at that level was apparent. The only significant
psychophysiological group difference found so far is smaller heart rate
variability in the patients, suggesting a cholinergic deficit. The low SC
activity could be attributable to this mechanism as well. More subjects will
be needed to confirm the group trends. Correlations of the individual
differences in ANS activity with behavioral, neuropsychological and brain
imaging data will be done.
e. Only six subjects with low resting plasma norepinephrine (NE) and
five subjects with high NE were available for testing. Aside from a trend
(p = .07) for a higher resting HR in the high NE group the physiological
results were essentially negative. This confirms our previous findings using
a within-subject design that changes in plasma NE are related to changes in HR
but not in other aspects of ANS activity.
3. Studies on Normals
a. We have done preliminary analyses on data from 22 subjects at
genetic risk for affective disorder (Risk group) and 27 controls, 15 to 25
years old. Since there is some evidence in the literature of low SC activity
In endogenous depression this channel was examined carefully. The results
show no significant differences in SC levels under any condition. The SC
orienting response showed nonsignificant differences opposite to the expected
direction: More controls (14%) than high risk subjects (9%) were
nonresponders, while 40% of the Risk group vs. 25% of controls failed to
habituate. There were no differences in SC response amplitudes to Innocuous
tones. Significant electrodermal hyperresponslvity was seen in the Risk
subjects in their response to the more stressful aspects of the protocol — the
instruction and practice period for a reaction time task and during a mental
arithmetic task. In addition, there were significant differences in SC
laterality: The Risk group had larger SC responses to significant stimuli on
the left hand and controls on the right hand. Similar differencves in
affectively ill patients have been reported. These results suggest that low
SC activity is not a likely genetic marker for bipolar affective disorder.
Rather, persons at risk show ANS hyperresponslvity to mild stress and
lateralized information processing differences from controls. The mood scale
showed highly significantly greater ratings of depression during the stressful
part of the session in the Risk group, compared to controls but no differences
in anxiety or aggression ratings.
b. Preliminary results for the first 10 subjects in the learned
helplessness study were reported last year. The major findings were greater
elevations of ACTH, electrodermal activity and dysphoric mood following the
nonescape procedure. A paper on these subjects has been submitted for
publication. More extensive data analysis on these and more controls, 18
patients with diagnoses of affective disorder and several patients with other
diagnoses is in progress.
c. The major results were reported previously and have now been
ZOl MH 00484-27
published.
Significance to Biomedical Research and the Program of the Institute
Investigations of ANS activity and attention in psychiatric disorders,
especially schizophrenia, have produced promising results which suggest that
these processes may play fundamental roles in the etiology and expression of
the disorders. Limitations on inferences to be dravm from measures of ANS
activity come from incomplete understanding of their biological and
psychological determinants. One of the main goals of this research is to
increase this understanding by Investigations of biological and psychological
correlates and improving measurement techniques. The dynamic nature of these
measures permits the study of processes such as adaptation, habituation,
response to and recovery from stress, and effects of single stimuli through
noninvasive techniques. Thus, further understanding of their mechanisms could
greatly increase their utility in investigations of psychopathology.
Continued investigations of the diagnostic specificity of these processes and
of their relationships to other clinical features and to prognosis are
necessary to confirm and extend our previous results and to test the limits of
their generality.
Proposed Course
Analysis of data will continue for the completed project on
schizophreniawith the goals of determining the relationship of ANS variables
to diagnosis, task performance J and a number of clinical and biological
variables available on these patients. Concept modeling by confirmatory
factor analysis may be extended to this group.
Collection and analysis of data will continue for current projects on
schizophrenic and nonschizophrenic psychopathology and in normal controls. We
hope to have sufficient data this year to make a formal test of the diagnostic
specificity of ANS activity.
We are starting to test obsessive children on pharmacological treatments
in order to determine if ANS effects of the treatments are related to their
clinical effectiveness. This will attempt to replicate and extend a previous
similar study with adult obsessives from our laboratory.
Tests on children in the Hopkins preventive intervention project whom LPP
is negotiating to bring in. We will use the same test protocol as we used
previously with hyperactive, obsessive, and anxious children, and currently
with children having a diagnosis of conduct disorder (see ZOl MH 00486 LPP).
This study should determine how ANS activity relates to the extensive
classroom behavior observations on these children and how these and other
observations may compliment each other in predicting future psychopathology or
behavior problems.
We are planning to design a new protocol for schizophrenics by which to
test the hypothesis that schizophrenics and controls exhibit differential
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ZOl MH 00484-27
effects of increases in arousal on attention. Arousal will be manipulated by
changes in posture and variations in stimulus intensity and significance. We
also plan to obtain measures of smooth pursuit eye tracking on schizophrenic
patients, since Dr. Hommer of NSB, who had been doing this has left the
program. There is evidence that smooth pursuit eye movement dysfunction may
be a genetic trait linked to schizophrenia, so it is important to obtain this
measure on these patients.
Publications
Goldstein, D. S., Bonner, R. F. , Zimlichraan, R. , Zahn, T. P., Cannon, R. C,
III, Rosing, D. R., Stull, R. , and Keiser, H. R.: Indices of sympathetic
vascular innervation in sympathectomized patients. J. Auton. Nerv. Syst., 15:
309-318, 1986.
Zahn, T.P., Schooler, C, and Murphy, D.L.: Autonomic correlates of sensation
seeking and monoamine oxidase activity: Using confirmatory factor analysis on
psychophysiological data. Psychophysiology , 23: 521-531, 1986.
Zahn, T. P., Rumsey, J. M., and Van Kammen, D. P.: Autonomic nervous system
activity in autistic, schizophrenic, and normal men: Effects of stimulus
significance. J. Abnorm. Psychol. , 96: 135-144, 1987.
690
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 00486-15 LPP
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Psychophysiological Effects of Stimulant Drugs In Children
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aHiliation)
PI:
Other:
Theodore P. Zahn, Ph.D. Research Psychologist LPP, DIRP, NIMH
Judith Rapoport, M.D.
Martine Flament, M.D.
Marcus Kruesi, M.D.
Chief
Guest Researcher
Clinical Associate
CHP, NIMH
CHP, NIMH
CHP, NIMH
COOPERATING UNITS (If any)
Child Psychiatry Branch, NIMH
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH/ADAMHA, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
0.3
PROFESSIONAL
iL2_
JLJ_
CHECK APPROPRIATE BOX(ES)
^ (a) Human subjects
H (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Tests of the effects of acute and chronic administration of caffeine on
autonomic nervous system (ANS) functioning have been carried out to evaluate the
role of ANS activity in the behavioral and subjective effects of this drug. A
test of attention using a reaction time method is included.
The test protocol involves recording peripheral Indicators of ANS activity
such as skin conductance (SC), heart rate (HR) , and skin temperature during a
session consisting of a rest period, presentation of a series of simple tones to
which no response is required, and the reaction time task. Studies have been
carried out on the effects of the acute administration of two doses of caffeine
and a placebo in 6-13 year old boys and in men, and a study of chronic (2 week)
caffeine intake in children.
The effects of both acute and chronic administration of caffeine were
increases in SC indices of arousal but some trends toward decreases in HR. The
SC results are consistent with the hypothesis that caffeine can be considered a
pharmacologic model for anxiety, but the HR effects suggest the model is
imperfect.
The most recent study, an acute dose protocol with caffeine was conducted on
children with anxiety disorders and controls. This tested the hypothesis, for
which there is evidence in adults, that patients with anxiety disorders are more
sensitive to caffeine than controls.
Another current study compares ANS activity and attention in boys with
diagnoses of Conduct Disorder and Attention Deficit Disorder.
691
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GPO BI4-SII
ZOl MH 00486-15 LPP
Project Description
This project has evolved from the study of hyperactivity in children (now
called Attention Deficit Disorder) to the study of stimulant drugs —
dextroamphetamine and caffeine — in children and adults.
Results of the caffeine studies with normal subjects have been presented
in previous annual reports. The acute dosage studies were published this
year. The pattern of ANS results for caffeine was different from that of
another "stimulant drug" — dextroamphetamine — in that caffeine produced very
consistent and strong increases on SC activity but minimal or opposite effects
in HR, while amphetamine dramatically increased HR and had less consistent
effects on SC activity (although it also generally increased it).
The recent study of anxiety disorders, mentioned above, used the same
protocol as in some of our acute studies. Children were tested at baseline,
then in three sessions where they receive, randomly, 0, 3, and 10 mg/kg of
caffeine one hour before testing. Children with a diagnosis of anxiety
disorder and normal controls have been tested. Results on eight patients and
eight controls show that, as in our previous studies, caffeine increased SC
activity and decreased HR. Contrary to expectation, caffeine effects on SC
activity were not greater in the anxious children. On the contrary, caffeine
produced more electrodermal activation in the controls, significantly so in
some cases. These effects could not be accounted for by a difference in
placebo values as these were not generally much different. In contrast, the
anxious subjects showed a somewhat greater decrease in heart rate than
controls, but this might be due in part to higher heart rate on placebo in the
anxiety group. Side effects were not different in the two groups (see ZOl MH
00161-08 CHP).
Testing of boys with diagnoses of conduct disorder (CD) and attention
deficit disorder (ADD) is being done with two general objectives in mind. One
is to look for ANS markers of diagnosis. Previous research in this laboratory
and others has shown that ADD boys do not differ from normals in indices of
arousal but have generally lower ANS responsivity to stimuli. The literature
on psychopathic personality in adults — a possible outcome of CD — shows
evidence of low SC levels and diminished SC (but not HR) reactivity to
nonsignal stimuli. Our ANS protocol will allow tests of these differences in
the boys. A second objective is to explicate the attention deficit in ADD
children and to test whether CD boys have similar deficits. A battery of
simple and choice reaction time tasks, similar to those given to
schizophrenics are being used for this purpose (see ZOl MH 00484-25 LPP,
1984-85).
Significance to Biomedical Research and the Program of the Institute
The ANS effects of caffeine consistently found In these studies partially
resemble those seen In anxiety states and other psychopathology. Since
caffeine effects are thought to be mediated by blockade of adenosine
receptors, these studies may help determine the mechanisms involved in those
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ZOl MH 00486-15 LPP
aspects of ANS activity that are components of anxiety states. Our finding
that children with anxiety disorders are not especially sensitive to caffeine
effects is surprising in terms of the usual models of caffeine effects in
adults with anxiety disorders and suggests either that these models do not
apply to children or that they generally need revision.
The study on CD and ADD boys may also help determine the mechanisms of ANS
measures through correlation with the extensive neurobiological data being
obtained in this group. It should also provide evidence of a possible
biological basis for the diagnostic distinctions. This study is relevant to a
major objective of LPP to develop a taxonomy of attention disorders.
Proposed Course
Continued collection of data on the CD-ADD project is planned. The
analysis of the data will include correlation of the ANS and attention data
with metabolites of biogenic amines from CSF.
Publications
Zahn, T.P. and Rapoport, J.L.: Acute autonomic nervous system effects of
caffeine in prepubertal boys. Psychopharmacology, 91: 40-44, 1987.
Zahn, T.P. and Rapoport, J.L.: Autonomic nervous system effects of acute
doses of caffeine in caffeine users and abstainers. Int. J. Psychophysiology,
5: 33-41, 1987.
693
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00491-11 I.PP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 chertclen Of less. Title must lit on one line between the borders.)
Personality Factors and Psychophysiological Responses to Changing Stimulus Tnput
PRINCIPAL INVESTIGATOR (List other prolessionel personnel below the Principal Investigator) (Name, title, laboratory, and institute attilialion)
PI: Theodore P. Zahn, Ph.D. Research Psychologist LPP, NIMH
Other: Thomas N. Robinson, Jr. Guest Researcher LPP, NIMH
COOPERATING UNITS (It any)
NIH Normal Volunteer Office.
UkB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NTMH, ADAMHA. Bethesda, Maryland 2Q892
TOTAL MAN-YEARS:
PROFESSIONAL
-OOL
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The objectives of this project are to investigate relationships among
differences in personality, sensory thresholds, and autonomic nervous system
(ANS) activity in normal humans and to study racial differences in ANS
activity. Bilateral skin conductance and heart rate have been recorded in two
sessions in which constant and variable intensity tones and lights are
presented and auditory and two-flash thresholds (TFT) determined by methods
which permit signal detection analyses. A procedure for determining the
uncomfortable loudness level has also been used. Several standardized
personality tests were also given. These include scales of sensation-seeking,
extraversion, neuroticism, psychoticism, field dependence and anxiety. In
addition comprehensive measures of lateral dominance have been given as well as
a measure of "torque" (clockwise drawing of circles) which has been
hypothesized to reflect a neurointegrative deficit and be related to risk for
future psychopathology. A procedure for manipulating ANS arousal
experimentally with minimal distracting effects — a change in posture from
supine to standing — is being used to study such problems as the effects of base
levels on responsivity, the effects of arousal on performance, and the effects
of personality variables on this relationship. This project allows testing of
several theoretical models of the relationships of ANS activity, sensory
sensitivity, and personality, some of which have implications for the etiology
of psychopathology. Tests of the relationships between laterality In skin
conductance variables and behavioral laterality will also be done to see if
inferences about lateralized brain function can be made from such variables.
695
PHS 6CMQ (Rhv 1/M)
ZOl MH 00491-11 LPP
Project Description
A. Objectives
A large body of psychological literature postulates that an important
dimension of individual differences in behavior or personality is reflected in
the reactions of the nervous system to sensory stimulation. Pavlov's original
conception of "strong" and "weak" nervous types has been modified and extended
by Western theorists to reflect such personality dimensions as "extraversion-
introversion," "sensation-seeking," and "field dependence," each of which can
be measured by a questionnaire or other test procedures. The theoretical
models that have been built up from these concepts have implications for
interrelationships among personality, autonomic nervous system (ANS) base
levels and responsivity to stimulation, and sensory sensitivity. There are
also implications for psychopathology, in that schizophrenics have been
considered to be extremely "weak" nervous types in the Pavlovian system (i.e.,
overreactive to weak stimulation and underreactive to strong
stimulation — "transmarginal inhibition"). Another development is the more
recent delineation by H. Eysenck of the dimension of "psychoticism."
The major objective of this project is to test some of the implications of
these models of personality by interrelating the personality measures with
sensory thresholds and sensitivity, and ANS activity in normal humans. Other
objectives are to assess racial differences in ANS activity and in its
relationships to the other variables in the study and to explore relationships
of differences in the laterality of skin conductance activity with behavioral
assessments of laterality, and to test the effects on ANS activity increasing
arousal by means of a postural change.
B. Methods Employed
Over 200 normal volunteers have been assessed on several personality
dimensions, including the three Eysenck scales of extraversion, neuroticism,
and psychoticism in addition to, field dependence, sensation-seeking,
impulsivity, ego strength, and anxiety, assessed for degree of lateral
dominance, and given tests of ANS and sensory functioning in the various
protocols described earlier. Since not all subjects have received all
procedures, the results presented below are based on partially overlapping
subsets of subjects for different comparisons.
In another protocol a fixed foreperiod reaction time procedure is Included
similar to that used with patients in ZOl MH 00484 LPP.
C. Major Findings
In previous annual reports, relationships between questionnaire-defined
personality variables, ANS activity, and sensory thresholds have been
described. In general, subjects with high scores on the Eysenck personality
scales of extraversion, psychoticism, and, surprisingly, neuroticism tend to
696
ZOl MH 00491-11 LPP
have low ANS activity and reactivity. Low ANS activity was also found in
subjects with high scores on a scale of schizotypal personality. Subjects
high on sensation-seeking were also very responsive autonomically to novel
stimuli. Low sensory senslvity was shown by subjects high on psychoticism and
those showing a "torque" (clockwise) pattern of drawing a circle.
Recent analyses of this large data set have attempted to test the utility
of the concept of strength of the nervous system to unify personality, sensory
sensitivity, and physiological data. According to the theory, subjects with a
"weak" nervous system type should be high on sensory sensitivity and have a
low threshold for aversiveness as stimulus intensity increases, have high ANS
activity, and be low on sensation seeking, extraversion, and ego strength, and
high on anxiety and field dependence.
Results show that there were many confirmatory results for the model, many
neutral results, and few disionfirmatory ones, such that some predictions from
the model fared better than others. The ego strength measure was related to
all three sensory measures in the predicted direction but not to the
physiological data, while sensation seeking, extraversion, and field
dependence showed some expected relationships with the ANS data but not to
auditory sensitivity or aversiveness. Sensation seeking and intraversion were
predictive of a low two-flash threshold, however. ANS activity was related to
auditory sensitivity and TFT but not to aversive level. Thus the general
pattern of the results seems to fit the model reasonably well, but there are
some inconsistencies in the individual measures. Possibly there is more than
one dimension in the strength construct.
A preliminary analysis of the reaction time data in the latest protocol
shows that introverts had slower reaction time than extraverts and showed more
slowing as the length of the preparatory interval increased. This is similar
to the usual finding in schizophrenia.
Significance to Biomedical Research and the Program of the Institute
The construct of the strength of the nervous system, whatever it may be
thought to denotate in terms of neuronal functioning, has a traditional
relevance to psychopathology in that a weak nervous system is said to be
characteristic of schizophrenia. The association with the Western measures of
introversion and low ego strength is also relevant in this context, as are the
associated phenomena of enhanced sensory sensitivity and elevated ANS
activity. Although there are wide subtype differences among schizophrenics,
the concept of a schizophrenic characterized by social withdrawal, weak ego
boundaries, and high arousal who is overwhelmed by environmental stimuli has
been associated with the early stages of the illness. The data from this
project appear to support the existence of a similar syndrome in normal
subjects, albeit to a lesser degree. Thus this approach may be considered as
an alternative to the increasingly popular "high risk" study using scales
designed to measure some form of schizotypy by assessing symptom-like
phenomena more directly.
697
ZOl MH 00491-11 LPP
Increased understanding of the relationships among autonomic, perceptual,
and personality variables in normal subjects should be of great assistance in
interpreting the autonomic and perceptual results from studies on
psychopathology in which similar methods are used. This project has been very
useful in the development of protocols for studies of psychopathology.
Proposed Course
A priority is to attempt to delineate further the strength of the nervous
system construct. To this end multivariate analyses such as multiple
regression and/or confirmatory factor analysis will be applied to this large
data set. We also plan to do more data collection and analysis with the newer
reaction time protocol.
We have some as yet unanalyzed psychophysiological data collected in two
"high risk." studies in which extremely poor or good performance on either the
Continuous Performance Task or pendulum eye tracking (both of which are
impaired in schizophrenia) were selection variables. These data are obviously
quite relevant to the questions discussed here and we plan to analyze them.
We are planning also to develop a new battery of tests that vary in their
sensitivity to arousal, making use of some of the recent developments in the
field of cognitive psychology.
Publications
None.
698
PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
ZOl m 00503-07 LPP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line beOveen the borders.)
Human Clinical Studies of Attention Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, latxyatory, and institute affiliation)
Pi: Allan F. Mirsky Chief LPP, NIMH
COOPERATING UNITS (if any)
Epilepsy Branch, NINCDS; Clinical Neurosciences Branch, NINCDS; Laboratory of
Clinical Sciences, NIMH; Neuropsychiatry Branch, NIMH; Boston University
UAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.0
PROFESSIONAL
1.25
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects D (b) Human tissues D (c) Neither
Q (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This research comprises three related areas of investigation concerned
with specifying neuropsychological factors underlying clinical conditions in
humans in which disturbed attention is a major symptom. A major emphasis is
on (1) illuminating the nature of brainstem pathophysiology, if any, in such
entities as petit mal or absence epilepsy, infantile autism, schizophrenia,
and related diseases; (2) an additional major emphasis is on extending the
neurobehavioral analysis of attention loss in absence epilepsy so as to
facilitate developing alternative treatment strategies for such patients. Both
of these projects form part of a larger effort which is aimed at (3)
developing a comprehensive and systematic taxonomy of attentional disorders in
humans. This latter study will eventually comprise study of patients with
cerebral lesions, seizures, dementing diseases, and metabolic illnesses of the
brain.
699
PHS 6040 (Rev. 1/84)
SPO «I4-SII
ZOl MH 00503-07 LPP
Project Description
A. Other Personnel
Connie C. Duncan, Ph.D.
Walter H. Kaye, M.D.
Richard Coppola, D.Sc.
Theodore P. Zahn, Ph.D.
Roger Porter, M.D.
Debbi Fein, Ph.D.
Daniel R. Weinberger, M.D.
Senior Staff Fellow
Associate Professor
Senior Engineer Officer
Research Psychologist
Chief
Assistant Professor
Chief
LPP, NIMH
Univ. of
Pittsburgh
CBD Branch
NIMH
LPP, NIMH
EBB, NINCDS
Boston Univ.
CBD Branch
NIMH
B. Project Description
1. Brainstem Mechanisms in Attention Impairment
Current approaches to the neuropsychology of attention Impairment have
emphasized that the system responsible for the maintenance of attention or
consciousness within the brain is most likely represented at a variety of
levels of the neuraxls. From an evolutionary point of view, it is clear that
the capacity for sustained attentive behavior is present in many species which
do not possess more than a rudimentary forebrain or telencephalon. Maclean's
analysis of the R-complex within the human brain leads to the view that this
"clump of ganglia," which constitutes virtually all of the reptilian brain,
can support a variety of ritualistic, repetitive behaviors which could be
characterized as sustained and attentive. Evolution progressed and the brain
developed additional complexity and volume. Additional capacity for
attentive behavior was thus overlaid on the more primitive, although in many
aspects thoroughly adequate, brainstem system of the reptile. Therefore,
although the system for maintenance of attentive behavior in the human (or
higher primate) Includes limbic and neocortical components, the brain stem
remains a key component and possibly the keystone of the entire system.
Authors such as Hughlings Jackson and Penfleld and Jasper recognized this in
their conceptions, respectively, of "highest-level seizures" and the
"centrencephalon. " In their theorizing, consciousness was either localized In
or regulated by deep brainstem structures. Without reviewing all of the
evidence that led to those views of the hierarchical organization of attention
and consciousness within the brain, we nevertheless point to the extremely
deleterious effects on such capacities of small lesions in the brainstem
region of the third and fourth ventricles. In the last ten years, a new
technological refinement of evoked-potentlal methodology has made possible an
other-than-theoretical exploration of the role of brainstem structures in
certain clinical states. This "far field" or BAER (for brainstem
auditory-evoked responses) technique makes it possible to assess the integrity
of auditory (and somatosensory) relay nuclei within the brain stem of humans.
Although the technique has probably had most utilization in the diagnosis of
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ZOl MH 00503-07 LPP
demyelinatlng disease, it has also been used in the study of other
neurological and, recently, psychiatric disorders. There may or may not be
any specific interest in these sensory systems (auditory, somatosensory) in
studying a particular clinical entity (i.e., absence seizures, infantile
autism); nevertheless, the possibility of evaluating the functional integrity
of certain systems within the brain stem is extraordinarily valuable, and many
clinical investigators are using these techniques. We have published work
indicating that there are disturbances (prolonged transmission time) in the
processing of auditory information in the brain stem in infantile autism and
in schizophrenia. We have also shown that in absence seizures (spike-wave
activity), both naturally-occurring and experimentally-induced, there may be
perturbations of auditory brainstem functioning. Several years ago, we
completely revised the hardware and computer software used for analyzing BAERs
in our laboratory. The data are now clear, clean, reliable, and repeatable
and we have run some control studies of normal subjects using parametric
variations of intensity, etc.
BAERs are now routinely gathered on our patient subjects and we hope
within the coming year to have publishable quality data from schizophrenic
subjects and other clinical populations. We are also planning to gather BAERs
in patients with head injuries, as well as possibly recalling some autistic
subjects for retesting. BAERs are also included in the alcohol protocol being
conducted by Dr. Frances Gabbay, a Guest Researcher from John Hopkins
University.
2. Neurobehavioral Studies in Absence Epilepsy
We have for a number of years been studying the absence attack in patients
with petit mal/centrencephalic/absence seizures (the terms are more or less
interchangeable) as a model state to understand the phenomenon of
consciousness/attention. Some. of these studies have involved comparing the
behavioral capacities of patients suffering from petit mal — as opposed to
focal seizure disorders; other studies have involved detailed comparison and
contrast between the behavioral and the electroencephalographic S5niiptoms/signs
of the disorder. Most recently, these investigations have: (1) used evoked
potentials in the visual and auditory modalities as indices of the sensory
effects of generalized seizure activity of the symmetrical and synchronous
wave and spike variety, and (2) examined changes in the EEG power spectrum
prior to WS bursts as prodromal signs which may be used to predict (and
ultimately to control) WS bursts. We propose to continue this line of
neurobehavioral investigation, using event-related potentials of various types
as well as other behavioral and physiological tools, to refine further our
understanding of the nature of altered consciousness in absence (petit mal)
epilepsy.
A group of approximately eight subjects with absence epilepsy has now been
studied with a full battery of tests, including a complete neuropsychological
examination and a number of ERP paradigms requiring varying amounts of
attention. Untreated cases with absence epilepsy are difficult to find and
persons with good medication control of their seizures are reluctant to serve
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ZOl MH 00503-07 LPP
as subjects. Nevertheless, we now have a sufficiently large group to be able
to make some additional contributions to the study of attention in absence
epilepsy. Analysis of the neuropsychological data is underway. Preliminary
results indicate that although this is a high-functioning group of absence
patients, they demonstrate the expected impairment in attention in the
interictal period, as assessed by the Continuous Performance Test (CPT).
Further, it was found that significantly greater impairment was seen in the
auditory version of this task than in the visual version.
Analyses of the ERPs to CPT stimuli revealed impairment in information
processing which paralleled the behavioral data. In addition, new insights
into the response failures in absence epilepsy were provided by this analysis,
which is reported in more detail in protocol ZOl MH 00509-05 LPP.
3. A Taxonomy of Attentional Disorders
The goal of this project is to develop a comprehensive and coherent
account of the relation between symptoms of altered or disturbed attention or
consciousness as they appear in various clinical entities, the other
behavioral and clinical characteristics of the several disorders, and the
specific central nervous system damage or disturbance in each disorder. The
attentive capacities of the patients are assessed by a number of attention
tests Including the CPT (continuous performance test), a measure of sustained
visual attentive behavior. The ultimate goal will be to describe the precise
attentive deficit (as opposed to cognitive losses) and the nature of the
neuropathophysiology associated with each of the following clinical entitles:
cerebral lesions (frontal, parietal, temporal lobe, or brainstem);
centrencephalic/absence epilepsy; schizophrenia; infantile autism; dementing
diseases (Alzheimer's, Korsakoff's, Huntington's); and metabolic diseases
(Phenylketonuria, Uremia, Anorexia Nervosa and related Illness).
We will attempt, as well, to relate these changes where possible to
standardized measures of mnemonic and other cognitive function, and to
autonomic indices of attention, arousal, and habituation. Reasonable amounts
of data have now been collected on a number of these populations and the work
continues .
During the past year, a theoretical model of the elements of attention has
been proposed in a number of publications. This model is based on a factor
analysis of the data from nearly 100 subjects. In addition, it Incorporates
Information from neuroanatomical and neurophysiologlcal sources. It suggests
that "attention" comprises a series of behavioral components or elements
Including the capacities to focus, encode, sustain, shift and execute.
Further, it is suggested that those elements are best assessed by different
groups of neuropsychological tests (which are incorporated in our LPP test
battery). Additionally, it is speculated that these behavioral elements are
supported by different regions of the central nervous system.
The elements of a attention model, it is hoped, will provide a useful
heuristic device for organizing studies and analyzing data, and will
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facilitate the development of a taxonomy of attention disorders. The model is
discussed further in ZOl MH 00508-05 LPP, which also describes its use in
screening for attention disorders in a population of second grade public
school children.
Significance to Biomedical Research and to the Program of the Institute
Since attention disturbance is a characteristic of many significant
psycho- and neuropathological disorders, it is essential to have a clear
empirical and theoretical account of the role and pathophysiological
significance of this symptom. Such a theoretical model will aid in
understanding the etiology and course of these illnesses and may aid in
improving their treatment.
Proposed Course
We have a substantial group of schizophrenic, epileptic, and brain-injured
patients tested on our laboratory procedures (i.e., CPT, brainstem
auditory-evoked potentials, various tests of cognition and memory, autonomic
indices of attention, etc.). We are in the process of preparing the results
of these studies for publication.
We have completed an edited book on petit mal epilepsy which is in press
at this time.
Publications
Mirsky, A.F., and Ray, C: Studies in the Neuropsychology of Attention
Impairment: Human Sjnnptoms and Animal Models. In Galbraith, G.C., Kletzman,
M.L., and Donchin, E. (Eds.): Neurophysiology and Psychophysiology:
Experimental and Clinical Applications. Hillsdale, N.J. , Lawrence Erlbaum
Associates, 1987, in press.
Kaye, W.H., Gwirtsman, H.E. , George, D.T., Weiss, S.R., and Jimerson, D.C.:
Relationship of mood alterations to bingeing behavior in bulimia. Brit. J.
Psychiatry, 1987, in press.
Kaye, W.H., Obarzanek, E. , George, D.T., Jimerson, D.,C., and Eber, M.H.:
Caloric intake for satisfactory maintenance in anorexia nervosa: Non-bulimics
require greater caloric intake than bulimics. Amer. J. Clin. Nutrition, 1987,
in press.
Kaye, W.H. : Opioid Antagonist Drugs in the Treatment of Anorexia Nervosa. In
Garfinkel, P.E., and Gardner, D. (Eds.): The Role of Psychotropic Drug Use
for Treating Eating Disorders, 1987, in press.
Kaye, W.H.: Neuroendocrinology in anorexia nervosa: Into the central nervous
system with gun and camera. Int. J. Eating Disorders, 1987, in press.
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ZOl MH 00503-07 LPP
Coppola, R.: Topographical Representation of Spike-Wave Activity. In
Myslobodsky, M.S. and Mirsky, A.F. (Eds): Elements of Petit Mai Epilepsy, New
York, Peter Lang, 1987, in press.
Mirsky, A.F. and Grady, C: Toward the Development of Alternative Treatments
in Absence Epilepsy. In Myslobodsky, M.S. and Mirsky, A.F. (Eds.): Elements
of Petit Mai Epilepsy, New York, Peter Lang, 1987, in press.
Mirsky, A.F.: Behavioral and Psychophysiological Effects of Petit Mai
Epilepsy in Light of a Neuropsychologically Based Theory of Attention. In
Myslobodsky, M.S. and Mirsky, A.F. (Eds.): Elements of Petit Mai Epilepsy,
New York, Peter Lang, 1987, in press.
Duncan, C.C.: Application of Event-Related Brain Potentials to the Analysis
of Interictal Attention in Absence Epilepsy. In Myslobodsky, M.S. and Mirsky,
A.F. (Eds.):: Elements of Petit Mai Epilepsy, New York, Peter Lang, 1987, in
press.
Bridge, T.P. , Mirsky, A.F., and Macdonald, D.I.: Acquired immunodeficiency
syndrome (AIDS): Neuropsychologic and psychoimmunologic aspects. In press.
Mirsky, A.F. and Duncan, C.C.: An Introduction to Modern Techniques of
Clinical Neuropsychology . In Wise, T.N. , and Fava, G. (Eds.): Advances in
Psychosomatic Medicine. New York, Karger, 1987, pp. 167-184.
Mirsky, A.F. and Ray, C: Studies in the Neuropsychology of Attention
Impairment: Human symptoms and Animal Models. In Galbraith, G.C., Kietzman,
M.L., and Donchin, E. (Eds.): Neurophysiology and Psychophysiology: Basic
Mechanisms and Clinical Applications. Hillsdale, N.J. , Lawrence Erlbaum
Associates, in press.
Mirsky, A.F. Neuropsychological Manifestations and Predictors of HIV
Disease in Vulnerable Persons. Paper presented at the Conference on AIDS in
Washington, D.C. May 28-29, 1986. In press.
Mirsky, A.F. and Rosvold, H.E.: The Case of Carolyn Wilson — A
Thirty-Eight-Year Followup of a Schizophrenic Patients with Two Prefrontal
Lobotomies. In Goldberg, E. (Ed.): Festschrift to Alexandr R. Luria. New
York, IRBN Press, in press.
Mirsky, A.F.: Behavioral and psychophysiological markers of disordered
attention. Environmental Health Perspectives, 1987, in press.
Mirsky, A.F.: [Review of: The frontal lobes by D.T. Stuss and D.F. Benson].
In: Journal of Clinical Neurophysiology. 4: 89-90, 1987.
Mirsky, A.F.: The Neuropsychology of Attention: Elements of a Complex
Behavior. In Perecman, E. (Ed.): Integrating Theory and Practice in Clinical
Neuropsychology . New York, IRBN Press, in press.
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ZOl MH 00503-07 LPP
Freedman, R. and Mirsky, A.F.: Evoked Potentials: Exogenous Components. In
Zubin, J., Steinhauer, S.R. and Gruzelier, J.H. (Eds.): Handbook of
Schizophrenia. Volume 4, Experimental Psychopathology , Neuropsychology and
Psychophysiology . Amsterdam, Elsevier, in press.
Mirsky, A.F. and Duncan, C.C: Attention Impairment in Human Clinical
Disorders: Schizophrenia and Petit Mai Epilepsy. In Sheer, D.E. and Pribram,
K.H. (Eds): Attention; Theory, Brain Functions and Clinical Applications.
Hillsdale, N.J., Erlbaum, 1987, in press.
705
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00504-07 LPP
PERIOD COVERED
October 1. 1986 to September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Models in the Monkey of Generalized Seizures of the Absence Type
PRINCIPAL INVESTIGATOR (List other professional personnel t>elow the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI:
Others;
Allan F. Mirsky, Ph.D.
Eva Bakay Pragay, Ph.D.
Chief
Guest Researcher
Richard Nakamura, Ph.D. Guest Researcher
Michael Myslobodsky, M.D., Ph.D. Professor,
Univ. of Tel Aviv
Richard Coppola, Ph.D. Engineer
LPP, NIMH
Vienna
Austria
Israel
CBD Branch
NIMH
COOPERATING UNITS (It any)
Tel-Aviv University, Israel
LjAB/BHANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH. ADAMHA. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
0.7
PROFESSIONAL
0.7
0.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use startdard unreduced type. Do not exceed the space provided.)
Generalized seizure activity with the electrographic appearance of
absence epilepsy (bilaterally symmetrical and synchronous paroxysmal
three-per-second spike and wave discharges) can be elicited in the monkey by a
variety of methods. These include electrical stimulation of various locations
within the brain, injection of convulsant drugs and other substances, and
administration of compounds which may alter normal inhibitory mechanisms
within the cell. Model seizure states created in these ways are studied in
order to test hypotheses about pathophysiological seizure mechanisms, sensory
processing and attentional capacities during absence seizures, effects of
spike-wave activity on cellular activity, and effects of techniques or
maneuvers which may modify or reduce convulsive activity. Most recently this
project has involved the following work: we studied the behavioral and
electrographic effects of a GABA-enhancer and surveyed the attention-related
cells in the frontal lobes of the monkey. Other studies of "attention" cells
in inferior parietal and preoccipital cortex have been completed as well.
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ZOl MH 00504-07 LPP
Project Description
The putative neurotransmitter GABA (y-aminobutyric acid) is thought to be
involved in the central neural processes of inhibition whose perturbation can
result in generalized seizure disorders. Two compounds which are
metabolically related to GABA are GBL and GHB. y-butyrolactone (GBL) and the
pharmacologically active product of its hydrolytic cleavage, y-hydroxybutyrate
(GHB), produce several central effects of potential significance for therapy
and experimental pathology. Winters and Spooner identified GHB effects as
epileptogenic or related to "non-convulsant epilepsy." Other research in
rodents and monkeys added to the conviction that GHB causes
electroencephalographic and behavioral effects akin to petit mal epilepsy.
We attempted to assess the petit mal-like effects induced by GBL by
studying the attention-related performance and EEG responses in monkeys
administered a single dose of the drug. All animals had been trained to
perform a go/no-go visual attention task similar to the Continuous Performance
Test (CPT) used in studying human subjects with petit mal. The criterion
performance was 80% correct responses.
While there were individual differences in responding after administration
of 125 mg/kg of GBL, a dose of 200-250 mg/kg caused a reliable suppression of
responding in all subjects. When the testing began 30 minutes following the
drug, animals initially responded rapidly and reliably but soon ceased
responding altogether. However, they remain sufficiently alert to groom and
react to environmental stimuli. Some occasionally resumed responding for
several minutes. Offered water, all animals drank it eagerly.
If tested immediately following administration of the drug (200-250
mg/kg), they were able to perform at 60-70% correct; at about 40 minutes the
responding came to a complete halt. Here again, monkeys remained competent
perceptually and motorically for some time thereafter.
EEG monitored during the task performance showed a build-up of generalized
hypersynchronous activity in some areas when performance deficit was
noticeable. A pattern resembling 3 cps wave-spike discharges typical of petit
mal was never seen either during this period or at the end of the study.
These effects are not typical of either petit mal or petit mal status and may
be explained by the development of frank sleep. These effects seem related to
the general anesthetic properites of GBL (described by some investigators)
rather than to its potential (if any) as a model of petit mal.
Significance to Biomedical Research and to the Program of the Institute
This protocol provides information concerning the nature of the
attention-support system in the primate brain, the role of various
neurotransmitter substances in consciousness and in generalized seizures and
contributes to the current efforts to produce an accurate primate-based model
of the pathophysiological processes in absence epilepsy.
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Proposed Course
The work described here has been accepted for publication in Behavioral
Brain Research. Additional findings from this project will be published in
the future. We have recently completed a book, which is currently in press,
that includes chapters reviewing the recent developments in the biochemistry,
electrophysiology and genetics of absence epilepsy. The book will incorporate
much of the material germane to this project.
Publications
Mirsky, A.F., and Pragay, E.B.: Brainstem Mechanisms in the Processing of
Sensory Information: Clinical Symptoms, Animal Models, and Unit Analysis. In
Sheer, D.E. and Pribram, K.H. (Eds.): Attention: Cognitive, Brain Function
and Clinical Applications. 1987. In press,.
Pragay, E.B., Mirsky, A.F., and Nakamura, R.K. : Attention-related unit
activity in the frontal association cortex during a go/no-go visual
discrimination task. Exp. Neurol. 96: 481-500, 1987.
Myslobodsky, M. , Sharon, D. , and Novis, B. : Pattern-reversal evoked
potentials in hepatic chirrosis. Hepato-Gastroenterology . 33: 145-147, 1986.
Myslobodsky, M.S. and Mirsky, A.F.: Theoretical Summary. In Myslobodsky,
M.S. and Mirsky, A.F. (Eds.): Elements of Petit Mai Epilepsy. New York,
Peter Lang, 1987, in press.
709
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1'
PROJECT NUMBER
ZOl MH 00508-05 LPP
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Neuropsychological Evaluation of Psychiatric and Neurological Patients
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI:
Connie C. Duncan, Ph.D. Chief, Unit on Psychophysiology
LPP, NIMH
COOPERATING UNITS frf any; Biological Psychiatry Branch, Laboratory of Clinical Science,
NIMH; Developmental and Metabolic Neurology Branch, NINCDS; Chestnut Lodge
Hospital; Johns Hopkins University; Maryland Head Injury Foundation; Division
of Neuropsychology,' Department of Psychiatry, Medical College of Pennsylvania .
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAL
0.7
1.0
CHECK APPROPRIATE BOX(ES)
(El (a) Human subjects
H (a1) Minors
S (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A set of comprehensive neuropsychological test hatteries is used to
provide a complete assessment of various cognitive and sensory functions that
can be related to damage or dysfunction in different regions of the brain.
The adult battery comprises tests designed to tap the following aspects of
behavior; attention, executive functions, language , memory, motor functions,
orientation, selected sensory and perceptual functions, vigilance, and
visual-spatial functions. ' In addition, adults are given a test of general
inte 1 ligence and a personality inventory. In some studies, subjects are
administered a structured psychiatric interview. Modified batteries have
been developed for the assessment of infants, preschool children, children
ages 5-8, and children ages 6-16. The data provided by these batteries are
being used to construct neuropsychological profiles of the neurological and
psychiatric diagnostic groups under study in the LPP. The LPP has been
particularly interested in disorders involving impaired attention, including
schizophrenia , complex partial seizures, eating disorders, affect ive
disorders , and head injuries. Comparisons are being carried out between the
neuropsychological profiles of various groups of psychiatric patients and
those of patients with known cerebral lesions in specified brain regions. Our
data are also being used to delineate neurobehaviorally-def ined subgroups
within diagnostic categories, an undertaking aimed at reducing variability in
psychiatric diagnosis, treatment, and outcome. The data provided by this
protocol provide a complete behavioral assessment that may be integrated with
concurrently gathered neurophysiological , neurorad iological , and biochemical
inf ormat ion.
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CPO BI4-SI(
Project Description
A. Other Personnel
Allan F. Mirsky, Ph.D.
Barbara P. Jones, Ph.D.
Bruno J. Anthony, Ph.D.
Emile Brouwers, Ph.D.
Mary Beth Ahearn, M.A.
Robert Post, M.D.
David C. Jimerson, M.D.
John Fink, M.D.
Elkhonon Goldberg, Ph.D.
C. Wesley Dingman, M.D.
Sheppard G. Kellam, M.D.
William W. Eaton, Ph.D.
B. Objectives
Chief
Special Expert
Senior Staff Fellow
Visiting Associate
Graduate Associate
Chief
Chief
Clinical Associate
Director
Assistant Clinical
Director
Chairman
Associate Professor
ZOl MH 00508-05 LPP
LPP, NIMH
LPP, NIMH
LPP, NIMH
LPP, NIMH (to 11/1/86)
Department of Mental
Hygiene. Johns Hopkins
University
BPB, NIMH
SBP, LCS, NIMH
DMNB, NINCDS
Division of
Neuropsychology ,
Department of
Psychiatry,
Medical College
of Pennsylvania
Chestnut Lodge Hospital
Department of Mental
Hygiene, Johns Hopkins
University
Department of Mental
Hygiene, Johns Hopkins
University
This project has as its goal the investigation of neurobehavioral
functioning in neuropsychiatrlc patients, such that: (1) Neuropsychological
profiles of diagnostlcally distinct groups can be obtained, and differences in
the profiles among groups can be used as an indication of specific organic
Influences in the psychopathology of these patient groups; (2) Neuro-
psychological profiles of patients within a heterogeneous diagnostic
classification can be obtained. Differences between patients can provide
neurobehaviorally-deflned subgroups that might reduce variability in diagnosis
and treatment, as well as Improve outcome; and (3) The obtained comprehensive
neurobehavioral data can be correlated with electrophysiological, biochemical,
and neuroradiological data that are being collected concurrently on these
patients to provide a link between pathophysiology and behavior.
C. Methods Employed
The standard adult neuropsychological battery, used for many of our
studies, is presented below. Cognitive and sensory functions are presented in
tabular form along with the test(s) used to assess them. Modified batteries
have been assembled for some of our studies, and we have assembled special
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batteries for the assessment of infants and children in three different age
groups from 2 to 16. Administration of the adult battery takes 8-12 hours and
occurs over several days. Administration of the infant and child batteries
takes from 1/2 hour to 6 hours, depending on the age of the child, and is
divided into as many sessions as needed to avoid overtaxing the subject.
LPP Adult Neuropsychological Test Battery
FUNCTION
TEST
Executive
Sequencing, Attention
Attention
Perception and Reasoning
Concept Formation and Abstraction
General Intelligence
Trail Making Test
Stroop Test
Letter Cancellation Test
Raven Standard Progressive
Matrices
Wisconsin Card Sorting Test;
Category Test (Halstead)
Wechsler Adult Intelligence Scale-
Revised
Language
Initiation
Lexical
Written
Comprehension
Auditory Discrimination
Controlled Oral Word Association
Test
Boston Naming Test
Boston Diagnostic Aphasia
Examination — Narrative Writing
Token Test (Spreen & Benton)
Semantic Aphasia Test (Goldberg)
Wepman Auditory Discrimination
Test
Memory
Global
Recent Verbal Memory
Recent Visual-Spatial Memory
Remote Memory
Motor Functions
Orientation
Wechsler Memory Scale I
Buschke Selective Reminding Test
Rey Auditory Verbal Learning Test
Recurring Figures Test (Kimura)
Complex Figure Test
(Rey-Osterrieth)
Boston Famous Faces Test (Short
Form)
Television Test
Boston Recall Test (Short Form)
Purdue Pegboard Test
Boston Apraxia Test
Temporal Orientation Test (Benton)
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LPP Adult Neuropsychological Battery (continued)
Personality
Sensory and Perceptual
Minnesota Multiphasic Personality
Inventory
Bioptor Vision Tests
Dvorine Pseudo-isochromatic Plates
Titmus Stereo Tests
Harris Test of Hand Dominance
Eye Dominance Test
Vigilance Continuous Performance Test
Visual-Spatial Hooper Visual Organization Test
Embedded Figures Test (Witkin)
Butters' Embedded Figures Test
D. Major Findings
The test battery or parts thereof has been administered to a total of 302
subjects in our laboratory (107 males, 195 females), of which 70 were control
subjects (22 males, 48 females). In addition, 77 follow-up test sessions have
been conducted in our laboratory for protocols involving two or more serial
neuropsychological evaluations. Outside the laboratory, 485 subjects have
been tested in the collaborative study with the Johns Hopkins School of
Hygiene and Public Health. The total number of subjects tested, both in and
outside of the laboratory and including follow-up testing, is 864.
1, Eating Disorder Patients
Testing of patients with eating disorders and matched normal controls has
been continued as a result of the intriguing results of last year's data
analyses. To date, 28 underweight anorexic patients (11 restrictors and 17
bullmarexics) have been assessed, and 18 have been retested after short-term
weight restoration. In addition, we have tested 40 normal-weight bulimics and
16 long-term weight-restored anorexics (7 former restrictors and 9 former
bullmarexics). Reanalysis of the data adding the subjects tested within the
past year has, for the most part, confirmed our previous findings. In many
cases, the reanalysis has strengthened those findings. First, with regard to
psychopathology, we .found, as we had previously, that both hospitalized
patient groups (underweight anorexics and normal-weight bulimics) had
significantly more pathological scores on the MMPI compared to either normal
controls or long-term weight-restored anorexics. Specifically, the
underweight anorexics had higher scores on the Hypochondriasis, Depression,
Hysteria, Psychasthenia, and Social Introversion scales compared to the
normal-weight bulimics. On the cognitive tests, underweight anorexics and
normal-weight bulimics showed deficits on a number of tasks assessing aspects
of attention (Continuous Performance Test, Letter Cancellation Test). In
addition, some deficits were seen on tests of verbal memory; these memory
deficits disappeared, however, when the contribution of attentional problems
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In these tasks was controlled statistically. On a number of tests, however,
significant deficits remained: The underweight anorexics performed
significantly worse than all other groups on the Comprehension subtest of the
WAIS-R and worse than the normal controls on the Similarities subtest; on the
Arithmetic subtest of the WAIS-R, the normal-weight bulimics were impaired;
and finally, on non-verbal learning, as assessed by the Recurring Figures
Test, the underweight and long-term weight-restored anorexics were impaired,
particularly those who had lost weight by restricting caloric intake
(restrictor type) as opposed to those who had engaged in binge eating and
vomiting or using laxatives (bulimic type).
Upon retest following weight restoration, the elevated scores on the MMPI
exhibited by the underweight anorexics were significantly reduced; however,
differences from the normal control group remained. On most cognitive tests,
improvement was seen, some of which could have been due to the effects of
practice.
2. Affective Disorder Patients
Recent developments in the study of affective disorders and their
treatments have suggested a rationale for examining the similarities and
differences between patients with bipolar affective illness and patients with
complex partial seizures. Post, Ballenger, and their colleagues have proposed
a kindling model of affective illness and temporal lobe epilepsy. Briefly, it
is postulated that repeated seizures (in the case of temporal lobe epilepsy)
or repeated biochemical and/or psychological stresses (in the case of
affective illness) produce cumulative bioelectrical changes, which in turn
result in abnormal limbic neuronal sensitization and major psychiatric
disturbances. Both disorders are thought to involve temporal lobe and limbic
system abnormalities, and both disorders have been shown to respond to
treatment with anticonvulsant agents, including carbamazepine .
We are in the process of examining the neuropsychological profiles of
patients with complex partial seizures, patients with bipolar affective
illness, and normal controls. Preliminary analyses of the neuropsychological
test data of 11 complex partial seizure patients, 13 patients with bipolar
affective illness (tested on placebo during their first test session), and 15
normal controls show that the complex partial seizure patients perform
significantly more poorly than either patients with bipolar affective illness
or normal controls on a number of neuropsychological measures. These measures
include the Trail Making Test, the Complex Figure Test, the Purdue Pegboard
Test, the Buschke Selective Reminding Procedure, and the Stroop Test (Word,
Color, and Color-Word measures). In the results for the Trail Making Test, in
addition to the significant group differences, there was also a significant
interaction between task and group. There were no significant group
differences on the Continuous Performance Test, the Halstead Category Test, or
the Stroop Interference measure. However, because there were significant
group differences on Full Scale and Verbal IQ (with the complex partial
seizure patients scoring lower than the other two groups) and on Performance
IQ (with the complex partial seizure patients lower than the controls), we
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performed analyses of covariance using Full Scale IQ as the covariate to see
what group differences in cognitive measures remained when the (probably bona
fide) IQ differences were controlled. When these analyses of covariance were
performed, significant main effects of group remained only for the Trail
Making Test. These findings are of interest and merit further investigation.
We plan to increase the sizes of our samples, and we are particularly
interested in testing more high functioning complex partial seizure patients
in order to see how these patients might differ from our rather high
functioning bipolar affective disorder group.
3. Children with Attentional Disorders
This project is a collaborative effort with the Prevention Intervention
Research Center (PIRC), a program of the Department of Mental Hygiene of the
Johns Hopkins School of Hygiene and Public Health and the Baltimore City
Public Schools. The PIRC study involves children in the first and second
grades of the Baltimore Public School System who are at risk for the
development of substance abuse, delinquency, and/or psychopathology , as well
as a large group of normal controls. Two cohorts (approximately 2400
children) of first-grade children attending 19 elementary schools in Eastern
Baltimore are being assessed periodically through teacher ratings, peer
nominations, independent behavior time sampling, and structured self-reports
coupled with information on school progress.
Our interest in this population has been the neuropsychological assessment
of attention. The correlation between attention deficit disorder and the
later development of substance abuse, delinquency, and various forms of
psychopathology has been documented in the literature. Furthermore, the
taxonomy of attention and the relationships between impairments of the various
components of attention and classroom learning, behavior, and the later
development of psychopathology are of considerable interest in their own
right. During the past year, we have developed, tested, and implemented a
battery of neuropsychological tests of attention. This battery has been
designed not only to assess attention but also to separate more global
"attentiveness" into different components of attention. Previous work from
the LPP, examining attentional components in adults, delineated a
focus/execute aspect, tapping perceptual-motor speed, a sustain component
involved in vigilant behavior, an encode factor, capturing numerical-mnemonic
qualities of attention, and the flexibility aspect of attention or the ability
to shift focus. In developing the battery for children, we borrowed some
tasks used in the adult work, adapted others for use with children, and added
some new instruments. In its final form, the battery consisted of three
versions of the X Task of the Continuous Performance Test (standard, using
auditory distraction, using degraded visual stimuli). Digit Cancellation (with
and without auditory distraction), the Wisconsin Card Sorting Test, three
subtests of the WISC-R (Coding, Arithmetic, Digit Span), and the Peabody
Picture Vocabulary Test.
We piloted the one-hour battery on 50 second graders in a school not
participating in the PIRC program. Preliminary analysis of the data indicates
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ZOl MH 00508-05 LPP
the presence of a sustain factor, reflected by Continuous Performance Test
accuracy and a shift factor comprised of performance on the Wisconsin Card
Sorting Test. In adults, the focus aspect of attention was intertwined with
an execute, or speed, component. In children, these components were found to
be distinct, reflected by Digit Cancellation performance and speed of
Continuous Performance Test responses, respectively. Of interest was a major
impulsivity factor that appeared in the child data, consisting of errors of
commission on the Continuous Performance Test and Digit Cancellation Test. In
the spring of 1987, this battery was administered to a representative sample
of 435 second graders participating in the PIRC project. The data are
currently being scored, coded, and prepared for analysis. These data will
provide the first information on the incidence of neuropsychologically-
evaluated attentional deficits in a population-based sample. In addition,
plans call for the data to be correlated with other information collected on
these children to assess the relationship of such deficits to teacher ratings
of attention, learning, and behavior in the classroom and to various
maladaptive behaviors. Also, we plan to follow these children to examine the
usefulness of the battery in the prediction of future disordered behavior.
4. Inherited Metabolic Disorder Patients
A project is underway to study the neuropsychological status of children
with inherited metabolic disorders. Two disorders are currently under study:
Gaucher's disease. Type I and III; and cystinosis, infantile type.
A subset of the LPP neuropsychological test battery has been selected and
is being administered serially to assess the presence, if any, of intellectual
deterioration over time. Also to be studied are the neuropsychological
profiles of the various groups, as well as the presence of signs consistent
with focal brain lesions. Control subjects for the Gaucher's disease group
will be drawn from the LPP-Johns Hopkins PIRC collaborative project, as our
largest group of subjects in this study is in the age range of 5 to 9 years.
A number of different clinical subgroups can be distinguished in Gaucher's
disease. We are studying those forms in which a slow, progressive
deterioration in the central nervous system (CNS) has been identified, namely.
Types I and III. Within Type I, a further distinction is made between Jewish
and non- Jewish patients, since thus far there is no evidence of CNS
deterioration in Jewish patients. So far, 17 patients with Gaucher's disease
have been studied once; follow-up data are not yet available on any of the
patients.
We have studied 8 cases of cystinosis of the infantile type who have had
kidney transplants following chronic renal failure. There is some evidence
that slow, progressive neurologic deterioration may occur following long-term
survival with this disease. This is thought to be due to CNS pathology.
Preliminary analysis of our data does not reveal evidence of deterioration and
thus suggests that the incidence of intellectual loss is low.
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5. Closed Head Injury Patients
The LPP has begun a study of patients with closed head injuries who are
referred by the Maryland Head Injury Foundation. This study is designed
primarily to provide heuristic models for psychiatric illnesses based upon
information from subjects with cerebral lesions. In addition, the data will
allow validation of the cerebral localizing value of tests in our
neuropsychological battery. Portions of the neuropsychological battery have
been shown in previous studies to be sensitive to frontal-lobe damage,
temporal-lobe damage, brainstem damage, and left- and right-hemisphere damage,
respectively. However, cross-validation studies are needed; and, to this end,
we are seeking to test six classes of patients at this time:
a. bilateral frontal-lobe damage
b. bilateral temporal-lobe damage
c. bilateral f rontotemporal-lobe damage
d. brainstem damage
e. diffuse right-hemisphere damage
f. diffuse left-hemisphere damage
It had been our original plan to test patients with damage confined to one
lobe of the brain (e.g., left or right frontal, left or right parietal, etc.);
however, it has become clear that the nature of closed head injuries is such
that damage confined to one lobe of the brain is very rare. At the same time,
patients with more widespread damage are of considerable interest in
themselves and of considerable relevance to the study of psychopathology ,
since the presumed cerebral dysfunction underlying a number of forms of
psychopathology is likely to involve more than one lobe of the brain. In
addition, although much is already known about the performance of these kinds
of patients on neuropsychological tests, we have not as yet had the
opportunity for a cross-validation study using our particular battery of
neuropsychological measures. This study will provide such an opportunity.
Thus far we have completed neuropsychological testing of 7 closed head-
injury patients: 3 with bilateral f rontotemporal damage, 1 with bilateral
frontal-lobe damage, 1 with brainstem damage, and 2 with diffuse right
hemisphere damage. As a preliminary look at these data, we have compared the
neuropsychological test performance of the 3 patients with bilateral
frontotemporal damage and the patient with bilateral frontal-lobe damage to a
group of hospitalized schizophrenic patients, since it has been hypothesized
by some that the locus of cerebral dysfunction in schizophrenia is in
bilateral frontotemporal areas. Preliminary analyses reveal that, as
expected, the schizophrenics had more elevated scores than the head-injury
patients on the MMPI, with significantly higher scores on the Frequency,
Hypochondriasis, Psychasthenia , Schizophrenia, and Social Introversion
scales. However, the schizophrenics also performed significantly worse than
the head-injury patients on a number of cognitive measures, including the
memory passages from the Wechsler Memory Scale, part B of the Trail Making
Test, immediate and delayed recall of the Complex Figure Test, the Color and
Word conditions of the Stroop Test, WAIS-R Performance IQ, and the WAIS-R
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subtests Arithmetic, Picture Completion, and Object Assembly. Finally, the
schizophrenics performed significantly worse than the bifrontal or bilateral
frontotemporal patients on three Wisconsin Card Sorting Test measures (total
errors, perseverative errors, and conceptual level responses). We will be
increasing the sample sizes of these groups in order to determine whether
these very preliminary but interesting and somewhat unexpected findings hold
up.
Proposed Course
We are planning to increase our sample sizes for several of the ongoing
studies, in order to provide greater power to our analyses. These studies
include those of eating disorders; Gaucher' s disease; normal children and
children at risk for the later development of substance abuse, delinquency,
and/or psychopathology in the LPP-Johns Hopkins collaborative study;
schizophrenia; and patients with closed head injuries. In addition, we plan
to increase the number of normal controls assessed using all forms of the
neuropsychological batteries to facilitate comparison with the various patient
groups. A number of normal controls will also receive follow-up
neuropsychological testing in order to allow for the analysis of data in
studies that involve repeated neuropsychological testing. During the coming
year, we plan to begin a more intensive study of the correlations between the
neuropsychological test data and electrophysiological data that are gathered
concurrently on many of these patient populations as well as the relationship
to biochemical measures.
Publications
Joffe, R.T., Rubinow, D.R., Squillace, K. , Lane, C.H., Duncan, C.C, and
Fauci, A.S.: Neuropsychiatric aspects of AIDS. Psychopharm. Bull. 22:
684-688, 1986.
Mirsky, A.F., and Duncan, C.C: An introduction to modern techniques of
clinical neuropsychology. In Fava , G.A. , and Wise, T.N. (Eds.): Research
Paradigms in Psychosomatic Medicine. Basel, Karger, 1987, pp. 167-184.
Jones, B.P.: Advise and dissent [Review of Clinical Application of
Neuropsychological Test Batteries]. Contemp. Psychol. , in press.
Jones, B.P.: Updating for clinical neuropsychologists [Review of Neuropsycho-
logical Assessment of Neuropsychiatric Disorders]. Contemp. Psychol., in
press.
Jones, B.P., Henderson, M. , and Welch, C.A.: Executive functions in unipolar
depression before and after electroconvulsive therapy. Int. J. Neurosci. ,
in press.
719
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00509-05 LPP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must tit on one /me between tt)e borders.)
Attention Disorders As Assessed by Event-Related Brain Potentials
PRINCIPAL INVESTIGATOR (Ust other profess/ona/ personnel below the Principal Investigator) (Name, title, laboratory, and institute attillatlon)
PI: Connie C. Duncan, Ph.D., Chief, Unit on Psychophysiology , LPP, NIMH
COOPERATING UNITS rtf any; Clinical Psychobiology Branch, Neuropsychiatry Branch,
Laboratory of Clinical Science, Child Psychiatry Branch, NIMH; Chestnut Lodge
Hospital; Developmental Neurology Branch, Medical Neurology Branch, NINCDS;
Maryland Head Injury Foundation.
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
4.0
PROFESSIONAL
0.8
3.2
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
Q (a1) Minors
Q (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The aim of this project is to investigate the roles of event-related brain
potentials, attention, and information processing and their interrelationships
in the etiology, pathology, and prognosis of psychiatric and neurological
disorders. Major emphasis is on the diagnostic specificity of disorders of
attention and cognition and the identification of the specific aspects or stages
of information processing underlying observed decrements in performance.
Concurrently recorded event-related brain potentials and performance on
cognitive tasks are used to define mechanisms of cognitive failure in subjects
with diagnoses of seasonal affective disorder, schizophrenia, eating disorders,
learning disorders, seizures , and closed head injury. Event-related brain
potentials are also used to investigate the role of altered neurochemical
mechanisms by comparing drug-induced electrophysiological and behavioral effects
with those seen in the various disorders. Psychological correlates are
investigated by relating the data to extensive neuropsychological, psychiatric,
and personality measures as well as to performance on behavioral tasks.
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PHS 6O40 (Rev. 1/84)
SPO >i4-»ia
Project Description
A. Other Personnel
Allan F. Mirsky, Ph.D.
Barbara P. Jones, Ph.D.
Edward Turner, M.S.W.
Norman E. Rosenthal, M.D.
Robert G. Skwerer, M.D.
Darrell G. Kirch, M.D.
Ralph W. Fawcett, M.D.
C. Wesley Dingman, M.D.
David C. Jimerson M.D.
Timothy D. Brewerton, M.D.
Judith M. Rumsey, Ph.D.
Martha B. Denckla, M.D.
William H. Theodore, M.D.
B. Objectives
Chief
Special Expert
Research Social Worker
Chief, Unit on Out-
patient Studies
Medical Staff
Fe 1 1 ow
Associate Clinical
Director
Medical Staff Fellow
Assistant Clinical
Director
Chief
Medical Staff Fellow
Senior Staff Fellow
Chief
Acting Chief
ZOl MH 00509-05 LPP
LPP, NIMH
LPP, NIMH
LPP, NIMH
CPB, NIMH
CPB, NIMH
NPB, NIMH
NPB, NIMH
Chestnut Lodge
Hospital
SBP, LCS, NIMH
SBP, LCS, NIMH
CPB, NIMH
ABD, DNB, NINCDS
CES, MNB, NINCDS
The major objective of this project is to yield data that will illuminate
the neurophysiological bases of the cognitive and attentional deficits in the
clinical disorders of seasonal affective disorder, schizophrenia, eating
disorders, dyslexia, epilepsy, closed head injury, and other forms of brain
pathology. Defining the specific ways in which information processing can
fail may provide new diagnostic strategies for more effective evaluation and
treatment of patients with attentional and cognitive impairments. A related
objective of this project is to differentiate state versus trait attributes of
these disorders to increase understanding of their etiologies. Concurrently
obtained event-related brain potentials (ERPs) and measures of performance
during active cognitive processing are used to define the mechanisms of
attention failure in these syndromes. Defining and understanding the
different determinants and forms of attentional and cognitive failure is
diagnost ically important as well as useful in characterizing the nature of the
psychobiology of attention disorders. Finding differences in overall response
levels between normal subjects and patients is a necessary first step;
however, the goal is to use the knowledge to lead to new approaches to
classification and treatment, to increased understanding of etiology, and to
effective preventive interventions.
C. Methods Employed
1 . Electrophysiological Assessment
The general methods of these studies include recording the EEG, using the
International 10/20 system, from frontal (FPz, Fz, F3, F4), central (Cz, C3,
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C4), parietal (Pz, P3, P4), and occipital (Oz) scalp sites referred to linked
ears, while stimuli are presented to the subject. Stimulus presentation and
data collection are controlled by a PDP-11/34 or PDP-11/73 computer. Eye
movements are monitored during the recordings, and trials contaminated by
artifacts are discarded. The EEG is averaged to yield ERPs. Since these
scalp-recorded electrical waves are associated in time with either an event in
the environment, such as the presentation of a stimulus, or with an internal
cognitive event, they are called event-related brain potentials.
Brainstem auditory evoked responses (BAERs) are used to measure, directly
and noninvasively , the progress of a sensory signal through brainstem to the
cortex, and thereby obtain a measure of the integrity of brainstem
functioning. BAERs are obtained by presenting click stimuli to the ears.
Evaluation of endogenous components of the ERP, associated with
higher-level processes such as selective attention, learning, memory, and
decision-making, yields information on the attentional and cognitive
functioning of the subject. The ERPs are elicited by trains of auditory or
visual stimuli presented in the context of an attentional or cognitive task.
The selection of tasks, which use reaction time techniques as well as recall
and recognition of stimulus material, allows for the measurement of a pattern
of cognitive behaviors and associated ERPs, where different components reflect
different aspects of information processing. Using ERPs, it is possible to
get an indication of the subject's processing of all environmental stimuli,
both relevant and irrelevant, and thus to assess, for example, the
differential processing that is the hallmark of selective attention.
A major focus of our investigations is the "P300" component of the ERP.
This scalp-derived electrical potential appears 300 msec or longer after an
event that engages the interest or attention of a subject and is a positive
voltage as recorded on the scalp; hence the name P300. The amplitude of the
P300 component depends on the amount of processing capacity invoked by a
stimulus and reflects stimulus-evaluation and decision-making activity. It is
also a sensitive indicator of orienting reactions to novel, surprising, or
incongruous stimuli and a predictor of the memorability of events. Moreover,
P300 allows a direct evaluation of the subjective probabilities that a subject
assigns to event outcomes and may reflect the extent to which a stimulus is
encoded. The latency of P300 indexes the time required to classify and
evaluate a stimulus independent of response-production factors. ERPs can thus
help to clarify the timing and order of neural events in information
processing activities and to identify the aspects or stages of information
processing responsible for observed decrements on cognitive tasks in a variety
of clinical populations.
2. Neuropsychological Assessment
Normal volunteers are screened by a psychologist who uses the lifetime
version of the Schedule for Affective Disorders and Schizophrenia (SADS-L) to
exclude those with past or current psychopathology or with first-degree
relatives with such a history. Many patients and normal volunteers are
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evaluated on an extensive neuropsychological battery of cognitive and sensory
functioning. When appropriate, tests of formal thought disorder are
administered. We plan to correlate the neuropsychological and
electrophysiological data to aid in the classification of disorders
characterized by attentional deficit and cognitive failure.
3. Biological Assessment
In collaboration with other laboratories, patients are assessed for
treatment responsiveness. In some studies, blood, urine, and/or
cerebrospinal fluid measurements reflecting neurochemical activity are
correlated with electrophysiological, neuropsychological, and behavioral
data. We also plan to use X-ray transmission tomography (CT scan) to measure
ventricular size. These data will be correlated with electrophysiological
data to yield information on the relation between ERPs and cerebral structures.
D. Major Findings
We are using a variety of attentional paradigms, which tap visual and
auditory information processing systems, to investigate patients with seasonal
affective disorder, schizophrenia, eating disorders (anorexia nervosa and
bulimia), adult dyslexia, absence epilepsy, and closed head injury. These
tests provide a differential assessment of specific types of attention,
including the ability to initiate, select, inhibit, shift, and sustain
attention. The protocol also includes evaluation of automatic and controlled
cognitive processes. The rationale for the approach of using the same
paradigms, which tap specific cognitive processes, on different patient groups
is to allow inferences about which processes are uniquely impaired in one
group in comparison with other groups. To determine whether ERPs can serve as
sensitive yet specific markers of disorder, patients with diverse
symptomatologies and diagnoses are compared. The ERP measures are correlated
with concurrently recorded behavioral responses, including reaction time, and
in some studies, with performance on neuropsychological tests.
A number of ERP studies have been completed during the past year, and the
results are now either in press or are being analyzed in preparation for
publication. The studies include investigations of seasonal affective
disorder, schizophrenia, eating disorders, the alpha-2 adrenergic agonist
clonidine, dyslexia, and absence epilepsy. The results of each of these
studies are summarized briefly below.
1. Seasonal Affective Disorder
Seasonal affective disorder (SAD) is a syndrome characterized by recurrent
depressions during the fall and winter months, when daylight is at a minimum.
The symptoms of SAD, including decreased activity, sadness, irritability,
anxiety, and appetite changes, can be treated with bright artificial light.
Moreover, the depression remits spontaneously when daylight increases in the
spring. We used ERPs to investigate whether improvement in clinical state
following phototherapy is associated with an increase in the attentional
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resources allocated to the processing of stimuli in the visual modality. We
wished to learn whether P300 changes would reflect clinical improvement. Such
a result might increase our understanding of the pathophysiology of this
disorder.
Subjects were 21 patients (15 female) who met the criteria for SAD. The
patients were tested twice, once following nine or more days of phototherapy
("On Lights") and once either preceding phototherapy or nine or more days
following its cessation ("Off Lights"). Order of the two tests was
counterbalanced across subjects. Light therapy comprised two 2 1/2-hour
sessions of 2500-lux lights per day, one in the morning and another in the
evening. ERF testing followed immediately after the morning phototherapy. A
subset (n = 11) of the control subjects was also tested twice under the same
experimental conditions. Clinical response was assessed with the Hamilton
Rating Scale for Depression.
Between-subject correlations on the patient data from the first year of
the study were computed to determine the relation between the change in P300
amplitude and the change in clinical ratings following phototherapy. SAD
patients who exhibited the most clinical improvement showed the greatest
increase in P300 amplitude in the visual modality (r = -.85, £< .005). In
contrast, clinical response was uncorrelated with changes in P300 to auditory
stimuli (_r = .24). Moreover, neither the speed nor accuracy of performance
correlated significantly with changes in the clinical ratings. No changes in
P300 were apparent in either modality in the two tests of the controls. It
thus appears that light treatment increases the amplitude of the P300
component in direct proportion to its antidepressant effect.
The time course of this effect was evaluated in 6 patients and 2 controls
who were tested several times during light therapy as well as at baseline.
The enhancement of P300 was found to occur as early as 2 days after the
initiation of light treatment in SAD patients. This finding is of
considerable interest in that it provides, for the first time, an objective,
sensitive index of the antidepressant effects of light. The results suggest
that the response to phototherapy in SAD patients is highly correlated with an
increase in the attentional resources that are mobilized to process
visually-guided information. It may be that the favorable clinical response
to phototherapy is in part mediated by enhanced cognitive capacities of SAD
patients.
2. Schizophrenia
During the past 15 years, a number of studies have shown that the
amplitude of the P300 component of the ERP is reduced in schizophrenic
patients. The amplitude of P300 has been shown to be a sensitive index of
attention deployment, so that its reduction in schizophrenic patients is
consistent with the behavioral findings. However, since the P300 is derived
from cerebral electrical activity, it offers the potential to study dynamic
brain function. Thus, the P300 component is an attractive tool to investigate
putative neurobiological mechanisms underlying the attention deficit in
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schizophrenia. We extended and broadened the finding of attenuated P300 in
schizophrenic patients by evaluating the relative effects of stimulus modality
and probability.
A total of 58 patients (including 24 unmedicated) who met DSM-III Criteria
for schizophrenic disorder were matched on age, sex, and education to a group
of 24 normal controls. Preliminary analysis of the data showed that, as in
previous studies, the P300 was smaller in the schizophrenic patients than the
normal controls. However, this difference was significant only for low
probability stimuli in the auditory modality and was not found to be
significant in the visual modality, suggesting that schizophrenic patients
have a greater deficit in auditory than in visual processing. This finding
may provide a clue to the underlying pathophysiology and is reminiscent of the
relative prevalence of auditory as compared with visual hallucinations in
schizophrenic symptomatology.
We sought to determine whether this P300 reduction observed in
schizophrenic patients is a reflection of a core deficit, independent of
clinical state, or whether it is a reflection of clinical symptomatology.
That is, is the reduced P300 a trait as contrasted with a state marker of the
disorder? To address this question, 11 schizophrenic patients were tested
twice, once when they had been free of medication for at least 4 weeks and
once when they had been stabilized on neuroleptics for at least 6 1/2 weeks.
To control for the effects of repeated testing, 7 matched normal controls were
also tested twice, at approximately the same intervals as the patients.
Clinical state was assessed with the Brief Psychiatric Rating Scale. The
relation between the change in P300 amplitude and the clinical response to
medication was assessed in the first several patients. Patients who exhibited
the most clinical improvement showed the greatest increase in P300 to visual
stimuli (i^ = -.88, £ <.0l). In contrast, clinical response was uncorrelated
with changes in P300 to auditory stimuli (_r = .13). No change in P300 was
apparent in either modality between the first and the second evaluation of the
controls.
The increase in visual but not auditory P300 amplitude is consistent with
the hypothesis that successful neuroleptic treatment enhances a patient's
capacity to process visual but not auditory information. Because auditory
P300 amplitude was not correlated with clinical state, it remains a candidate
for a vulnerability trait marker of schizophrenia. Our data, in fact, suggest
that auditory P300 appears to be significantly more sensitive to differences
between schizophrenic and normal persons than is visual P300. Moreover, it is
clear that mere treatment with neuroleptic medication alone, without
symptomatic change, is insufficient to alter visual P300 amplitude. It is
conceivable that the core deficit in schizophrenia is more closely related to
relatively invariant impaired auditory information processing and that
fluctuations in clinical symptomatic state are reflected in visual
processing.
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3. Eating Disorders
Persons with eating disorders appear to be characterized by altered
cognitive processing. In particular, there have been reports that patients
with anorexia nervosa may be impaired in automatic but not in controlled
processing. We used ERPs to assess these aspects of information processing.
All patients were women who met DSM-III criteria for anorexia nervosa
(n = 24) or normal weight bulimia (n = 34). A group of 27 matched normal
controls was also studied. Auditory and visual versions of four reaction time
tasks, which varied along the automatic-controlled dimension, were employed to
elicit ERPs.
The results indicate separation on the ERP measures between anorexic and
control and between anorexic and bulimic subjects. Specifically, anorexic
patients showed disturbances in automatic processing, as indexed by a
component reflecting an automatic cerebral mismatch process. Altered
controlled processing, as measured by P300 amplitude, was also seen in the
anorexics; the difference increased with increasing task demands. Bulimics
were not distinguishable from controls on the measures. Preliminary findings
after long-term weight restoration in 15 anorexic patients indicate reversal
in all of the ERP abnormalities except one: The Slow Wave component following
P300 was significantly enhanced in these patients, indicating a relatively
permanent disturbance in controlled information processing.
4. Clonidine
A number of investigations have found that malnourished, underweight
patients with anorexia nervosa have decreased concentrations of norepinephrine
or MHPG in urine, plasma, or CSF. More recently, several studies have
suggested that underweight anorexic patients have increased alpha-2
adrenoceptor activity. Such findings suggest that the functional activity of
noradrenergic systems is reduced in these patients. A major CNS noradrenergic
pathway originates in the locus ceruleus, which, by virtue of its widespread
cortical and subcortical connections, appears to play a role in the
maintenance of such functions as attention, sleep, and wakefulness. We have
demonstrated that there is an attentional deficit in women with anorexia
nervosa that is reflected in changes in the ERP. This study was designed to
explore the possible role of locus ceruleus pathophysiology in the attentional
disturbance in anorexia nervosa. Clonidine, a relatively specific alpha-2
adrenoceptor agonist, is reported to decrease locus ceruleus activity. This
drug was given to healthy, normal women to determine whether it would produce
ERP changes similar to those observed in anorexic patients. Such changes
would support the hypothesis of a disturbance in locus ceruleus function in
this disorder.
Eight healthy young women were administered, on separate days, three doses
of clonidine (0.5, 1.0, and 2.0 micrograras/kilogram) and two saline placebos
infused intravenously in a counterbalanced order under double-blind
conditions. To assess effects on attention, ERPs were recorded from a variety
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of standard scalp placements during auditory discrimination tasks.
Results indicate that clonidine produced changes in the ERP that resemble
some of the alterations observed in anorexic patients, namely, decreases in
the amplitude of the P300 component. The data thus support, in part, the
hypothesis of altered locus ceruleus function in anorexia nervosa. However,
the lack of effect of clonidine on the early negative component suggests that
neurochemical systems other than the noradrenergic locus ceruleus system may
underlie the reduction in this component in anorexic patients.
5. Dyslexia
Sixteen dyslexic adult men, including 7 with a probable history of
Attention Deficit Disorder (ADD) and 8 with no history of Attention Deficit
Disorder (NONADD), and 15 matched normal controls were studied with a battery
of paradigms designed to elicit ERPs . The goal of the study was to discover
whether dyslexic adults differ from controls in visual and/or auditory
information processing.
Smaller P300s were observed over the left hemisphere for both patient
groups in comparison to controls. Over the right hemisphere, P300 amplitude
of the NONADD group was equal to that of the controls, while the ADD subjects'
P300s remained relatively attenuated. No group differences in modality were
found. P300 latency in the parietal areas was longer for both the ADD and
NONADD subgroups relative to the controls, but these differences were not
specific to one modality. The amplitude of the auditory Slow Wave was smaller
over both hemispheres for ADD than for NONADD or control subjects, while the
NONADD subjects exhibited larger visual and auditory Slow Wave components over
the right hemisphere. This difference between groups diminished with
decreasing attentional load.
The reduced amplitudes of the P300 and Slow Wave components for the ADD
group suggests a generalized deficit. Although these results must be
considered as preliminary, they suggest that different etiologies may underlie
dyslexias with and without concomitant attention deficit. Both dyslexic
groups showed evidence of compromised left hemisphere functioning; however,
the possibility exists, as well, of greater right hemisphere (and right
parietal) involvement in dyslexia with ADD. Such considerations suggest two
different types of pathophysiological processes underlying the two
disordersand may ultimately lead to different strategies of remediation in the
two groups of patients.
6. Absence Epilepsy
The ERP technique was used to study information processing in the
interictal period in a group of 8 patients with absence seizures and a matched
group of 8 normal controls. Event-related brain potentials were recorded
during performance of auditory and visual versions of the Continuous
Performance Test (CPT) of sustained attention.
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Reaction time differences on the CPT between the absence patients and the
normal controls confirm previous findings: such patients are strikingly
impaired in their ability to perform tasks of sustained attention. This study
extended the previous findings by demonstrating convincingly that the
attention deficit is observed in the auditory as well as the visual modality.
The behavioral differences seen on the CPT were paralleled in virtually
isomorphic fashion in the P300 amplitude data. Whereas the between-group
differences in visual P300s were somewhat smaller, the between-group P300
differences found in both versions of the auditory CPT were substantial.
Moreover, the between-group processing differences apparent in the ERPs to
target stimuli were also seen in the ERPs to at least one category of
nontarget trial—warned, no-go trials. These waveforms suggest, in fact, that
the control subjects' processing of this category of nontarget stimulus was at
least as complex as their processing of target stimuli: the P300 and Slow
Wave components elicited under these conditions were larger than in the other
conditions in this experiment. Such waveform complexity was either not
present in the absence patients or was present in a much attenuated form.
NlOO amplitude and latency were also evaluated as additional indices of
information processing. Whereas NlOO amplitude did not vary between groups,
NlOO latency was longer for the absence patients than the normal controls.
Although the possibility of a generalized deficit in the patients seems
unlikely, this possibility was tested by computing average ERP waveforms for
the three highest functioning patients only. These average ERPs were
essentially the same as those based on the remaining patients. Moreover,
analyses of the P300 data of this subgroup of patients and a matched subgroup
of three normal controls yielded the same results as the analyses based on the
full samples.
This suggests that the failure to respond efficiently on visual and
auditory forms of the CPT is due, at least in part, to the failure of absence
patients to mobilize and sustain attentional capacity. Whereas later NlOOs
are indicative of delayed perceptual encoding, the lack of difference in P300
latency indicates that the higher-level processes of identifying and
categorizing the stimulus proceed at an approximately normal rate. The
reaction time data indicate a significantly slower response to auditory
targets in the patients. The lack of a significant difference between groups
in P300 latency suggests that the slower response is not due to an increase in
stimulus processing time but to prolonged response processing in the
patients. Moreover, the lower percentage of correct responses in the patients
indicates that this prolonged response processing is not due to the use of a
more cautious response strategy. Rather, it appears that the execution of the
response itself is delayed. Thus, by using P300 in conjunction with reaction
time, we were able to show that the delay in responding on the auditory CPT-AX
task exhibited by patients with absence epilepsy occurs subsequent to stimulus
evaluation.
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Significance to Biomedical Research and the Program of the Institute
Since attentional deficit and cognitive dysfunction are characteristic of
many psychopathological and neuropathological disorders, it is important to
develop a precise empirical and theoretical account of these symptoms. The
scalp-recorded ERP is the only noninvasive technique available for studying
the dynamic neural activity associated with cognitive processing in human
subjects. The ERP provides information on mental events involved in selective
attention, stimulus evaluation and decision making, memory, learning, and
response preparation. The temporal resolution of ERPs can support inferences
about brain activity on time scales not possible in studies using tissue
assays or radioactivity. Because of the noninvasive character of ERPs,
patient state can be monitored often enough to assess the effects of specific
clinical or experimental variables. The appropriateness of evaluating ERPs in
studies of attention is apparent, as they may provide a dissection of the
various components involved and thereby permit more precise identification of
the types of information processing deficits responsible for poor performance
on attention tasks in a variety of patient groups. It is hoped that the
developing battery of ERP and neuropsychological tests applied to patients
characterized by attentional deficit and cognitive dysfunction will ultimately
provide a neurobiological profile of each disorder and lead to more refined
subcategorizations , as well as to more efficacious treatments.
Proposed Course
We are currently completing data collection on our studies of patients
with eating disorders. Data analysis is in progress on our studies of
seasonal affective disorder, schizophrenia, absence epilepsy, dyslexia, and
clonidine. We plan to continue our investigations of patients with seasonal
affective disorder, schizophrenia, and closed head injury. We plan to extend
our studies of affective disorder patients in depth as well as scope. In the
seasonal population, we are interested in whether an even more rapid change in
P300 may be measurable. If so, this would be of predictive value in
evaluating which patients are most likely to benefit from li-ght therapy. It
would also be of theoretical value in helping to evaluate the time course of
the neurobiological processes underlying the effect of phototherapy. In
addition, we plan to study P300 in other types of affective disorder patients
to assess the specificity of our findings. Our work is aimed at illuminating
the neurophysiological bases of the cognitive and attentional deficits in
affective disorder, schizophrenia, and other psychiatric disorders. Of
interest are the relation of ERP variables to diagnosis, diagnostic
symptomatology, severity of disorder, degree of formal thought disorder,
performance on tests of attention, memory, and intellectual functioning,
degree of improvement during treatment, and improvement on specific
treatments. We plan to expand our investigation of the interrelation among
ERP components and neuropsychological and neurochemical variables. To
increase our understanding of the etiology of schizophrenia and affective
disorder, we are planning additional studies to differentiate state versus
trait attributes of the disorders. The strategy we intend to use is to
compare normal controls with patients when they are actively symptomatic and
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when they are in remission. We plan to begin testing first-degree relatives
of psychiatric patients to determine whether the ERP is a marker of specific
disorder. Selected studies with head-injured cases are planned to test
hypotheses derived from various clinical groups concerning the involvement of
brain structures in the pathophysiology of psychiatric disorders. Electro-
physiological predictors of clinical response to psychopharmacological and
other forms of treatment will be sought, as patient availability allows.
Publications
Duncan, C.C.: P300 Applications to Research on Schizophrenia. In McCallum,
W.C, Zappoli, R., and Denoth, F. (Eds.): Cerebral Psychophysiology : Studies
in Event-Related Potentials. (EEG Supplement 38). Amsterdam, Elsevier, 1986,
pp. 420-424.
Joffe, R.T., Rubinow, D.R., Squillace, K., Lane, C.H., Duncan, C.C. and Fauci,
A.S.: Neuropsychiatric aspects of AIDS. Psychopharmacol. Bull. 22:
684-688, 1986.
Roth, W.T., Duncan, C.C, Pfefferbaum, A., and Timsit-Berthier , M. :
Applications of Cognitive ERPs in Psychiatric Patients. In McCallum, W.D.,
Zappoli, R. , and Denoth, F. (Eds.): Cerebral Psychophysiology: Studies in
Event-Related Potentials. (EEC Supplement 38). Amsterdam, Elsevier, 1986,
pp. 419-438.
Mirsky, A.F., and Duncan, C.C: An Introduction to Modern Techniques of
Clinical Neuropsychology. In Fava, G.A. and Wise, T.N. (Eds.): Research
Paradigms in Psychosomatic Medicine Basel, Karger, 1987, pp. 167-184.
Duncan, C.C: Application of Event-Related Brain Potentials to the Analysis
of Interictal Attention in Absence Epilepsy. In Myslobodsky, M.S. and Mirsky,
A.F. (Eds.): Elements of Petit Mai Epilepsy. New York, Peter Lang, in press.
Duncan, C.C, Perlstein, W.M. , and Morihisa, J.M. : The P300 metric in
schizophrenia: Effects of probability and modality. Electroencephalo. Clin.
Neurophysiol. , in press.
Mirsky, A.F., and Duncan, C.C: Attention Impairment in Human Clinical
Disorders: Schizophrenia and Petit Mai Epilepsy. In Sheer, D.E. and Pribram,
K. (Eds.): Attention: Cognition, Brain Function, and Clinical Application.
New York, Academic Press, in press.
Duncan, C.C, and Kaye, W.H. : Effects of clonidine on event-related potential
measures of information processing. Electroencephalog. Clin. Neurophysiol.,
in press.
Duncan, C.C: Current Issues in the Application of P300 to Research on
Schizophrenia. In Straube, E. and Hahlweg, K. (Eds.): Schizophrenia:
Models, Vulnerability and Intervention. New York, Springer-Verlag, in press.
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Duncan, C.C, Morihisa, J.M. , Fawcett, R.W, , and Kirch, D.G. : P300 in
schizophrenia: State or trait marker? Psychophamiacol . Bull., in press.
Duncan, C.C: Event-related brain potentials: A window on information
processing in schizophrenia. Schizophr. Bull., in press.
Rosenthal, N.E., Skwerer, R.G., Sack, D.A. , Duncan, C.C, Jacobsen, P.M.,
Tamarkin, L. , and Wehr, T.A. : Biological effects of morning plus evening
bright light treatment of seasonal affective disorder. Psychopharmacol .
Bull. , in press .
Skwerer, R.G., Duncan, C.C, Sack, D.A., Jacobsen, F.M. , Tamarkin, L. , Wehr,
T.A. , and Rosenthal, N.E. The biology of seasonal affective disorder and
phototherapy. J. Biol. Rhythms, in press.
732
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02288-03 LPP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 charecten or less. Title must lit on one line between the borders.)
Studies on Etiological Factors In Schizophrenia
PRINCIPAL INVESTIGATOR {List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
PI: Seymour S. Kety, M.D.
Others: Loring Ingraham, Ph.D.
Bjorn Jacobsen, M.D.
Fini Schulsinger, M.D.
Dennis Kinney, Ph.D.
Paul Wender, M.D.
Senior Scientist, NIMH
Staff Fellow, LPP, NIMH
Assoc Prof Psychiatry, Univ. of Copenhagen
Prof. Psychiatry, Univ of Copenhagen
Asst Prof Psychiatry, Harvard University
Prof Psychiatry, Univ. of Utah
COOPERATING UNITS (if any)
Psychological Institute, Copenhagen, Denmark; McLean Hospital, Belmont, Mass.
Harvard University; University of Utah.
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
2.0
PROFESSIONAL:
2.0
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects
D (a1) Minors
S (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies of the occurrence of mental illness in families have been useful in
identifying familial forms of the illnesses and in the development of hypotheses
regarding the form and strength of genetic and environmental factors in
etiology. Where these major variables are separated by the process of adoption,
specific etiologic hypotheses can be tested separately and in combination. A
total national sample of 14,500 adult adoptees in Denmark provides the basis of
the main components of this research. The research this year has focused on 46
classical schizophrenic adoptees identified in the national sample with a
comparable number of control adoptees with no history of mental illness. The
remarkable population registers in Denmark permit the identification of the
close biological and adoptive relatives of these adoptees. By search of mental
hospital registers and ultimately by personal interviews, information on the
psychiatric history and status of the relatives has been obtained. During the
past year these interviews and other records have been used to obtain consensus
diagnoses by two raters without knowledge of the relationship among relatives
and adoptees. The first phase of the analysis has now been completed with
results which confirm the findings on the first sample (restricted to Greater
Copenhagen) that classical chronic schizophrenia occurs almost exclusively among
the biological relatives of chronic schizophrenic adoptees and not In their
adoptive relatives. A marginal syndrome which has been variously designated as
latent or borderline schizophrenia or schizotypal personality was also found to
be significantly more prevalent in the biological relatives of chronic
schizophrenic adoptees, but was also more prevalent in the biological relatives
of adoptees with other major mental disorders or marginal schizophrenia.
The study of mental disorder in the relatives of adoptees with affective
disorder completed in the previous year has now been published.
1 Z33.
PHS 6040 (Rev. 1/84)
SPO 9I4-9I
ZOl MH 02288-03 LPP
Project Description
Objectives
The objective of this phase of the study of schizophrenic adoptees and
their families has been to extend the survey, initially confined to the city
and county of Copenhagen, to all of Denmark, evaluating the strength of
genetic and family-related environmental Influences, and to define more
explicitly the traits which comprise the syndrome of latent schizophrenia.
Adoption Study of Schizophrenia
Comprehensive interviews with the biological and adoptive relations of 34
schizophrenic adoptees and 34 non-schizophrenic control adoptees, rated
blindly, had found a significantly higher prevalence of schizophrenia and
schizophrenia-related disorder in the biological relatives of schizophrenic
adoptees than in controls. Moreover, eight of the characteristics found in
the relatives diagnosed by us as latent or uncertain schizophrenia permitted
us to characterize them with the DSM-III diagnosis of "schizotypal personality
disorder."
In the 9,000 adoptees outside of Copenhagen, 37 were identified as having
developed schizophrenia, of which 29 qualified as 'chronic schizophrenia' as
described in DSM-II or 'schizophrenia' in DSM III. In the course of the past
three years, interviews were completed on approximately 90 percent of the
relatives of these and their control adoptees who are alive and residing in
Denmark; these interviews were rated blindly by two experienced judges using
global evaluations based on the descriptions of Kraepelin and Bleuler.
During the past year those evaluations were completed and complemented by
a review of abstracts of hospital records and reports of incomplete
interviews. As was the case in the Copenhagen sample, over- 90% of the
relatives alive and residing in Denmark, Sweden or Norway participated in a 36
page interview covering a social, psychological and medical history and a
complete mental status examination. Since the research design called for
control adoptees with no history of serious mental Illness, the initial group,
selected as having no record of hospitalization for mental illness, was also
interviewed and rated blindly along with the relatives. The 26 control
adoptees thus selected gave complete Interviews and no mental disorder more
serious than anxiety neurosis or mild depression was found.
The study in Copenhagen found a significant concentration of schizophrenia
and schizophrenia-like disorders (chronic, acute, latent and probable
schizophrenia, and schizoid personality - the 'schizophrenia spectrum of
disorders') in the biological relatives of the chronic schizophrenic
adoptees. The same finding occurred in the present or Provincial sample
representing the rest of Denmark outside of Copenhagen, (34 among 173
identified biological relatives of chronic schizophrenic adoptees versus 11
among 162 such relatives of the control adoptees, p = 0.0004). Further
734
ZOl MH 02288-03 LPP
breakdown of the schizophrenia spectrum in the present sample gave results
which confirmed those from the Copenhagen study: 'acute schizophrenia' (a
diagnosis similar to DSM-III schizophreniform disorder) and 'schizoid
personality' did not occur significantly more often in the biological index
relatives than in those of the controls; if these two categories are excluded
from the spectrum, its preponderance in the biological relatives of the
chronic schizophrenic (index) adoptees is considerably enhanced as it was in
the Copenhagen sample (24/173 vs 3/162 p = 0.00003). Chronic schizophrenia
appeared only in the biological index relatives (5/173 vs. 0/162, p = 0.036).
Probable chronic schizophrenia as well was absent from the relatives of the
controls and if the results for definite and probable schizophrenia are added
the results are even more striking (9/173 vs 3/162 p = 0.008). Latent
schizophrenia was found three times more frequently in the relatives of latent
schizophrenic adoptees than in those of chronic schizophrenic adoptees.
These vaguer syndromes are not limited to the index relatives, but, in
contrast to chronic schizophrenia, are found in comparable prevalence in the
biological relatives of adoptees who could not be interviewed or were rejected
as controls by virtue of a history of serious mental disorder (major
affective, hysterical psychosis, or a schizophrenia spectrum disorder). This
was also true for the Copenhagen sample.
In contrast to chronic schizophrenia, the syndromes of latent and probable
latent schizophrenia are not limited to the index relatives, but are also
found in the biological relatives of adoptees rejected as index or control
probands by virtue of a history of psychiatric disorders other than
schizophrenia. For example, among the biological relatives of adoptees with a
history of major affective Illness, 12% (5/30) were found to have latent or
probable latent schizophrenia, while only 2% (3/162) of the relatives of
control probands received these diagnoses (p = 0.009). Taken in conjunction
with the finding above of a greater proportion of latent schizophrenia in the
relatives of latent schizophrenic adoptees than In relatives of chronic
schizophrenic adoptees, this finding raises the question of the specificity
the relatives of individuals with schizotypal personality disorder in the
current diagnostic nomenclature have to schizophrenia, and whether diagnostic
criteria can be developed for a marginal sjmdrome more specifically related to
schizophrenia.
Major affective disorders were not more prevalent in the index relatives
than in their controls, confirming the similar observation made in the
Copenhagen sample and supporting the genetic distinctiveness of schizophrenia
from manic-depressive illness. A significantly higher proportion of the
relatives of controls were found to be free of mental illness than any other
group of relatives, but neurosis is significantly more common in them (10.5%
vs 2.3% in the index relatives, p = 0.002), probably reflecting the absence of
more serious overriding diagnoses. In general, the only disorders found to be
significantly concentrated in the index relatives were chronic and latent
schizophrenia.
Counting by probands is the more conservative means of testing the genetic
735
ZOl MH 02288-03 LPP
hypothesis. In this sample 6 chronic schizophrenic adoptees had one or more
biological relatives with definite or probable chronic schizophrenia and none
among the control adoptees (p = 0.016), 10 families with latent schizophrenia
and 3 among the controls (p = 0.045), and 14 families with chronic or latent
schizophrenia compared with 3 among the controls (p = 0.0034). Similar but
less significant results were found in the Copenhagen study.
A deficiency in the Copenhagen sample was the relative absence of
biological full siblings of the probands (there were only 3 in the index and 5
in the control families) so that the significant concentrations of
schizophrenic illnesses were found mainly in the biological half-siblings
rather than the first degree relatives. In the Provincial sample the bulk of
the first degree biological relatives were full siblings, which is
attributable to the greater number of stable monogamous relationships existing
in the towns and villages, with the result that the first degree relatives in
that sample account for most of the significant differences. In the 82 first
degree biological relatives of the chronic schizophrenic adoptees compared
with the 67 such relatives of the control adoptees there were 7 vs 0 diagnoses
of chronic schizophrenia, definite or probable (p = 0.014) and 2 vs 0 in the
second degree relatives. For chronic and latent schizophrenia, definite or
uncertain, the prevalence in first degree relatives was significantly higher
than that in their controls (p = 0.006) and less strikingly so for the second
degree relatives (P = 0.027). There is a greater preponderance of
schizophrenia and schizophrenia spectrum disorders in the first degree
relatives of this sample which is in accord with genetic expectations.
Absence of a concentration of schizophrenia in the index adoptive
relatives does not argue against the Importance of environmental factors in
tlie etiology of schizophrenia. In addition to the several hundred
environmental variables examined in the interviews, efforts vrere made to
obtain specific information relating to current hypotheses. These results are
presently under examination and analysis.
Significance to Biomedical Research and to the Program of the Institute
These findings confirm and extend the results obtained previously
indicating a strong and quite specific genetic influence in the transmission
of classical schizophrenia. They also support a genetic relationship between
a milder syndrome - latent schizophrenia of DSM-II or schizotypal personality
of DSM-III- and classical schizophrenia, although this milder syndrome is not
specific to the relatives of chronic schizophrenic subjects alone. These
observations also indicate that the well known tendency of schizophrenia to be
concentrated in families is the result of genetic rather than
family-associated environmental factors and validate the usefulness of family
studies of nonadopted schizophrenics for the examination of genetic
influences. A major implication of the operation of genetic factors in
etiology is the recognition of the importance of biological, and especially,
biochemical factors in this disorder, since the genes can only express
themselves through biochemical processes. Another important result of the
736
ZOl MH 02288-03 LPP
more conclusive genetic evidence derived from adoption studies in
schizophrenia, already realized, is the rejection of the widely promulgated
hypothesis of the 'schizophrenogenic' parent, freeing such parents from an
unwarranted and oppressive burden.
Proposed Course
Environmental variables pertaining to socioeconomic class, history of
infections and dietary habits, rearing practices, personality of rearing
parents, language patterns, communication deviance, expressed emotion, as well
as evaluation of cognitive function such as measures of thought disorder, a
psychobiological test of smooth pursuit eye movements and one biochemical
measure of were included in the new data gathered with the interviews in the
Provincial sample. These will be analyzed and correlated with the clinical
and demographic data at hand to examine existing hypotheses and possibly to
generate some new ones regarding the environmental influences operating in
schizophrenia .
The interview data, particularly the large number of items in the mental
status examination, will be analyzed to more accurately specify the symptoms
and manifestations found among the biological relatives of the chronic
schizophrenic probands, with the aim of identifying traits genetically
associated with schizophrenia and developing more specific characteristics of
syndromes genetically related to schizophrenia in the biological relatives of
chronic schizophrenic adoptees. The identification of such a syndrome would
aid in the search for clear pedigrees of schizophrenic illness by allowing
more individuals to be studied and tested for biological markers than the
current low number of biological relatives with frank schizophrenic illness.
As part of this search it will be important to differentiate such a syndrome
from the milder psychopathology which has been found in the biological
relatives of adoptees with other mental disorders such as major affective
illness.
A third sample of schizophrenic adoptees has been identified in Denmark,
representing adoptees with onset of illness and hospitalization after the
previous search through the adoption and psychiatric registers. The
biological and adoptive relatives of this sample, with suitable controls, will
be examined for mental illness on the basis of hospitalization alone,
deferring perhaps indefinitely the expense of the exhaustive psychiatric
interviews that have characterized the two previous samples. There Is reason
to believe on the basis of the results with these samples that in Denmark the
number of chronic schizophrenic individuals who never reach a mental hospital
is very small.
Publications
Wender, P.H. , Kety, S.S., Rosenthal, D. , Schulslnger, F. , Ortmann, J., and
Lunde, I.: Psychiatric disorders in the biological and adoptive families of
adopted individuals with affective disorders. Arch. Gen. Psychiatry, A3:
923-929, 1986.
737
ZOl MH 02288-03 LPP
Kety, S.S.: Cerebral circulation and its measurement by Inert diffusible
tracers. Israel J. Med. Sci., 23: 3-8, 1987.
Kety, S.S.: Neuroscience and psychiatry. Psychiatric Annals, 17: 412-416,
1987.
Kety, S.S. and Matthysse, S. Genetic and biochemical aspects of
schizophrenia. In Nicholi, A. (Ed.): Harvard Modern Guide to Psychiatry,
Cambridge, MA, Harvard University Press, in press.
Ingraham, L.J., Kety, S.S.: Schizophrenia spectrum disorders. In Handbook of
Schizophrenia, Vol. Ill; M. Tsuang, M. , & Simpson, J.C. (Eds.): Amsterdam,
Elsevier, in press.
738
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02295-02 TPP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less. Title rr^ust fit on one line between the borders.)
Genetic Factors in Response to Alcohol
PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
PI: Connie C. Duncan, Ph.D.
Co-PI: Frances H. Gabbay, Ph.D.
Others: Allan F. Mirsky, Ph.D.
T. Peter Bridge, M.D.
Chief, Unit on Psychophysiology T.PP/NTMH
Guest Researcher LPP/NIMH
Chief LPP/NIMH
Deputy AIDS Coordinator ADAMHA
COOPERATING UNITS (It any)
Department of Mental Hygiene, School of Hygiene and Public Health, Johns
Hopkins University
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.6
PROFESSIONAL
1.1
0.5
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
n (a1) Minors
13 (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this project is to assess the relative contributions of
genetic and environmental factors to alcohol drinking and response to alcohol
challenge. The project is composed of two studies. In the first, 100 male
monozygotic (MZ) and 100 male dizygotic (DZ) twin pairs will complete a
questionnaire describing their alcohol and other drug use and a 28-day record of
their alcohol intake. These data will permit estimation of the relative
contributions of genetic and environmental factors to frequency and amount of
alcohol consumption. In addition, self-reports of contact between cotwins will
permit estimation of the importance of shared environment on cotwin similarities
in alcohol drinking. Finally, comparison of the questionnaire estimates of
alcohol consumption with those derived from the 28-day record will permit
evaluation of the validity of the questionnaire method.
In the second study, 15 male MZ and 15 male DZ twin pairs will receive, in
separate testing sessions, a placebo and two doses of alcohol (0.40 and 0.80
g/kg of 95% ethyl alcohol). The protocol will consist of electrophysiological
measures (e.g., brainstem auditory evoked responses, resting EEG, and visual and
auditory event-related potentials), self-reports of affect, and a measure of
standing stability. The use of placebo and multiple doses of alcohol will
permit conclusions about the effects of alcohol on information processing,
response production, mood, and motor activity. The twin design will provide
information on the relative contributions of genetic and environmental factors
to variability in these measures in the drug-free state and following response
to alcohol challenge. Finally, conclusions regarding the stability of the
measures across time will be based on comparisons of baseline measures across
the three sessions.
739
PHS 6040 (Rev. 1/84)
SPO SI4-9lt
ZOl MH 02295-02 LPP
A. Objectives
The primary and most significant objective of this project is to estimate
the relative contributions of genetic and environmental factors to alcohol
drinking and response to alcohol challenge. In addition, the project will
test the effects of alcohol on information processing and response production,
on mood, and on motor activity, and examine the stability of alcohol drinking,
electrophysiological activity, and response to alcohol across time within
individuals. Finally, these data will permit comparison of alternative
self-report methods of estimating alcohol intake.
B. Methods Employed
In the first study, 200 male twin pairs (MZ=100 and DZ=100) will complete
a comprehensive questionnaire describing their alcohol and drug use.
Estimates of monthly alcohol intake will be computed on the basis of these
data. The same twins will keep a 28-day record of their alcohol intake. From
these "diaries," seven variables describing twins' alcohol use will be
computed (e.g., grams of alcohol consumed per weekend day, number of days
drinking, maximum amount consumed in a day). Intraclass correlations will be
used to assess intrapair similarity, and these estimates will be used to
calculate the heritability of alcohol intake (i.e., percent variance accounted
for by genetic factors). A self-report measure of "twin closeness" will be
taken to estimate the extent to which cotwins interact. This estimate will be
compared to estimates of within-pair similarity in alcohol drinking patterns
to determine to what extent this component of shared environment accounts for
similarity in drinking. Finally, a Pearson correlation will be computed to
compare the two methods of estimating alcohol intake.
In the second study, 30 male twin pairs (MZ=15 and DZ=15) will be studied
in the laboratory. Over the course of three experimental sessions, they will
ingest a placebo, 0.40 g/kg, and 0.80 g/kg 95% ethyl alcohol. The test
protocol will include electrophysiological measures (brainstem auditory evoked
responses, resting EEG, and visual and auditory event-related potentials),
self-reports of affect, and a measure of standing stability. Baseline
recordings will be made; and, after the beverage, ERP measurements will be
taken once again, while resting EEG, BAERs , standing stability, and the
affective measure will be repeated twice. Breath samples will be taken every
10 minutes to estimate blood alcohol levels. Intraclass correlations will be
computed to assess intrapair similarity in these measures in the drug-free
state and following alcohol challenge. Repeated measures analyses of variance
will be used to test the effects of alcohol on these measures. Pearson
correlations will be used to assess the relationships among the various
measures (e.g., EEG and affect) and to estimate the stability of these
measures across time within subjects.
C. Major Findings
Both studies are still in progress. Preliminary results from the first
study indicate that MZ twins are strikingly similar in the amount of alcohol
740
ZOl MH 02295-02 LPP
consumed and in their patterns of consumption. Intraclass correlations for
the variables derived from the 28-day diaries range from .77 to .91. In
contrast, estimates of within-pair similarity for DZ twins indicate that they
are, on the average, far less similar than MZ pairs in their patterns of
alcohol consumption. This suggests that for males, genetic factors play a
role in moderate alcohol consumption patterns, a finding that is consistent
with previous research. The pattern of MZ-DZ correlations (e.g., DZ
correlations that equal less than half the corresponding MZ correlation)
suggests that the genetic contribution is not strictly additive.
Over 40 pairs of twins have been recruited through advertising in the
Washington, D.C. Metropolitan area, and these twin pairs are currently being
screened for participation in the laboratory study. Computer programs that
will permit the recording, analysis, and display of resting EEC, self-reported
affect, and standing stability have been developed. Non-twin pilot subjects
have been run in the alcohol protocol, and the following results (based on
visual inspection of the data, not on statistical analysis) are reported:
1. There appears to be a detectable alcohol effect on the measures we are
using. Alcohol caused a slight delay of peaks I through V of the brainstem
auditory evoked response (BAER), indicating that transmission time in the
brainstem is slowed by alcohol. Further, alcohol appears to cause a decrease
in P300 amplitude of the event-related potential and an increase in its
latency, suggesting that alcohol also slows cognitive processes which occur
later. Spectral analyses of the resting EEC suggests that the EEC is
characterized by increased power in the lower frequency range (i.e., theta
band) following alcohol ingestion, consistent with the known soporific effects
of the drug. Conclusions about the affective change and its relationship to
the EEG frequency changes await further analyses.
2. Of great importance is the finding that, while the alcohol effects
appear in some subjects, and generally appear in the averaged data, the
effects are not equally strong and in the same direction for all subjects.
For example, one subject showed a decrease in the latency of BAER peaks. This
is consistent with previous findings by the Co-PI, as well as other
investigators; but because of statistically significant effects in grouped
data, these individual differences typically have not been emphasized in
research reports.
3. The electrophysiological measures appear to be very stable within
subjects across time. This retest stability has implications for the analysis
of alcohol effects and for the estimation of heritability. Within-subject
variability increases error variance and decreases the chances of detecting
alcohol effects, while retest reliability puts an upper limit on heritability
(i.e., cotwin similarity will be limited by the extent to which the trait is
stable within a single subject). Thus, their retest stability reinforces the
decision to include these measures in this study and suggests they will prove
useful in future pharmacogenet ic research.
4. Time course effects observed in the early data from this study point
741
ZOl MH 02295-02 LPP
to important methodological issues as well as provide potentially important
information on the mechanism of drug action. Recordings were made once before
and twice after alcohol ingestion. There were differences across subjects and
across variables in the course of effects. In some cases, depressant-like
effects occurred immediately, while in others effects were not observed until
the second test. As has been reported previously, stimulant-like effects were
observed in some cases during the ascending blood alcohol curve, with
depressant effects occurring later. Failure to repeat assessment over time
would result in incorrect conclusions about the effects, or lack of effects,
of alcohol.
D. Significance to Biomedical Research and the Program of the Institute
The significance of the first study lies in its power to estimate the
relative contributions of genetics and environment to alcohol drinking in a
sample of nonalcoholic males. To date, no such studies based on samples drawn
from within the U.S. have been published. Moreover, the use of multiple
measures of alcohol intake will increase the confidence in the validity of the
findings. Finally, the estimation of social interaction between cotwins and
the use of that estimate to account for within-pair similarity in alcohol
drinking will provide information on the nature (rather than just the size) of
the environmental contributions.
The significance of the second study derives from its use of powerful
electrophysiological measures of brain activity within the context of a
pharmacogenetic design. This project will shed light on information
processing and response production, on disruptions in these processes
resulting from alcohol ingestion, and on the heritability of these aspects of
brain functioning. Moreover, the finding of individual differences in both
baseline electrophysiological characteristics and in response to alcohol will
be of great importance in the study of alcoholism. These measures will now
need to be employed in studies of individuals at risk for the disorder in
order to determine whether these differences are associated with differences
in vulnerability to alcoholism.
E. Proposed Course
Data collection for the first study (questionnaires and "diaries) will be
completed by the end of 1988. Collection of laboratory data on the twin
sample will commence in October and proceed as twin availability allows. Data
analysis and preparation of the results for publication and presentation will
be conducted as the data are collected.
F. Publications
None
7A2
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
:;01 MH 02404-01 LPP
PERIOD COVERED
July 21, 1987 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit or> one line between the borders.)
Psychophysiological Investigations of Preattentional and Attentional Function.
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Bruno J. Anthony, Ph.D. Senior Staff Fellow LPP, NIMH
COOPERATING UNITS (if any)
Department of Mental Hygiene, Johns Hopkins School of Hygiene and Public
Health; Baltimore City Public Schools.
LAB/BRANCH
Laboratory of Psychology and Psychopathology
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
PROFESSIONAL:
CHECK APPROPRIATE BOX{ES)
S (a) Human subjects
13 (a1) Minors
H (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project applies a new methodology to examine dysfunction in
preattent ive and attentive mechanisms involved in the regulation of
information processing within the central nervous system. Actions of these
regulatory mechanisms are assessed through patterns of blink reflex
modification, cardio-respiratory change and behavioral measures during simple
two-stimulus paradigms. These measures are used to assess preattentive
inhibitory and excitatory effects on sensory processing, the integration of
different neural systems (sensory, motor and autonomic), and different
components of attention including intensity, maintenance, breadth (focus or
selectivity), and resistance to distraction. Reports of studies with normal
infants, children and adults have been prepared and serve as the groundwork
for clinical studies with groups exhibiting different types of regulatory
dysfunction. Laboratory facilities and procedures have been developed,
protocols have been designed, and pilot work is in its initial phases.
743
PHS 6040 (Rev. 1/84)
ZOl MH 02404-01 LPP
Project Description
A. Other Personnel
Allan F.
Connie C.
Sheppard
Mirsky, Ph.D.
Duncan, Ph.D.
G. Kellam, M.D.
William W. Eaton, Ph.D.
Mary Beth Ahearn
Chief
Senior Staff Fellow
Chairman, Department of
of Mental Hygiene
Associate Professor,
Dept. of Mental Hygiene
Graduate Assistant,
Dept. of Mental Hygiene
LPP, NIMH
LPP, NIMH
Johns Hopkins
University
Johns Hopkins
University
Johns Hopkins
University
B. Objectives
The project is designed to differentiate patterns of regulatory
dysfunction among different patient groups by measuring attentive functions
and preattentive , automatic processes that underlie adequate attention. The
project will be principally concerned with, but not confined to, studies of
infants and children. In general, patterns of deficits will be examined in
infants characterized as hyperreactive to stimulation, irritable and difficult
to calm, children and adults with specific neuropsychiatric disorders
(Pervasive Developmental Disorder, Attention Deficit Disorder, Stereotyped
Movement Disorders and Anxiety Disorders) or with neurological impairments
(seizure disorders, brain damage) as well as those individuals at risk because
of familial psychopathology or symptomology related to specific disorders. In
addition, groups of infants and children will be followed longitudinally to
examine the developmental course of attentive and preattentive deficits, and
the relationship between these deficits and the development of
neuropsychiatric problems.
C. Major Findings
A major thrust of the proposed work is to study dysfunction in
preattentive functions. Organisms have a limited ability to carry out
processing that requires attention. Preattentive processes often lay the
groundwork for further analysis by attentive processes. As a result,
disruption of attention-demanding communicative, social and cognitive
activities may be secondary to a distortion of preattentive processing. Such
a distortion has been suggested as contributory to a variety of
neuropsychiatric disorders; but it has been difficult to assess preattentive
processes with traditional, information processing methods. However, the
psychophysiological methods employed in the present work — startle blink and
autonomic responses — allow for a more direct and powerful way to assess
preattentive control of processing. Changes in these measures as a result of
stimulation have been shown to reflect the adequacy of important inhibitory
(gating), excitatory (arousal), and sensory integrative functions operating
without attentional or conscious control.
744
ZOl MH 02404-01 LPP
A summary of the work supporting the usefulness of the measures in
examining preattentive processes across the developmental span will appear in
a forthcoming volume of Advances in Infancy Research. Also, a report of
previous studies with normal volunteers detailing the surprisingly uneven
development during childhood of preattentive, inhibitory processes was
prepared and submitted for publication. Another report of a series of studies
utilizing the blink reflex to document important developmental changes in the
processing of brief or transient stimuli was published this year.
Investigations of the effects of temporal characteristics of stimulation on
responsiveness deserves systematic investigation because problems in the
processing of transients appear to be involved in perceptual difficulties of
the aged, of dyslexic and dysphasic children, of autistic children, and in the
dysfunction following lesions of sensory cortex.
The psychophysiological measures employed in this project are also
sensitive to attentive processes and, in conduction with behavioral measures,
can be used to distinguish different components of attention. We are
currently preparing chapters summarizing a series of previous studies with
infants, children and adults which investigated the ability to mobilize
attentional resources, distribute these resources selectively and to resist
distraction. These chapters will appear in a book that will integrate central
and autonomic nervous system approaches to the study of human information
processing.
In the first few months of work on this protocol, we have revised and
developed computer programs for stimulus delivery and timing as well as data
collection. In addition we have purchased, installed and tested additional
equipment for the measurement of autonomic and skeletal muscle responses as
well as the more precise control of acoustic and visual stimulation. We are
currently involved in pilot work with normal volunteers to examine the quality
of the data and to set stimulus parameters for work with clinical populations.
A major part of this project will be the longitudinal examination of
children attending second and third grade in the Baltimore City Public Schools
who will be selected on the basis of their performance on a battery of
neuropsychological tests. The neuropsychological battery was administered to
a representative sample of 435 children in the Spring of 1987. In the past
few months we have begun the examination of these data, and preliminary factor
analysis has revealed distinguishable components of attentive functioning.
Beginning in the Fall of 1987, a group of children deemed at highest risk from
this battery and a representative sample of the population who do not appear
at risk will be brought to the LPP to participate in this project. The goal
of this study is to examine and describe neurobiological indices of attention,
and of the processes that underly attention and, further, to examine their
relation to behavioral and neuropsychological indices. Moreover, we hope to
follow these children longitudinally to examine the stability of these
psychophysiological indices and their usefulness as predictors of later
maladaptive behavior.
745
ZOl MH 02404-01 LPP
Significance to Biomedical Research and to the Program of the Institute
Many children who do poorly in school and/or exhibit learning difficulties
or significant neuropsychiatric disturbances appear to have attentional
deficts. In addition, the severity of learning problems increases as the
child grows older. Attention problems are also associated with behavioral
disturbance in childhood. One-half of the referrals to the nation's mental
health clinics are for attention-related problems. Also, it is becoming
increasingly clear that attention disorders can no longer be considered only
as problems of childhood. One quarter to one half of adolescents with
Attention Deficit Disorder are referred to the courts for theft and truancy
and one quarter are later diagnosed as Anti-social Personality Disorder.
Moreover, attention problems may represent a factor which increases the
vulnerability of those children already at genetic risk for serious
psychopathology such as schizophrenia. This work contributes to an
understanding of the neurobiologic dyfunction underlying attention disorders,
to the separation of more global "attent iveness" into different components,
and to the role of attention problems in the development of neuropsychiatric
problems.
Proposed Course
Besides the longitudinal examination of 2nd and 3rd graders outlined
above, we plan to embark on two other studies. The first will compare a
sample of normal boys with three groups of boys each of which exhibits
different behavioral patterns of regulatory dysfunction: Attention Deficit
Disorder with Hyperactivity, anxious-inhibited behavior and Tourette's
Syndrome. The second study will examine groups of 4-month-old infants at risk
for the development of regulatory problems because of familial attention
disorders or because of temperament features. The latter group will consist
of infants characterized as overly active, easily agitated, difficult to calm,
and hyperreactive to stimulation. Basic aspects of regulation, such as
inhibition, arousal, orientation and attention are relevant to an integrated
transaction between the infant and the environment, and difficulties with
these functions may place the infant at risk. As the potential for early
intervention increases, it becomes more important to evaluate children as
early as possible with an eye to preventative and therapeutic approaches. The
infant studies will examine neurobiological indices of dysregulation, the
stability of these indices, and their predictive validity for later
development of neuropsychiatric difficulties.
Publications
Anthony, B. J., Zeigler, B. L. , & Graham, F. K. : Stimulus duration asan
age-dependent factor in reflex blinking. Dev. Psychobiol., 20: 285-297, 1987.
Anthony, B. J., Graham, F.K., & Balaban, M.T. : Mechanisms of selective
processing in development. In Rovee-Collier , C. (Ed.): Advances in Infancy
Research. In press.
746
ZOl MH 02404-01 LPP
Balaban, M. T. , Anthony, B. J., & Graham, F. K. : Prestimulation effects on
blink and cardiac reflexes of 15-month human infants. Dev. Psychobiol. In
press.
Anthony, B. J. : Developmental changes in information processing. In
Jennings, J. R. & Coles, M. G. H. (Eds.): Psychophysiology of information
processing: An integration of central and autonomic nervous system
approaches. Sussex, England: John Wiley. In press.
747
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT .NUMBER
ZOl MH 00672-22 LSES
PERIOD COVERED
nrtnhpr 1 1 qHf, t-hT-n.,gh q^ptoTiiber "'O, 1QS7
TITLE OF PROJECT (80 characters or less. Title must fit on one iine between tl
Social Psychological Correlates of Occupational Posil-ion
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute attiliation)
PI: C. Schooler, Acting Chief, Laboratory of Socio-environmental Studies, NIMH
OTHER:
C. Schoenbach
M. Kohn
Social Science Analyst
Guest Researcher
LSES
LSES
NIMH
NIMH
COOPERATING UNITS (H any)
None
LAB/BRANCH
Laboratory of Socio-environniRnral Sfnrlip.q
INSTITUTE AND LOCATION
NTMH, ADAMHA- NTH^ Rpth
RHa, Maryland 2089?
TOTAL MAN-YEARS:
5.00
PROFESSIONAL;
.50
OTHER:
4.50
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects
n (a1) Minors
^ (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The object of this study is to assess the reciprocal effects of occupational con-
ductions and psychological functioning (in particular, values, self -concept ions,
social orientation, and intellectual flexibility). Structured interviews were
conducted in 1964 with a sample of 3101 men, representative of all men employed in
civilian occupations throughout the United States. The study was extended into a
longitudinal study in 1974, with the reinterviewing of a randomly-selected one-
fourth of the original sample, together with their wives and, where appropriate,
one of their children. Replications of this research have been carried out in
Poland and Japan.
749
PHS 6040 (Rev. 1/84)
GPO SI 4-«ia
Project No. ZOl MH 00672-22 LSES
Project Description:
The central aim of this study Is to examine how the psychological
effects of the different occupational conditions faced hy people in various
positions in the social hierarchy help explain the differences found in
their psychological functioning. It began in 1964 with structured
interviews with a sample of 3100 men, representative of all men employed in
civilian occupations throughout the United States. In 1974 follow-up
interviews were conducted with a randomly selected one fourth of the
original respondents, as well as with their wives and with one of their
children.
The central findings have been that self-directed work leads to
intellectual flexibility and to a self-directed orientation to self and
society and that oppressive working conditions lead to distress. These
findings have been found to hold true under a variety of different
conditions (paid employment, school work, women's housework) and at
different stages in the life span. They have also been replicated in
separate studies carried out by the Laboratory and its collaborators In
Poland and Japan.
The most important accomplishment this year has been the completion of a
major paper directly comparing the effects of position in the social class
structure on psychological functioning in the United States, Japan, and
Poland. This work was carried, out by Melvin Kohn, presently a guest
researcher in the Laboratory, and Carrie Schoenbach and Carrai Schooler,
together with Atsushi Naoi of the University of Osaka and Kazlmierz
Slomczynski of the University of Warsaw, both former Visiting Scientists in
the Laboratory. For each of the three disparate countries, social classes
were conceptualized and indexed in terms of ownership and control of the
means of production and control over the labor power of others. This was
done in ways that took into account the particular historical, cultural,
economic, and political circumstances of each country. We found, as we had
hypothesized, that men who are more advantageously located in the class
structure of their society would be more likely to value self-direction in
their children, to be Intellectually flexible and to be self-directed In
their orientations than are men who are less advantageously located. We
also found, as we had also hypothesized, that occupational self-direction
plays a crucial role In explaining the psychological impact of social class
in each of these quite disparate countries.
We also uncovered relationships between social class and a sense of
distress. Their pattern, however, differed by country: In the United
States, managers have a strong sense of well-being and manual workers are
distressed; in Poland, just the opposite; and in Japan managers have a
strong sense of well-being, but it is the white collar workers, not the
blue collar workers, who are most distressed. Both a self-directed.
Intellectually flexible mode of psychological functioning and a sense of
distress can affect the individual's mental health. Both can be affected
by social structure and culture. Our findings indicate that although the
750
Project Noc ZOl MH 00672-22 LSES
way social structure affects psychological self-directedness and
flexibility is generally the same in quite different countries, the
determinants of distress can be a function of country-specific cultural and
historical factors.
Other analyses on the effects of social class that were done this year
were those by Schooler and Schoenbach examining the relationship between
Income and social class in the three countries. A central finding is that
class has a greater affect on income in communist Poland than it does in
the two capitalist countries.
This year also saw the continued analysis of the thorny problem of
isolating the effects of parental behavior in the familial transmission of
various modes of psychological functioning. Also examined, was the
question of how traditional culture and social conditions interact to
affect Japanese women's feelings towards the interdependence of family
members. This was explored by examining how these factors affect their
feelings of responsibility for taking their own and their husbands' aged
parents into their households. Taking care of the elderly in this manner
is seen in traditional Japanese culture as a central duty. Our initial
analyses suggest that not only do such attitudes continue to exist, but
also that they are correlated with the woman's working in a traditional
industry, doing relatively simple and nonself directed work both on the job
and at home, and coming from a rural and lower social economic status
background, as well as with having a husband with the same general social
characteristics. It also appears that these relationships are even
stronger for the women's beliefs about their childrens' responsibilities
towards taking them in when they themselves are aged. This suggests that,
at least in the Japanese context, change may be occurring more in terms of
wanting increased independence for oneself when one is older, than in
rejecting traditional norms about one's responsibilities towards one's own
elders.
Significance of the Research:
Because the characteristics of both more and less complex levels of
phenomena affect intermediate ones, to understand human psychological
functioning we have to have a basic knowledge of both the human central
nervous system and the human social system. Recognizing the importance of
the social level of phenomena in determining human behavior means that the
study of the human social system cannot be totally neglected without
seriously imperiling the search for the root causes of psychological
dysfunction. Although the examination of social level phenomena thus
represents a legitimate basic research goal, the LSES, in light of its
limited resources, has focused on those aspects of the social environment
which we have reason to believe most directly affect psychological
functioning. Where we have studied social level phenomena in themselves,
it has been with the aim of knowing more about those aspects of the social
environment that may affect people psychologically. In fact, almost all of
our research has been concerned with the causal connections between the
751
Project No. ZOl MH 00672-22 LSES
social and cultural systems and the individual's psychological functioning.
In particular, we have focused on how individuals' positions in the social
system affect their cognitive functioning, interrelationships with others
and ability to cope with life's stresses. These three areas, besides being
essential for our basic understanding of human nature, are clearly
implicated in mental illness. Schizophrenia, for example, is characterized
by cognitive dysfunction, social withdrawal and an inablility to cope with
social and other forms of stress. In addition, direct evidence of the
relevance of social level phenomena to schizophrenia can be found in the
fact that for reasons that are still not clear, there is a negative
relationship between social class and the incidence of schizophrenia.
Recent, carefully controlled research suggests that noisome, dangerous,
and uncomfortable occupational conditions, which are more likely to be
characteristic of lower status jobs, play a part in the development of
schizophrenia. Research carried out by the Laboratory indicates that
people at lower stratification levels have less effective cognitive and
other psychological mechanisms for coping with stress and uncertainty and
more rigid and conformist orientations towards others. Thus oppressive job
conditions, cognitive and other forms of coping mechanisms, and the nature
of the individual's orientation to others are linked to social status in
ways that suggest that they may play a part in the etiology of
schizophrenia. If the problem of schizophrenia is to be truly faced, basic
socio-environmental reseach on these problems must accompany basic research
on the molecular and neurological level.
Beyond NIMH's concern with mental disorder is the Institutes mandate to
study the conditions that facilitate and those that interfere with
effective psychological functioning. This research demonstrates that job
conditions have appreciable effects on cognitive performance, self
conceptions and orientations to the outside world and thus are directly
linked to the mental health and effective psychological functioning of the
individual .
Proposed course of further research:
Data has been collected on the psychological effects of occupational and
other social structural conditions on a sample of Japanese fathers, mothers
and children that parallels the data we have collected in the United
States. The analyses of the data on the Japanese men have already been
completed and reported. We now plan to analyze the women's and childrens
data to investigate the interaction of culture and social structure on the
individual's intellectual and interpersonal functioning. This Japanese
data will also be used to examine the nature of the intraf amilial
transmission of psychological functioning, and of orientations to self and
society. The results of these analyses will serve as a basis of comparison
with those of similar, presently ongoing analyses of our American data.
Such comparisons will permit us to assess the cross-cultural
generalizability of our results as V7ell as the way in which culture
interacts with social structure and parental influence to affect the
psychological functioning of the individual.
752
Publications :
Kohn, M.L. : Unresolved Issues in the Relationship between Work and
Personality. In Kai Erikson (Ed.): Working. New Haven: Yale
University Press, in press.
Kohn, M.L. , and Schoenbach, C: Social Stratification and Parental
Values: A Multi-national Assessment. In Tominaga, K. and Treiman,
D. (Eds.): Social Stratification and Mobility in Japan and the
United States. Connecticut, Greenwood Press, in press.
Kohn, M.L. , and Schooler, C.
Personality.) Tokyo, Japan:
Shigoto to Personality. (Work and
Saiensu-sha Publishing Co., 1987, in press.
Miller, Karen A., Kohn, M.L. and Schooler, C.
direction and personality. Am. Soc. Rev . , 5;
; Educational self-
372-390, 1987.
Miller, K.A. , and Kohn, M.L.: The Reciprocal Effects of Job Conditions
and the Intellectuality of Leisure-time Activity. In Tominaga, K. and
Treiman, D. (Eds.): Social Stratification and Mobililty in Japan and the
United States. Connecticut, Greenwood Press, in press.
Roberts, B.
Soc. Psych.
: A Confirmatory Factor-analysis Model of Alienation.
Q. , in press .
Schooler, C: Psychological and Social Perspectives on Status Attainment.
In Tominaga, K. and Treiman, D. (Eds.): Social Stratification and Mobility
in Japan and the United States. Connecticut, Greenwood Press, in press.
Schooler, C. and Naoi , A.: The Psychological Effects of Traditional
and of Economically Peripheral Job Settings in Japan. Am. J. Sociol. ,
in press.
Schooler, C: Using Linear Structural Equations Analysis to
Estimate the Reciprocal Effects of Job Complexity and Ideational
Flexibility. J. Voc. Beh. , 31: 1987, in press.
753
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
PROJECT NUMBER
ZOl MH 0067 9-07 LSES
Orfnhpr 1, 1 Q«ft t-hrnngh f^ppte^mh^-r -^0 ^ 1 Q«f^
TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.)
Sfriirrnral F.niial-inn MnHpIg in Mip Analygig nf T)ata t.ttI-H MpasiirpTnpnf Frrnr
1INCIPAL INVESTIGATOR (List other professional personnel below the Pnncipal Investigator) (Name, title, laboratory, and institute atfiliati
Ronald J. Schoenberg, Research Sociologist
PI:
OTHER :
C. Schooler Acting Chief
LSES
LSES
atfiliation)
NIMH
NIMH
COOPERATING UNITS (it any)
None
LAB/BRANCH
Laboratory of Socio-environmental Studies
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.10
PROFESSIONAL:
1.10
CHECK APPROPRIATE BOX(ES)
£] (a) Human subjects
n (al) Minors
K\ (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this work is to further develop the methods and techniques for the
specification and estimation of the parameters of structural equation models of
survey data that contain random and nonrandom measurement error. Included in
this are methods for the identification of the models, estimation of the means of
unobserved variables, the determination of model condition, and the treatment of
polytomous variables.
755
PHS 6040 (Rev 1/84)
SPO SI 4-aiB
Project No. ZOl MH 00679-07 LSES
Project Description :
Diagnosis is an important medical problem. It amounts to a problem in
the assignment of people to classes or categories on the basis of symptoms.
This assignment can be accomplished quite easily if the pathology as well
as its physiological relationship with the symptoms, are well understood.
But in some cases neither the disease nor its relationship to symptoms is
very well understood. This is the case with schizophrenia.
Drawing on the statistical literature on "latent class analysis"
Ronald Schoenberg and Carmi Schooler are developing statistical models of
the relationship of schizophrenia to symptoms. These models are similar to
"cluster models", which are well developed in psychometric research, but
were not designed for categorical or discrete variables which is the form
of most measures of symptoms.
Latent class models can be used in an exploratory way to assist in
discovering first, the dimensionality of the underlying latent constructs,
in this case, the dimensionality of schizophrenia, and second, the
relationships of the latent constructs to the observed measures or
symptoms. And when a specification has been decided upon then the latent
class analysis can be used to produce a method for the classifying
individuals into latent categories, that is, to diagnose.
The method of latent class analysis is being applied to items in the
Diagnostic Interview Schedule (DIS) data in the Epidemiological Catchment
Area study conducted for NIMH that purport to measure schizophrenia. Early
results of this analysis supports a multidimensional explanation for the
DIS symptoms.
A long recognized problem in non-experimental research at NIMH has
been the issue of sampling. Most subjects of studies at NIMH are found
through referrals from clinics, hospitals, etc., or are volunteers. And
the subjects actually studied must pass through a stringent qualifying
process that usually excludes people on medication or with chronic
diseases. This produces two problems: first, a problem in generalization,
and second, a problem in determining the outcome without bias.
The first problem is generally well known to researchers but the
second is not so well known. If the factors that determine whether a
subject will be included in the study are related in some way to the
dependent variable in the study, then the usual methods for calculating the
outcome — , multiple regression, for example — will produce biased estimates
of the outcome. Thus sample selection can attenuate or even reverse
findings. Solving the second problem, then, can be critical for many
nonexperimental studies that do not randomly sample from a population.
756
Project No. ZOl MH 00679-07 LSES
The solution for both of these problems requires that models be
constructed for the sample selection process. That is, models — in the
simplest case regression models — ^must be developed that predict the
probability that a subject vjith given characteristics will be included in
the study. Adjustments then can made in the analysis that will remove the
bias in estimating the outcome due to the nonrandom sample selection, and
permit generalization of the outcome to the general population.
Ronald Schoenberg, using data from the Epidemiological Catchment Area
study, is developing models for predicting the use of mental health
facilities which is the first step a subject must take to become included
in a study at NIMH. This model will then be used as a part of a further
analysis of the adjustments that can be made to studies conducted at NIMH
to correct for sample selection bias and to permit generalization to the
general population.
Signif icanc e of the Research :
The development of the latent class models of schizophrenia has helped
to establish the raultidimensionality of schizophrenia. The specification
of a measurement model for schizophrenia has implications for the diagnosis
of schizophrenia — it will now be possible to provide a rigorous method for
diagnosis on the basis of observed symptom. This model may also provide
some clues for further investigation into the etiology of schizophrenia.
Proposed Course of Further Research:
The measurement model of schizophrenia will be replicated in other
data sets to test its validity. The sample selection models will be
further developed and tested on clinical sample data to determine their
usefulness in enhancing generalization and undoing the effects of sample
selection bias.
References :
Schooler, C: A Statistical Interaction Approach to Problems in Cross-
Cultural Epidemology. To be published in the proceedings of the
International Symposim on Psychiatric Epidemiology, Taipei, Taiwan, in
press.
Schooler, C. : On Levels of Research: The Prospective of a Psychologist
Practicing Psychology. In Matilda Riley (Ed.): Social Structures and
Human Lives. 1986. Presidential Papers, American Sociological Association,
Washington, D.C. , in press.
Zahn, T.P., Schooler, C, and Murphy. D.L. Autonomic Correlates of
Sensation Seeking and Monoamine Oxidose Activity: Using Confirmatory
Factor Analysis on Psychophysiological Data. Psychophysiology , 23:
521-531, 1986.
757
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00680-05 LSES
PERIOD COVERED
Ortnhpr ^, 1 Q8fi thrnuoh Rppt-PmViPT- -^0^ 1 q«7
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Work Experiences and rVip Dpi n st- 1 fn f innal i zpH Mpnfally Til
PRINCIPAL INVESTIGATOR (Ust other proleasional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aHlllation)
Elliot Liebow, Guest Researcher, LSES, NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Socio-environmental Studies
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda,
Maryland ?aM7.
TOTAL MAN-YEAHS:
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
D (al) Minors
^ (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The objective of this exploratory, participant observation study is to examine
the work experience of the deinstitutionalized mentally ill over time and to
seek out ways in which job characteristics, symptoms, and social relationships
interact with one another to effect the course of recovery from psychiatric
disorder and reintegration into the community. Field work was carried out
with residents of halfway houses, participants in community-based psychosocial
and transitional work programs, and with "unattached" deinstitutionalized men
and women.
759
PHS 6040 (Rew. 1/84)
QPO >i4-»ia
Project No. ZOl MH 00680-04 LSES
Project Description :
Four years ago, Elliot Liebow, on detail to the Laboratory from
the Extramural Program, began an exploratory, participant-observer study
of the relationship between work experience and recovery from mental
illness. The goal of this research was not to test hypotheses but
rather to grasp the dynamics of the interaction between work experiences
and recovery from mental illness.
In 1984, while still collecting data on deinstitutionalized persons
in halfway houses and psychosocial programs in Montgomery County, Md . ,
Liebow was stricken by two successive major illnesses. He retired on
disability in September of that year but remained as a guest researcher in
order to try to salvage some of the data he had already collected.
These were somewhat too thin to serve their original purposes but were
potentially useful nonetheless.
In November of 1984, Liebow began collecting data as a participant
observer in a shelter for homeless women in Rockville. Many of the two
dozen women who are "regulars" (in the sense that they stay at the
shelter night after night, month after month) as well as the more casual
users who come for a night or two, have a history of mental illness
and/or institutionalization. Through these participant observation
experiences, Liebow plans to contrast the dynamics and outcomes of two
post-institutionalization life-styles: (a) the highly structured,
tightly supervised group living of halfway houses and psycho-social day
programs versus (b) the relatively unstructured, free floating life
style of shelters and soup kitchens.
This research will focus primarily on the two dozen women who are
"regulars" at the shelter. Liebow has now followed them intensely for more
than 24 months. Data collection is completed, coding categories have been
developed, and analysis and writing are underway.
Significance of the Re sea rch :
This project is directly pertinent to our understanding of
rehabilitation of the deinstitutionalized mentally ill.
Proposed Course of Rese arch:
Formal data collection has been completed. The investigator is
working on the analysis and writing up of this data as a Guest Researcher
at a moderate pace.
760
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT (MUMBER
ZOl MH 00681-01 LSES
PERIOD COVERED
October 1, 1986 rn SpptPrnhpr TO, 1 QSV
TITLE OF PROJECT (80 characters or less. Title must lit on one line lietween the tmrders.)
Reciprocal Effects of 5ip1 f-F,.qfpppi and Ppprpt^ginn
PRINCIPAL INVESTIGATOR (List Other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: M. Rosenberg, Guest Research, Laboratory of Socio-environmental Studies, NIMH
OTHER: C. Schooler
C. Schoenbach
Acting Chief
Social Science Analyst
LSES
LSES
NIMH
NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Socio-environmental Studies
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.05
PROFESSIONAL:
.55
.50
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects
D (a1) Minors
Bk (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Although low self-esteem has been shown to be associated with a number of non-
pschotic disorders among children, adolescents, and adults, the causal connection
among these variables has not been established. In order to do so, we plan to
make use of a longitudinal data set. This is the Youth in Transition study, a
five-wave national probability sample of adolescents. Through the use of
instrumental variables and structural equation models, we will seek to specify
the reciprocal effects of global self-esteem and a number of mental health and
behavioral variables.
761
PHS 6040 (Rev. 1/84)
SPO 9I4-*I(
Project No. ZOl MH 00681-01 LSES
Project Description:
The aim of this project is to explore the reciprocal effects of global
self-esteem and selected mental health and behavioral measures. Past
research dealing with the relationship between self-esteem and other
variables has been unable to answer the question: Which is cause and what
is effect? For example, although many studies show a strong relationship
between global self-esteem and depression, we do not know whether
self-esteem is responsible for depression or depression is responsible for
self-esteem. To be more exact, we do not know the extent to v/hich each
variable affects the other. The same basic question can be asked of the
relationship between self-esteem and many other important mental health and
behavioral variables.
Our purpose is to explore such questions by means of reciprocal
effects analyses. In order to do so, we plan to make use of a large scale
longitudinal data set containing a wide range of relevant variables. This
is the Youth in Transition study, a five-wave national probability sample
of 2213 adolescents. The use of such longitudinal data enhances the
possibility that structural equation models can be developed that will
enable us to specify the reciprocal effects of these variables. Dr.
Schoenberg , an expert in structural equation modeling, is available to help
us solve the complex statistical problems that such analysis entails.
Significance of the Project:
The relevance of global self-esteem for mental health has been
repeatedly demonstrated in the literature, and is supported both by survey
and experimental data. Studies conducted with children, adolescents, and
adults show that low self-esteem is associated with depression, somatic
symptoms of anxiety, psychological anxiety, irritability, loneliness,
resentment, hypersensitivity, external locus of control, and low life
satisfaction. Low self-esteem is also associated with interpersonal
inhibition, apathy, withdrawal, and other behavioral difficulties. What is
unknown is the reciprocal effects of self-esteem and these other variables.
Such findings have implications for therapeutic strategies. They can
suggest, for example, whether it is advisable to attempt to deal with
depression by altering self-esteem or whether, for this purpose, this
strategy is a waste of effort. Similar questions can be asked with respect
to many other correlates of self-esteem.
Proposed Course of Project:
Initial problems of coding and data organization have now been solved
and a workable data tape is available. The reciprocal effects analyses are
now in process.
762
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00682-01 LSES
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders )
Environmental Determinants of Cognitive Functioning
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institiAe affiliation)
PI: L. Caplan, Staff Fellow LSES NIMH
OTHER: C. Schooler, Acting Chief
LSES
NIMH
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Socio-environmental Studies
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
1.60
PROFESSIONAL:
1.35
.25
CHECK APPROPRIATE BOX{ES)
@ (a) Human subjects
n (a1) Minors
EJ (a2) Interviews
D (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this study is to investigate the effects of task complexity on
cognitive functioning. Subjects were trained to use a computer program under
different conditions of complexity. After training, the following were assessed;
1) knowledge structure development, 2) flexibility of problem solving, and 3)
planning ability.
763
PHS 6040 (Rev 1/84)
QPo ai4-sie
Project No. ZOl MH 00682-01 LSES
Project Description :
Over the last 20 years, Melvin Kohn and Carmi Schooler have published a
series of studies which have demonstrated that environmental complexity
(e.g., complexity of occupation, schoolwork, housework) encourages the
development of intellectual flexibility. The results have been reliably
demonstrated in a number of different populations: American men, American
women, Japanese men, Polish men, American schoolchildren.
Such effects are almost certainly the result of cognitive development
which occurs as individuals attempt to deal effectively with their
envirarments (see Schooler, 1984). Until recently, however, the exact
nature of the cognitive changes involved has not been examined. The
project described here is designed to test a number of hypotheses which may
account for the intellectual flexibility results obtained in earlier
studies. In particular, the experiment described below was designed to
test the following hypotheses about the effects of environmental
complexity:
1. Environmental complexity encourages the development
of highly detailed and elaborated knowledge structures,
which in turn enable an individual to engage in flexible
problem solving.
2. Environmental complexity encourages the development of
metacognitive planning skills, i.e., those skills
which enable individuals to plan and regulate
their own cognitive activities.
3. Environmental complexity is most likely to facilitate
the development of flexible problem solving when it occurs
in a task which is inherently meaningful.
The experiment involved training people in the use of a microcomputer
drawing program - a task similar to those they encounter in daily life.
The training conditions involved manipulating: 1) the degree to which
training materials were organized, 2) whether or not a concrete model of
the program's functioning was provided, 3) the degree of visual complexity
of the stimuli, and 4) the degree of decisional complexity required by the
task. Following training, tests were administered which assessed subjects'
ability to engage in flexible problem solving with the program, and the
development of mental representations of the program.
Data from the complete sample of 128 individuals have been collected.
In addition to data from the experimental task described above, we have
also collected demographic information, information about occupational
7ft4
Project No. ZOl MH 00682-01 LSES
complexity, and scores from two subtests of the Wechsler Adult Intelligence
Scale from each subject.
Significance of the Research:
The results of this study should provide some initial explanations of
the effects of environmental complexity previously demonstrated in this
laboratory. If, in fact, individuals exposed to the complex training
provided in this study develop more elaborated mental representations of
the program, or more advanced metacognitive planning abilities, then these
results will support the hypothesis that increased environmental complexity
facilitates the development of qualitatively different cognitive abilities
than those developed in response to simpler environmental demands. Such
findings would both explain the earlier results from Kohn & Schooler data,
and open up new avenues for research on the specific cognitive effects of
environmental complexity.
In addition, the results of this study will be a significant
contribution to the literature on human problem solving in cognitive
psychology. Much of this literature has focused on relatively simple
problems, with well-defined questions and clear solutions. The type of
problem-solving required by this task, as by many problems encountered in
everyday life, involves learning to deal with problems which are not
well-defined. The results of this project, therefore, should provide
cognitive psychology with a description of the problem-solving processes
involved in more ecologically valid domains.
In related work, Schooler is involved in the organization of a series
of conferences aimed at linking cognitive psychology, to the study of the
effects of social structure on individual functioning over the life course.
Proposed Course of Research:
The next step of this research project involves data analysis and
writing the results up for publication.
Publications :
Schooler, C. and Schaie, (Eds.): Cognitive Functioning and Social
Structures over the Life Course. New Jersey, Ablex Publishing Co.
1987.
Schaie, K. W. , and Schooler, C. (Eds.): Social Structure and Aging:
Psychological Processes. Hillsdale, New Jersey, Erlbaum, in press.
Schooler, C. : Social Structural Effects and Experimental Situations:
Mutual Lessons of Cognitive and Social Science. In Schaie, K. W. and
Schooler, C. (Eds.): Social Structure and Aging. Hillsdale, New Jersey,
Erlbaum, in press.
765
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00424-12 LCB
PERIOD COVERED
October 1. 1986 through September 30. 1987
TITLE OF PROJECT (80 characters or less. We must tit on one line between the borders.;
Bi ol ogically Active Peptides in the Brain
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, latmratory, and institute atfiliatlon)
P.I.S
Brownstein, Chief, Laboratory of Cell Biology, NIMH
Special Expert, Laboratory of Cell Biology, NIMH
Michael J.
T. Bonner.
C. Gerfen, Sr. Staff Fellow, Laboratory of Cell Biology, NIMH
M, Palkovits, Visiting Scientist, Laboratory of Cell Biology, NIMH
W. S. Young, Sr. Staff Fellow, Laboratory of Cell Biology, NIMH
(see attached)
COOPERATING UNITS fffanw Univ. Zuerich; Tulane Univ; Neurol .Sci . Inst. , Portland;
MD NIDDKD; CNG NIMH; BG NHLBI; ERR CH; Tufts U. Med. Sch; Westminster Hosp.
London; LVC NCI; LMG NINCDS; BPB NIMH; Karolinska Inst; LNC NINCDS
LAB/BRANCH
Laboratory of Cell Biology
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda. Maryland 20892
TOTAL MAN-YEARS:
10
PROFESSIONAL:
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have continued to study the organization and development of peptidergic
and non-peptidergic neurons in the central nervous system and periphery, bio-
synthesis of biological ly active peptides, factors that regulate peptide secre-
tion, and receptors and their second messenger systems.
767
PHS 6040 (Rev. 1/84)
SPO B14-ail
ZOl MH 00424-12 LCB
Other Professional Personnel Engaged on Project
H.
-U. Affolter
Ober Assistant
Univ. Zuerich
A.
Arimura
Professor
Tulane Univ.
N.
Barmack
Sr. Scientist
Neurol .Sci. Inst., Portland,
OR
W.
Benson
Biologist
LCB, NIMH
M.
Brann
Staff Fellow
MD, NIDDKD
N.
Buckley
Visiting Fellow
LCB, NIMH
J.
-M. Burgunder
Guest Researcher
LCB, NIMH
S.
Cetera
Sr. Staff Fellow
CNG, NIMH
T.
Gores
Scientist
Tulane Univ.
J.
Gutierrez
Chemist
LCB, NIMH
D.
Hilt
Sr. Staff Fellow
BG, NHLBI
S.
Horvath
Visiting Fellow
LCB, NIMH
L.
-S. Hsieh
Visiting Fellow
BG, NHLBI
F.
Huang
Staff Fellow
ERR, CH
K.
-P. Huang
Section Chief
ERR, CH
C.
Jelsema
Guest Researcher
LCB, NIMH
J.
King
Professor
Tufts U. Med. Sch.
D.
Kl igman
Staff Fellow
LCB, NIMH
K.
Koller
Staff Fellow
LCB, NIMH
M.
Konig
Microbiologist
LCB, NIMH
R.
Lad
Visiting Fellow
LCB, NIMH
R.
Lee
Guest Researcher
LCB, NIMH
S.
Lightman
Reader in Medicine
Westminster Hosp., London
S.
Lolait
Guest Researcher
LCB, NIMH
L.
Mahan
Staff Fellow
LCB, NIMH
L.
Matsuda
Guest Researcher
LCB, NIMH
E.
Mezey
Visiting Scientist
LCB, NIMH
W.
Modi
Geneticist
LVC, NCI
J.
-M. Muller
Visiting Fellow
LCB, NIMH
S.
O'Brien
Acting Laboratory Chief
LVC, NCI
H.
Okayama
Visiting Scientist
LCB, NIMH
P.
Padgett
Chemist
LMG, NINCDS
J.
Patel
Visiting Associate
BPB, NIMH
A.
Rokaeus
Asst. Professor
Karolinska Inst.
E.
Shepard
Microbiologist
LCB, NIMH
M.
Warden
Biologist
LCB, NIMH
S.
Wray
Sr. Staff Fellow
LNC, NINCDS
R.
Wolff
Biol . Lab. Tech.
LCB, NIMH
A.
Young
Chemist
LCB, NIMH
R.
T. Zoeller
NRC Fellow
LNC, NINCDS
768
ZOl MH 00424-12 LCB
Project Description
Peptide/protein isolation and characterization
Drs. Kligman and Pate! have purified a major 87kD substrate
of C kinase from brain extracts and, with the help of J.
Gutierrez, have the amino acid sequences of several tryptic
peptides derived from this protein. In collaboration with S.
Detera, they are attempting to clone a cDNA that encodes this
protei n .
Cloning and sequencing of cDNAs and genomic DNAs
T. Bonner, A. Young, N. Buckley, and M. Brann have shown
that there are at least four distinct muscarinic receptors by
isolating, sequencing, and expressing the DNAs that encode
them. Three of the receptors are of the Ml type, one is M2. The
receptors have unique distributions in the CNS suggesting that
they subserve different functions.
Drs. Bonner, O'Brien, and Modi are mapping the chromosomal
location of the four muscarinic receptor genes isolated to date,
and Drs. Bonner and Detera are looking for restriction fragment
polymorphisms to use to determine whether the receptor genes are
linked to mental illness.
T. Bonner, U. Affolter, and A. Young have cloned a rat cDNA
encoding the precursor of Jieurokinin B (neuromedin K) and
characterized its mRNA in rat brain. Dr. Bonner has also cloned
and completely sequenced the human neurokinin B gene and has
shown that two upstream exons proposed to be part of the gene are
the last two exons of an adjacent gene.
Drs. Bonner, O'Brien, and Modi have mapped the chromosomal
location of the human substance P and neurokinin B genes to 7q
21.1-22 and 12q 13.1-21.1, respectively.
Drs. Kligman and Hilt have cloned a full length cDNA that
encodes S1006, a neurite extension factor.
Dr. Koller isolated a cDNA that encodes the valocin
"precursor." She has shown that this protein is unlikely to give
rise to a peptide secretory product; it is found in the cytoplasm
of diverse cells. This is the first example of the use of
molecular genetics to eliminate a peptide as a transmitter
candidate.
Dr. Mezey has cloned the cDNA that encodes the rat
phenyl ethanol ami ne-N-methyl transferase so that she can construct
oligonucleotide probes and raise antibodies to use for in situ
hybridization histochemistry and i mmunocytochemi stry .
769
ZOl MH 00424-12 LCB
Dr. Buckley has developed a method for transferring colonies
of mammalian cells to polyester sheets. The "replicas" can be
screened for the presence of cells that are expressing receptors
of interest by means of receptor autoradiography.
number of methods have been developed for enriching
libr es for cDNAs encoding receptors including s i ze- sel ecti on
and eening with transient expression assays. Collaborations
have been established that will allow us to screen libraries by
injecting mRNA derived from our cDNAs into Xenopus oocytes. Very
big libraries with especially large inserts are being prepared in
a novel vector for this purpose. Finally, recent improvements in
transfection methodology (see Dr. Okayama's section) promise to
to contribute significantly to our ability to produce large
eukaryotic expression libraries.
At present Drs. Buckley, Lolait, Muller, Matsuda, Mahan,
Rokaeus, Young, and Koller are endeavoring to isolate receptor
cDNAs including those for substance P, angiotensin, VIP,
bradykinin, somatostatin, galanin, neurotensin, and CCK.
Dr. Young has recently isolated the gene for the rat, neural
nicotinic receptor and is testing the hypothesis that it
undergoes a novel alternative splicing in hypothalamic neurons.
Functional neuroanatomy
Drs. Young, Burgunder, Koller, Zoeller, Lightman, Mezey,
Gerfen, Buckley, and Lolait have continued to improve and exploit
the ISHH method.
Drs. Young and Burgunder are using ISHH to study factors
that regulate vasopressin and oxytocin mRNA levels in the
supraoptic nucleus.
M. Warden and her co-workers have mapped tachykinin mRNAs in
the CNS.
S. Young and S. Lightman have studied the regulation of
hypothalamic enkephalin mRNA levels.
770
ZOl MH 00424-12 LCB
S. Young and N. Barmack have used ISHH to examine the role
of inferior olivary CRF-cells in regulating eye movements.
Drs. S. Young, K.-P. Huang, and F. Huang have studied the
distribution and development of protein kinase C.
K. Koller, T. Zoeller, and R. Wolff have shown that thyroid
hormones influence the levels of TRH message and that TSH is not
required for these hormones to exert their effects on TRH mRNA.
T. Zoeller, S. Wray, and J. King have examined the role of
estrogen in controlling GnRH biosynthesis.
Dr. Buckley and his colleagues have used ISHH to map
muscarinic receptor subtypes in the CNS and in peripheral
tissues.
Dr. Gerfen has demonstrated that pharmacological
manipulation of the dopaminergic mesostriatal system
differentially regulates patch-matrix neuronal expression of
neuropepti des .
Dr. Lolait has used ISHH to look for neuropeptide mRNAs in
peripheral tissues.
I mm unocytochemi St ry
S. Horvath, M. Palkovits, T. Gores, and A. Arimura have
shown reciprocal connections between GRF- and somatostati n-
producing neurons in the hypothalamus.
L. Mahan, C. Jelsema and M. Palkovits have used
immunocytochemi stry to examine the nature and distribution of G
proteins in the retina and to study the role of somatostatin in
regulating the circadian rhythm in indoleamine metabolism in the
retina of X. laevis.
D
and de
and ne
the St
paral 1
throug
demons
do pa mi
system
D28kD
subset
thymi d
prior
neuron
manner
direct
into a
r. Ge
vel op
uroch
r i a t a
el in
h the
trate
n e r g i
expr
it wa
of d
i ne 1
to ma
s al 0
i n w
ly de
stroc
rf en
ment
e m i c a
1 pat
put-o
stri
d the
c sys
esses
s al s
opami
a b e 1 i
tri X
ng im
h i ch
monst
ytes .
and h
of th
1 tec
ch-ma
utput
atum
comp
terns .
the
0 sho
n e r g i
ng sh
neuro
munoh
the p
rated
Fol
1 s c
e ba
hni q
tri X
sys
to t
artm
Fu
bra i
wn t
c ne
owed
ns .
i sto
atch
the
1 owi
olle
sal
ues .
org
terns
he s
enta
rthe
n ca
hat
uron
tha
The
chem
-mat
tra
ng n
agues
g a n g 1 i
Thei
a n i z a t
which
ubstan
1 orga
rmore ,
1 c i u m
this p
s proj
t stri
async
i c a 1 1 y
r i X or
nsform
eurona
have
a by
r pre
ion r
conn
t i a n
n i z a t
as t
b i n d i
r 0 1 e i
e c t i n
atal
hrono
1 abe
gani z
a t i 0 n
1 mig
exami
means
V i 0 u s
ef 1 ec
ect t
i gra.
ion 0
he ma
ng pr
n al s
g to
patch
us mi
1 ed r
a t i 0 n
of s
ratio
ned
of
wor
ts t
he c
Cu
f me
tr i X
otei
0 d i
the
neu
grat
ad i a
dev
tri a
n th
the
neur
k sh
he e
ereb
rren
sost
str
n ca
sti n
matr
rons
i on
1 gi
el op
tal
i s
orga
oana
owed
xi st
ral
t wo
ri at
i ato
Ibin
guis
i X .
are
of t
ia s
s .
rad i
n i z a t i 0 n
tomi cal
that
ence of
cortex
rk
al
nigral
di n-
hes the
3H-
born
hese
how the
Studies
al gl i a
771
ZOl MH 00424-12 LCB
transformation process occurs to establish neuron-glia
relationships in the patches prior to the matrix.
Significance to Biomedical Research
N
one an
b i 0 s y n
result
neu ron
the sy
number
four 0
pept i d
pha rma
at pre
p e p t i d
outl i n
unders
eel 1 s ,
cau s i n
erve
othe
thes
i n
s i n
mpto
of
r fi
es .
col 0
sent
es r
ed a
tand
we
g di
eel
r an
is,
nerv
the
ms 0
pu ta
ve .
Our
gy 0
; in
ema i
bove
ing
can
seas
Is use
d with
rel ease
ous and
substa
f Parki
t i ve ne
Most 0
knowl e
f pepti
deed, i
n to be
is p r i
of cell
formul a
e .
c h e m i c a
other t
, and/o
mental
n t i a n i
nson ' s
urotran
f the n
dge of
dergi c
t i s cl
i s 0 1 a t
n c i p a 1 1
s . To
te bett
1 "t
arge
r me
d i s
gra ,
d i se
smi t
ewl y
the
neur
ear
ed a
y de
the
er h
ransm
t eel
tabol
order
for
ase .
ters
dete
anato
ons i
that
nd ch
voted
ex ten
ypoth
i 1 1 e r
Is.
ism h
s. D
examp
In t
has i
cted
my. P
s com
many
a rac t
to i
t tha
eses
s" to
Change
a ve be
eath 0
1 e , is
he las
ncreas
chemi c
h y s i 0 1
p a r a t i
b i 0 1 0 g
e r i z e d
m p r 0 V i
t we u
about
commu
s in
en su
f dop
asso
t ten
ed by
al me
ogy a
vely
i cal 1
. Th
ng ou
nders
their
m ca
tran
gges
ami n
c i a t
yea
a f
ssen
nd
i nco
y ac
e wo
r
tand
rol
t e with
s m i 1 1 e r
ted to
e r g i c
e d with
rs the
actor of
gers are
mpl ete
ti ve
rk
these
e i n
Proposed Course
The work outlined above is still in progress and will be
continued.
Publ i cat ions
Elekes, I., Patthy, A., Lang, T., and Palkovits, M.:
Concentration of GABA and glycine in discrete brain nuclei.
Stress- induced changes in the levels of inhibitory amino acid.
Neuropharmacology 25: 703-709, 1986.
Rokaeus, A. and Brownstein, M.J.: Construction of a porcine
adrenal medullary cDNA library and nucleotide sequence analysis
of two clones encoding a galanin precursor. Proc . Natl . Acad .
Sci . USA 83: 6287-6291, 1986.
Moskal , J. and Schaffner, A.E.: Monoclonal antibodies to the
dentate gyrus: i mmunocytochemi cal characterization and flow
cytometric analysis of hippocampal neurons bearing a unique cell-
surface antigen. J. Neu rose i. 6: 2045-2053, 1986.
Kiss, J.Z, and Mezey, E.: Tyrosine hydroxylase (TH) in
magnocel 1 ul ar , neurosecretory neurons: response to physiological
manipulations. Neuroendocr i nol ogy 43: 519-525, 1986.
Young, W.S. Ill: Periventricular hypothalamic cells in the rat
brain contain insulin mRNA. Neuropeptides 8: 93-97, 1986.
Bonner, T.I., Buckley, N.J., Young, A.C.
Identification of a family of muscarinic
genes. Science 237: 527-532, 1987.
and Brann, M.R.:
acetylcholine receptor
772
ZOl MH 00424-12 LCB
Brann, M.R. and Cohen, L.V.: Diurnal expression of transducin
mRNA and translocation of transducin in rods of rat retina.
Science 235: 585-587, 1987.
Kiss, A., PaUovits, M., Antoni, F.A., Eskay, R.L., and Skirboll,
L.R.: Neurotensin in the rat median eminence: the possible
sources of neurotensi n-l i ke fibers and varicosities in the
external layer. Brain Res. 416: 129-135, 1987.
Kligman, D. and Hsieh, L.-S.: Neurite extension factor induces
rapid morphological differentiation of mouse neuroblastoma cells
in defined medium. Dev. Brain Res. 33: 296-300, 1987.
Koller, K.J. and Brownstein, M.J.: Use of a cDNA clone to
identify a supposed precursor protein containing valosin.
Nature: 325: 542-544, 1987.
Palkovits, M. and Eskay, R.L.: Distribution and possible origin
of g-endorphin and ACTH in discrete brainstem nuclei of rats.
Neuropeptides 9: 123-137, 1987.
Palkovits, M., Leranth, C, Gores, T., and Young, W.S. Ill:
Corticotrop in-releasing factor in the olivocerebellar tract of
rats: Demonstration by light- and electron-microscopic
immunohi stochemi stry and in situ hybridization histochemistry.
Proc. Natl. Acad. Sci. USA 84: 3911-3915, 1987.
Rokaeus A.: Galanin: a newly isolated biologically active
neuropeptide. Trends Neurosci. 10: 158-164, 1987.
Young, W.S. Ill, Mezey, E., and Siegel, R.E.: Vasopressin and
oxytocin mRNAs in ad renal ectomi zed and Brattleboro rats:
analysis by quantitative in situ hybridization histochemistry.
Mol . Brain Res. 1 : 231-241, 1986.
Young, W.S. Ill, Walker, L.C, Powers, R.E., De Souza, E.B., and
Price, D.L.: Cort i cotropi n- rel ea s i ng factor mRNA is expressed in
the inferior olives of rodents and primates. Mol . Bra i n Res .
1: 189-192, 1986.
Young, W.S. Ill: Cort i cot ropi n- rel eas i ng factor mRNA in the
hypothalamus is affected differently by drinking saline and by
dehydration. FEBS Lett. 208: 158-162, 1986.
Young, W.S. Ill, Mezey, E., and Siegel, R.E.: Quantitative in
situ hybridization histochemistry reveals increased levels of
corti cotropi n-rel easi ng factor mRNA after adrenalectomy in
rats. Neurosci. Lett. 70: 198-203, 1986.
Young, W.S. Ill, Bonner, T.I., and Brann, M.R.: Mesencephalic
dopamine neurons regulate the expression of neuropeptide mRNAs in
the rat forebrain. Proc. Natl. Acad. Sci. USA 83: 9827-9831,
1986.
773
ZOl MH 00424-12 LCB
Koller, K.J., Wolff, R.S., Warden, M.K., and Zoeller, R.T.:
Thyroid hormones regulate levels of thyrotropi n- rel eas i ng hormone
mRNA in the paraventricular nucleus. Proc. Natl. Acad. Sci. USA
84 (in press), 1987.
Young, W.S. Ill, Warden, M.K., and Mezey, E.: Thyrosine
hydroxylase mRNA is increased by hyperosmotic stimuli in the
paraventricular and supraoptic nuclei. Neuroendocrinology ( i n
press), 1987.
Lightman, S.L. and Young, W.S. III.: Changes in hypothalamic
preproenkephal i n AmRNA following stress and opiate withdrawal.
Nature (in press), 1987.
774
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOirCE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02302-02 LCB
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (BO c/iacacfers Of less. Title must fit on one line fiefween the tmrders.)
Biochemical Studies on Myelin Basic Protein
PRINCIPAL INVESTIGATOR (Ust ottnr professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
R. E. Martenson
COOPERATING UNITS (if any)
LAByBRANCH
Laboratory of Cell Biology
INSTITUTE AND LOCATION
NIMH. ADAMHA. NIH. Bethesda. Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
□ (a1) Minors
n (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been discontinued because the principal investigator left
the Laboratory of Cell Biology.
775
PHS 6040 {Rev. 1/84)
SPO 914-SIS
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00422-16 LCB
PERIOD COVERED
October 1, 1986 through September 30. 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between rhs borders.)
Neuropharmacology of C1rcad1an Rhythms
PRINCIPAL INVESTIGATOR (Ust other professional personnel tielow the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: M. Zatz Section Chief SBP, LCB, NIMH
Others:
N. Harrison
M. Warden
Visiting Fellow
Biologist
LNP,NINCDS
LCB, NIMH
COOPERATING UNITS (if any)
LNP, NINCDS
LAB/BRANCH
Laboratory of Cell Biology
SECTION
Section on Biochemical Pharmacoloy
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
1.9
PROFESSIONAL
1.0
0.9
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Circadian rhythms and environmental lighting regulate a number of endocrine
and behavioral functions. Dispersed chick pineal cells remain rhythmic and
responsive to light in culture. Photosensitivity appears to reside in the
same cells as melatonin production. The mechanisms of phototransduction appear
to differ from those in retinal rod cells. Light, membrane potential,
norepinephrine, cyclic AMP, and calcium channels regulate melatonin rhythms in
these cells.
777
PHS 6040 (Rev. 1/84)
SPO B14.ail
ZOl MH 00422-16 LCB
Project Description
Objectives : To elucidate the biochemical mechanisms and
neuropharmacology of circadian rhythms; to elucidate the mechanisms
by which light suppresses and entrains melatonin rhythms.
Methods : Biochemical, pharmacologic, electrophysiologic,
histologic, cell culture, and radioactive trace techniques.
Major Findings: Several laboratories have demonstrated the
persistence of photosensitive rhythms related to melatonin secretion
in cultured chick pineals. We have developed a system using
dispersed chick pineal cells in static culture, which displays a
rhythm of melatonin release for at least two weeks under cyclic
lighting conditions, and for at least 4 cycles under constant red
light. Using a rapid and specific extraction assay for the -^^C-
melatonin formed (from ■'^^C- tryptophan ) and secreted by these cells,
we have examined the effects of perturbations on the amplitude,
period, and phase of the melatonin rhythm. With this approach,
simultaneous comparisons of the effects of multiple, independent
perturbations on virtually identical, cycling, photosensitive cells
can be made.
The period in constant red light was close to 20 hours, but in
constant white light (or 12:12 cycles) it was closer to 24 hours.
Four hour pulses of white light (in otherwise constant red light)
caused an acute fall in melatonin output, and phase-dependent phase
shifts of the rhythm relatfve to controls. Pulses of darkness (in
otherwise constant red light) tended to increase melatonin output,
and caused phase-dependent phase shifts. Elevated potassium
concentrations increased melatonin output and the amplitude of the
rhythm, but did not change the period. Four hour pulses of low (5.4
mM) potassium (in otherwise constant high potassium) mimicked the
acute effect of light, reducing melatonin output, but did not induce
appreciable phase shifts. Changes in membrane potential appear more
likely to be involved in the regulation of melatonin output (and
thus be regulated _b^ the pacemaker) than to be involved in
regulation oJ_ the pacemaker which generates the melatonin rhythm.
The acute suppressive effects and phase- sh i ft i ng effects of
light indicate a pathway from the photopigment to the pacemaker
generating the melatonin rhythm. One mechanistic model for such a
pathway derives from the retinal rod photoreceptor. There,
regulation of cyclic GMP levels is a critical step in
phototransducti on . In the chick pineal, addition of 8 Br-cyclic GMP
had minimal effects on the level, period, or phase of melatonin
production. In contrast, 8 Br-cyclic AMP markedly increased
melatonin production, without changing its phase or period. These
results suggest that light acts on the chick pineal by two
mechanisms, an acute suppressive effect involving cyclic AMP and an
effect on the pacemaker not clearly mediated by the cyclic
nucleotides. The mechanism of phototransducti on in retinal rods
does not appear to be used for either effect. Furthermore,
778
ZOl MH 00422-16 LCB
norepinephrine, acting through an al pha2-adrenoceptor , mimicked the
acute suppressive effect of light but did not cause phase shifts in
the rhythm of melatonin release. In view of other evidence
suggesting that norepinephrine's effect is mediated by the GTP-
binding protein which inhibits adenylate cyclase (G,-). these results
support the inference that cyclic AMP is involved in the regulation
of melatonin production by the pacemaker but not in the regulation
of the pacemaker by light,
Another question we addressed this year was whether the
photorecepti ve mechanism resides in the same cells as melatonin
production. Immunocytochemi stry revealed the presence of membrane-
bound opsin and al pha- transducin immunoreacti vi ty in virtually all
the non-f i brobl ast chick pineal cells in culture. These reults
suggest that the majority of pinealocytes in the dish are
photosensitive. Immunosta i ni ng with an antibody directed against
HIOMT, the final enzyme in melatonin biosynthesis, also showed
immunoreacti vity in virtually all the non-f i brobl ast cells. Taken
together, these results strongly suggest that regulation of
melatonin synthesis by light does not require intercellular
communication but is a cellular property of chick pinealocytes.
Significance to Biomedical Research: Circadian rhythms occur
in hormone levels, activity, mood, etc. and are primarily regulated
by light-dark cycles. The mechanisms generating and regulating
circadian rhythms are of broad clinical and biologic interest. This
photosensitive cultured cell system, with its biochemically
measurable output, has unique advantages for the investigation of
779
ZOl MH 00422-16 LCB
biochemical mechanisms regulating phototransduc t i on and circadian
rhythmi c i ty .
Proposed Course of Project: The relationship between cyclic
AMP and calcium influx in the regulation of melatonin biosynthesis,
will be explored. Whether the effects of light and norepinephrine
are mediated by GTP-binding proteins will be determined.
Pharmacological agents acting on the pacemaker will be sought;
agents acting on calcium and other ion channels, ion-exchange
mechanisms, phosphol i pases , and protein synthesis will be tested for
direct effects on the pacemaker and for interactions with the
effects of light. If feasable, mechanisms regulating melatonin in
chick and rat pineal will be compared. If feasable, dynamic
regulation of the photopigment and transducin will be sought at the
protein and mRNA levels.
Pub! i cat ions :
Zatz, M., Mullen, D.A., and Moskal, J.R.: Photoendoc r i ne
transduction in cultured chick pineal cells: Effects of light,
dark, and potassium on the melatonin rhythm. Brain Res., (in
press ) .
Zatz, M.: Pondering the pineal in chick vs. rat. In Sandler,
M. (Ed.): Proceedings of the 6th International Catecholamine
Symposi um . New York, Alan R~. L i s s , TTn press ) .
780
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00429-08 LCB
PERIOD COVERED
October 1. 1986
■Sppt.pmhpr ■•^0, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one tine between t/ie borders.;
Biochemistry of Membranes
PRINCIPAL INVESTIGATOR (Ust otlter protessional personnel t>elow the Principal Investigator) (Name, title, latmratory, and Institute attitiation)
PI: M. Zatz Section Chief SBP, LCB, NIMH
Others:
L.C. Mahan
Staff rellov.j
LCB, mi
COOPERATING UNITS (it any)
LAB/BRANCH
Laboratory of Cell Biology
SECTION
Section on Biochemical Pharmacology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
0.2
PROFESSIONAL:
0.1
0.1
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
□ (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
1.) Previous work explored the mechanisms by which lithium causes ACTH secretion
from anterior pituitary tumor cells. Departure of one of the investigators
and technical difficulties have delayed further work on this problem.
2.) Previous work demonstrated the acylation of rhodopsin by long chain fatty
acids in vivo and in vitro. Further work on this problem waits identification
of the opsin mediating photoreception in cultured chick pineal cells which will
provide a model system for the exploration of the role of this new class of
posttranslational modification in receptor function.
781
PHS 6040 (Rev 1/84)
ZOl MH 00429-08 LCB
Project Description:
Objectives : To determine the mechanisms by which lithium
stimulates and disensitizes ACTH secretion from cultured anterior
pituitary cells. 2) To elucidate the nature and function of
protein acyl ati on .
Methods Biochemical, chromatographic, enzymatic,
pharmacologic, cell culture, and radioactive trace techniques.
Major Fi nd i ngs : None this year.
Significance to Biomedical Research: 1) A stimulatory
effect of lithium, at therapeutic concentrations, is unusual.
Elucidation of the mechanism of action of lithium on ACTH
secretion could shed light on the therapeutic actions of lithium
as well as on the regulation of ACTH secretion. 2) Acylation of
membrane proteins provides a mechanism for pos ttransl a t i onal
modification of the 1 i poph i 1 i c i ty of receptors, ion channels,
etc. which could alter their function and regulate their
interactions with cell membranes, other proteins, or signal
mol ecu! es .
Proposed Course of Project:
1) Lithium's interactions with calcium and ion channels will
be investigated. 2) The role of acylation in regulation of
membrane receptor turnover" and function will be explored.
Publications:
Zatz, M. Translocation of protein kinase C in rat
hippocampal slices. Brain Res. 385: 174-178, 1986.
Zatz, M., Mahan, L.C., and Reisine, T.: Translocation of
protein kinase C in anterior pituitary tumor cells. J .
Neurochem. 48, 106-110, 1987.
O'Brien, P.J., St. Jules, R.S., Reddy, T.S., Bazan, N.G. and
Zatz, M.: Acylation of disc membrane rhodopsin may be non-
enzymatic. J. Biol . Chem. 262: 5210-5215, 1987.
Zatz, M, and Reisine, T.: Corticotropin (ACTH) secretion.
In Lithium therapy Monographs, Vol. II, Lithium and the Endocrine
System , F.N. Johnson, ed., Karger Medical Publishers, N.Y., (in
press ) .
Reisine, T. and Zatz, M.: Interactions between lithium,
calcium, d i acyl gl yceri des and phorbol esters in the regulation of
ACTH release from AtT-20 cells. J . Neurochem . , (in press).
782
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00427-10 LCB
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must lit on one line Ixtween the borders.)
On the Mechanism of Signal Transduction Through Receptors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfillatlon)
F. Hi rata
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Cell Biology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH. Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
n (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been discontinued because the principal investigator left
NIMH.
783
PHS 6040 (Rev. 1/84)
GPO 914-SII
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00434-06 LCB
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT ^80 characters or less. Title must tit on one line t>etween ttte tjorders.)
Molecular Mechanisms of Receptor-Mediated Signal Tran;;dijrtinn
PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, latjoratory, and
Guest Researcher
Guest Researcher
Guest Researcher
Staff Fellow
Guest Researcher
Guest Researcher
Biologist
Staff Fellow
PI:
Ju
lius Axelro
Others:
R.
Burch
B.
Conklin
C.
Felder
C.
L. Jelsema
A.
D. Ma
A.
L. Ma
L.
Mahan
institute aftiliationj
MH
MH
MH
MH
MH
LCB,N
LCB,N
LCB,N
LCB,N
LCB,N
LCB,N
LCB,N
LCB,N
COOPERATING UNITS (It any)
LAB/BRANCH
Laboratory of Cell Biology
INSTITUTE AND LOCATION
NIMH, ADAMHA. NIH, Bethesda. Maryland 20892
TOTAL MAN-YEARS:
5.0
PROFESSIONAL:
5.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
n (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
GTP-regulatory proteins, in particular transducin, mediate light-induced
stimulation of phospholipase A2 and C^ (PLA2, PLC) in rod outer segments (ROS) of
bovine retina. Studies using G protein-specific agents, cholera toxin and
pertussis toxin, in both light and dark-adapted ROS suggest a dual role for
3y subunits of
Both a and gy
G proteins in both activation and inhibition of PLA2 and PLC
transducin stimulated PLA2 while a subunits were inhibitory
subunits of transducin stimulated PLC, possibly through the inhibition of a in
hibitory G protein. In addition to light, stimulation of PLA2 and PLC by other
retinal neurotransmitters, in particular dopamine and somatostatin has been
characterized. Isolated a subunits from Gs, Gi , Go and transducin were phos-
phorylated vn vitro by cAMP-dependent protein kinase and protein kinase C.
Phosphorylated subunits were altered in their ability to stimulate phospholipase
activity and this may represent a biochemical mechanism of regulation of G protein-
mediated pathways. In retina from Xenopus laevis, both somatostatin and dopamine
inhibit the circadian rise in N-acetyl transferase (NAT), the rate limiting enzyme
in melatonin synthesis. These effects are mediated by G proteins although through
different transduction mechanisms. Somatostatin, acts through a non-cAMP dependent
mechanism. Receptors for somatostatin are regulated by Na"*" and GTP and co-localize
with G proteins, in particular Gq. by autoradiographic and immunocytochemical
analyses respectively. In addition, the ability to immunocytochemically detect G
protein subunits is markedly altered during peak periods of circadian activity.
Activation of PLA2 and PLC by bradykinin has been shown to occur through
distinct G proteins in Swiss 3T3 cells. Activation of PLA2 occurs via a pertussis
toxin-insensitive G protein. Evidence to support the existence of two distinct
bradykinin receptor subtypes coupled to the activation of phospholi pases was
obtained.
785
PHS 6040 (Rev. 1/84)
SPO >14'.«l(
ZOl MH 00434-06 LCB
Project Description:
Studies of phosphol i pase A^ and phosphol i pase C in receptor-
mediated signal transduction.
Dr. Jelsema has demonstrated that in the rod outer segments
of bovine retina, both phosphol i pase A2 and phosphol i pase C are
under regulation by GTP-binding proteins, including the retinal G
protein, transducin. The By subunits of transducin function in
activation of phos phol i pase A2 while the a subunit inhibits this
effect. The mechanism for phosphol i pase Ao stimulation by
the 3y subunits is currently under investigation. In the
modulation of phosphol i pase C, both the transducin a and 6y
subunits are stimulatory, and involve the inactivation of an
inhibitory G protein. Dr. Jelsema is currently attempting to
identify this inhibitory G protein and characterize the mechanism
whereby the transducin a and By subunits activate phosphol i pase
C. Since the By subunits are common to all G proteins, the
activation of phosphol i pase A^ and C by these subunits has
implications for all s i gna 1 - transduc i ng systems that employ G
proteins.
Dr. Jelsema, in collaboration with A.D. Ma, have
demonstrated the existence of three distinct phosphol i pase C
enzymes in rod outer segments of bovine retina, each of which
appears to be regulated by different G proteins as evidenced by
the different effects of light, GTPyS and pertrussis and cholera
toxin on the hydrolysis of phospha t i dyl i nos i tol ,
phosphatidyl inositol-4-phosphate and phosphatidyl inositol-4,5-
bisphosphate. In collaboration with Dr. S. Rhee (NHLBI), she is
currently attempting to identify these phos phol i pases using
specific antibodies. These studies, while revealing unexpected
complexities, are important to understanding the regulation of
this second messenger system.
Dr. Jelsema, in collaboration with A.D. Ma, has examined the
role of G protein subunits other than transducin in the
modulation of phos phol i pase A2 activity using a reconstituted
system that consists of isolated, purified G protein subunits,
porcine pancreas phos phol i pase A^ and phosphatidylcholine
vesicles. Future studies in collaboration with Dr. C. Felder are
designed to isolate the membrane- bound phos phol i pase Ap of the
rod outer segments for reconstitution with the G protein
subunits, as described above. It is only studies of this nature
will enable the identification of the G proteins responsible for
regulation of these phos phol i pases .
Dr. Jelsema, in collaboration with A.D. Ma and L. Mahan,
have investigated the role of retinal neurotransmitters, dopamine
and somatostatin, respectively in modulation of phos phol i pase A2
and C. Dopamine has recently been identified as a major
neurotransmitter in the retina and these studies may provide a
clue as to the action mechanism. G protein-dependent regulation
of these enzymes by D2 dopamine receptors is independent of its
786
ZOl MH 00434-06 LCB
inhibitory effect on adenylate cyclase, the classical mechanism
of action of D2 receptors. Preliminary observations indicate
that somatostatin also stimulates phosphol i pase ^2 ^nd C in these
membranes .
Dr. Jelsema, in collaboration with Dr. D. Rausch, have
discovered that nerve growth factor (NGF) inhibits phosphol i pase
Ao while stimulating phosphol i pase C activity in PC12
pneochromocytoma cells. They are currently examining the effect
that transfection with ras or src has on these two phosphol i pases
since these trans feet i ons , similar to NGF treatment, lead to
neurite extension and both ras expression as well as src-induced
kinase activity have been implicated in the regulation of these
two phosphol i pases . These studies should provide clues not only
to the role of phosphol i pases in the mode of action of these
oncogenes but also the role of phosphol i pases in neuronal
development and the mechanism of action of NGF.
Dr. Jelsema, in collaboration with Dr. D. Lewis (NINCDS), is
examining the effect of GTP-binding protein subunits on the
voltage-sensitive, G protein-regulated Ca++ channels present in
AtT20 mouse pituitary cells. They are also examining the role of
phosphol i pase A2 and the role of specific arachidonate
metabolites in the somatostati n-med i ated opening of the Ca++
channels. These studies are aimed at examining the mechanism
whereby G proteins regulate ion channels.
Studies of protein kinase C modulation of GTP-binding
protei ns :
Dr. Mahan has demonstrated the presence of somatostatin
(SRIF) receptors in retina from Xenopus laevis and shown that
SRIF can inhibit the circadian rhythm of N-acetyl trans ferse
activity and melatonin synthesis in in vitro eye cup
preparations. These receptors are coupled to a G protein that
mediates inhibition in a non-cAMP dependent fashion unlike the
inhibition by dopamine via D2 receptors in this tissue. Current
experiments are attempting to define this inhibitory transduction
^ 787
ZOl MH 00434-06 LCB
system and the role of SRIF in the regulation of the circadian
clock. In collaboration with Drs. Jel sema and Palkovits, studies
are being performed to identify the nature and distribution of
the 6 proteins in these retina during the circadian cycle.
In collaboration with Drs. Brownstein, Okayama, Buckley,
Lolait and Koller, Dr. Mahan has been screening mammalian cells
(mouse L(A9)) transfected with sized and unsized cDNA libraries
from anterior pituitary, AtT-20 and AR42J cells which are rich in
somatostatin receptors. This method utilizes a cell colony
repli cat i on/1 igand-binding assay capable of screening > 100,000
colonies in an assay. Dr. Mahan hopes to address whether the
multi-transductional nature of SRIF receptors and their
distribution in both peripheral and CNS tissues are the
consequence of a single gene product.
In collaboration with Drs. Koller and Bonner, Dr. Mahan has
begun to screen sub-libraries from the above mentioned cDNA
libraries by hybridization (southern) analysis using a 56-
oligonucleotide probe developed in this laboratory. This probe
has sequence homology to muscarinic and beta-adrenergic
receptors. These receptors as well are linked to G-protein-
mediated transduction systems and may make the use of this
"concensus" probe a valuable approach for other G prote i n- 1 i nked
receptors .
In collaboration with Dr. Zatz, Dr. Mahan has conducted
experiments on the mechanism of Li +- st imul ated secretion of ACTH
from AtT-20 cells.
Bradykinin stimulated phospholipase activation and control
of prostaglandin synthesis in Swiss 3T3 cells and CPAE
endothelial cells.
Dr. Ronald Burch has shown a dissociation of bradykinin-
induced activation of phospholipase A^ (leading to prostaglandin
formation) and phospholipase C (leading to phos pha t i dyl i nos i tol
turnover) in Swiss 3T3 cells. Further evidence was obtained
using GTP analogs which indicated that a pertussis toxin
insensitive G protein regulates phospholipase A2 activation by
bradykinin.
Bruce Conklin has characterized the differential effects of
several bradykinin analogs on the activation of phospholipases A^
and C in Swiss 3T3 cells and CPAE cells. The effects of these
bradykinin analogs on prostaglandin synthesis do not fit the
previously described Bj, B^ bradykinin receptor classification.
These findings indicate that there are at least two bradykinin
receptors which stimulate prostaglandin synthesis, one present in
Swiss 3T3 cells coupled to phospholipase A2 , and the other in
CPAE cells coupled to phospholipase C.
Significance to Biomedical Research: A clear understanding
of the action of psychoactive agents that mimic or interfere with
788
ZOl MH 00434-06 LCB
receptor-mediated functions in nervous tissue requires
elucidation of post-receptor mechanisms of signal transduction.
Guanine nucl eot i de-bi nd i ng proteins modulate a variety of
cellular responses to both hormones and neurotransmitters. The
role of G proteins in receptor-mediated stimulation and
inhibition of cAMP- dependent processes is best described. More
recently, these proteins have been shown to be involved in cAMP-
independent processes such as ionic conductance, Ca++
mobilization and phospholipid metabolism. These additional
mechanisms of signal transduction may act apart from or in
concert with cAMP to effect such diverse biological responses as
hormone secretion, circadian rhythm and cell growth. Alterations
in or loss of cellular responsiveness (e.g., desens i ti zation ) ,
once associated only with changes in receptor number, can also
reflect the state of coupling between receptor and the G protein
that mediates its biological response.
Proposed Course of Project:
1. Future studies on AtT-20 cells will focus on isolation
and characterization of SRIF receptors and the nature of the G
proteins that couple these receptors to cAMP-dependent and
-independent pathways of inhibition of ACTH secretion. Receptor
isolation by expression/cloning from selected cDNA libraries in
eukarystic vectors will continue. In addition screening of these
libraries with "consensus" G protein-linked receptor probes (30-
56 bp) has begun. Development of unique SRIF derivatives to
enable studies of receptor expression and regulation in the
intact cell will continue.
2. Future studies in retina will focus on the mechanism of
action for transduc i n-med i ated increases in phosphol i pases A2 and
C. In particular, Dr Jelsema will examine the effects of various
protein kinases on the phosphol i pase- stimul at i ng activities
associated with the transducin. Collaborations with Drs. J.
Axelrod, E. Jelsema and L. Mahan are continuing to describe the
interaction of various neurotransmitters with the phosphol i pases
of the ROS of bovine and frog retina. Activation of D2 dopamine
and somatostatin receptors mimic the stimulatory effect of light
in dark-adapted ROS. Using antibodies specific for transducin
and G protein subunits. Dr. Jelsema will focus on whether the
activation of the phos phol i pases by these neurotransmitters
similarly involves transducin or is coupled to other G
proteins. These antibodies are also being employed to identify
the G proteins involved in the inhibition of phosphol i pases A2
and C. Analysis of the modulating effect of various protein
kinases on G protein functions is being continued with Dr. S.
Jakens of the FDA.
Alternative inhibitory mechanisms of SRIF (e.g., Ca++
mobilization, arachiondic acid release, phos phoi nos i t i de
metabolism) in frog retina are currently being explored. In
addition, the role of G proteins in modulating the endogenous
circadian rhythm in these retina will be investigated.
789
ZOl MH 00434-06 LCB
Future studies in Swiss 3T3 cells and CPAE endothelial cells
will focus on the mechanism by which phorbol esters activate
phosphol i pase Ao. In addition investigations have been initiated
to study the effects of bradykinin on arachiodonic acid
metabolism in primary culture rate astrocytes.
Publications:
Burch, R.M. and Axelrod, J.: Di sassoc i at i on of bradykinin-
induced prostaglandin formation from phosphati dyl i nos i to!
turnover in Swiss 3T3 cell: evidence for G protein regulation of
phos phol i pase A2. Proc. Natl. Acad. Sci. USA, in press, 1987.
Mahan, L.C. and Reisine, T.D.: Molecular Mechanisms of
Somatostatin Inhibition of Hormone Release from AtT-20 Cells. In
Reich! in, S. (Ed.): Somatostatin: Basic and Clinical Status.
New York, Plenum Press, 1987, pp. 137-145.
Mahan, L.C, McKernan, R.M. and Insel, P. A.: Metabolism of
alpha-and beta-adrenergic receptors in vitro and in vivo. Ann .
Rev . Pharmacol . Toxicol . 27: 215-235, 1987.
Burch, R.M., Luini, A., Mais, D.E., Corda, D., Vanderhoek,
J.Y., Kohn, L.D., and Axelrod, J.: a^-adrenerg i c stimulation of
arachidonic acid release and metabolism in a rat thyroid cell
line. Mediation of cell replication by prostaglandin Ep. J.
Biol. Chem. 261; 11236-11241, 1986.
Burch, R.M., Luini, A., and Axelrod, J.: Phos phol i pase Ap
and phospholipase C activated by distinct GTP-binding proteins in
response to ai -adrenergi c stimulation in FRTL5 thyroid cells.
Proc. Natl. Acad. Sci. USA 83: 7201-7205, 1986.
Reisine, T.: Stress Hormones: Their Interaction and
Regulation. In Gass, G and Kaplan, E. (Eds.): Handbook of
Endocrinology. Boca Raton, CRC Press, 1987, pp. 167-180.
Reisine, T.: Pertussis Toxin Discriminates Somatostatin's
Regulation of ACTH Release Through Adenylate Cyclase and Non-
adenylate Cyclase Mechanisms. In Sekura, R., Moss, J., and
Vaughan, M. (Eds.): Pertussis Toxin Symposium. New York,
Academic Press, 1985, pp. 149-166.
Lewis, D.L., Weight, F.F., and Luini, A.: A guanine
nucl eoti de-bi ndi ng protein mediates the inhibition of voltage-
dependent calcium channels by somatostatin in a pitutary cell
line. Proc. Natl . Acad. Sci . USA 83: 9035-9039, 1986.
Luini, A., Lewis, D., Guild, S., Schofield, G., and Weight,
F.: Somatostatin, an inhibitor of ACTH secretion decreases
cytosolic free calcium and voltage-dependent calcium current in a
pituitary cell line. J. Neurosci. 6: 3128-3132, 1986.
790.
ZOl MH 00434-06 LCB
Reisine, T.: Somatostatin inhibition of cyclic AMP
accumulation and adrenocorti cotropin release: Mechanism of
action and mode of self regulation. CINP Congress, symposium on
"Receptors and specific binding sites in the brain ," Florence,
Italy, 1986, in press.
791
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02385-01 LCB
PERIOD COVERED
October 1, 1986
September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Genetic Control of Cell Differentiation, Growth and Transformation
PRINCIPAL INVESTIGATOR (List other professional personnel t>elow the Principal Investigator) (Name, title, laborator/, end institute affiliation)
PI: H. Okayama Visiting Scientist LCB, NIMH
Others:
M. Kawaichi
M. Eiden
A. Masuda
C. Chen
N. Nukiwa
Visiting Associate
Guest Researcher
Visiting Fellow
Biologist
Guest Researcher
LCB,
NIMH
LCB,
NIMH
LCB,
NIMH
LCB,
NIMH
LCB,
NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Cell Biology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3.4
PROFESSIONAL
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A highly efficient transfection protocol and a plasmid vector for the
construction and delivery into mammalian cells of cDNA expression libraries have
been developed. Many commonly used fibroblastic cell lines are stably transfected
at frequencies of >10% with the cDNA cloning vector that incorporates a neo
selectable marker. The system permits cloning of cDNAs on the basis of their
function expressed in mammalian cells.
The majority of human cancers seem to be induced by a recessive mechanism
(recessive oncogenes). To clone such a novel type oncogene, nitroso methyl urea-
transformed BHK cells were transfected with a cDNA expression library constructed
with the new vector and mRNA prepared from primary human fibroblast cells. After
neo selection and morphological screening, two flat revertants that were unable to
grow in soft agar were isolated. In secondary transfection with the genomic DNA
prepared from one of the two revertants, the flat phenotype co-transmitted with
neo resistance. The cDNA that induces flat reversion is being recovered in E.
coli for molecular cloning and characterization.
793
PHS 6040 (Rev. 1/84)
CPO 9I4'.SI(
ZOl MH 02385-01 LCB
Project Description:
Objectives : To elucidate the molecular mechanism of cell
differentiation, growth control and malignant transformation:
cloning of cellular oncogenes and genetic elements involved in
transformation, and genes for growth and differentiation factor
signal transduction pathways.
Methods empl oyed : Recombinant DNA, molecular cloning, cell
culture, and gene analysis techniques.
Major Findings:
1) Development of a high-efficiency transfection method and
a cDNA cloning expression vector with a selectable marker gene.
It is extremely important to develop a general method that
permits cloning of genes on the basis of their functions
expressed in appropriate host cells. Cellular genes that can be
detected with conventional probes (oligonucleotides synthesized
on the basis of the primary sequence of the protein product, or
antibody) are increasingly rare. Virtually no such probes are
available for genes that regulate cell growth and
differentiation. For this reason, we developed a method for
cloning full-length cDNA in an expression vector. This year we
have devised a calcium phosphate-mediated DNA transfection method
that achieves transformation frequencies of double-digit numbers,
and constructed pcD2, a neo marker-containing cDNA cloning
expression vector, which takes full advantage of this method.
This transfection- vector system is almost as efficient as
retrovirus vectors, yet more versatile, and is suitable for
delivering cDNA libraries into mammalian cells for expression
cloning of cDNA.
2) Molecular cloning of Recessive Oncogenes.
There is ample evidence that the majority of human cancer is
caused by a recessive mechanism (recessive oncogenes) unlike
virus-caused cancer, which is mediated by dominant oncogenes or
transforming genes incorporated in the viruses. One of the best
defined in vitro system to study transformation by recessive
mechanism is nitroso methylurea (NMU) transformed BHK cells
developed by Noel Bouk. et al. Treatment with nitroso
methylurea, a chemical mutagen, induces transformation of BHK
cells by inactivating a single gene. The resulting transformed
cells are phenotypi cal 1 y recessive. Upon fusion with normal BHK
or human fibroblast cells, the transformed cell property is
suppressed through genetic complementation. We have been using
this cell system to clone a recessive oncogene. A cDNA
expression library was constructed with the newly developed pcD2
vector and mRNA from primary human fibroblasts. The library was
transduced into NMU- trans formed BHK, and stably transfected cells
were selected in the presence of G418. The transfected cells
were screened for morphologically flat cells under a
794
ZOl MH 02385-01 LCB
microscope. Two flat reverants were isolated and both were found
to be unable to grow in soft agar. Secondary transfection of the
original transformed BHK with the total genomic DNA prepared from
one of the flat revertants yielded neo-resistant colonies with
similar flat phenotypes (unable to grow in soft agar), indicating
that the gene that induced the flat phenotype is physically
linked to the neo gene present in the vector and therefore, very
likely to be the integrated cDNA. Recovery in E . c o 1 i ,
expression assay, and structural analysis of the cDNA are
currently in progress.
3) Transformation of C3H10T1/2 cells by an epigenetic
mechanism.
Whether cells can be transformed by epigenetic mechanisms
has been one of the central questions in cancer research. During
the course of studies of chemically transformed C3H cells, we
have found that co-culturing normal C3H cells with
methyl Choi anthrene-transformed C3H(MB66) leads to morphological
transformation of the C3H cells. The transformation is partially
reversible and seems to be mediated by either a diffusible
factor(s) or factor(s) transmitted by direct cell-cell contact.
Conditioned medium obtained from the culture of the transformed
C3H is almost inactive. MB66 cells are reverse transcriptase-
negative, excluding the possibility that the factor is a
transforming retrovirus. We are currently identifying the
factor .
4) The transforming growth factor-beta (TGF-beta) signal
transduction pathways.
TGF-beta has been suggested to play a major role in the
control of cell growth, terminal differentiation and
transformation. TGF-beta has extremely diverse actions:
induction or suppression of differentiation, growth suppression,
and transformation, depending on what cells are used for assay.
At least three distinct receptors for TGF-beta have been
identified on cell membrane. In order to understand the TGF-beta
signal transduction pathways that induce such diverse biological
actions, we have begun to isolate TGF-beta insensitive cell
mutants for genetic studies of the pathways and for use as hosts
for complementation assay to clone genes that constitute the
pathways .
Significance to Biomedical Research: The development of the
gene cloning and transfection techniques should greatly
facilitate isolation and characterization of genes involved in
various fundamental cell functions. Studies of genes involved in
oncogenesis, cell growth and differentiation are essential to the
understanding of the mechanism of these cell functions and,
ultimately, the pathogenesis of various diseases caused by
disorders of these functions.
Proposed Course of Project: The possibility to use yeast as
795
ZOl MH 02385-01 LCB
an expression host to screen cDNA libraries will be explored.
The cDNA will be recovered from the flat revertant of NMU-
transformed BHK, and its gene and protein structure, its tissue-
or devel opment- s pec i f i c expression and the function of the coded
protein will be examined. The factor that induces transformation
of C3H cells will be identified.
Publ i cat ions :
Okayama , H., Development and application of a vector system
that permits cloning of cDNAs based on their functional
expression in mammalian cells. In Umeda, M., Koyama , H., Gishi,
M., and Minowada J. (Eds.): Biotechnology of Animal Cells .
Tokyo, Japan Scientific Societies Press, pp. 91-101, 1987.
Okayama, H.: Phage-mediated transduction of cDNA libraries
into mammalian cells. Methods Enzymol . 151: 434-444, 1987.
Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota,
T., and Arai, K.: High-efficiency cloning of full length cDNA;
Construction and screening of cDNA expression libraries for
mammalian cells. Methods Enzymol . 154: 3-28, 1987.
Chen, C., and Okayama, H.: High-efficiency transformation
of mammalian cells by plasmid DNA. Mol . Cell. Biol . (in press)
1987.
796
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl
02386-01 LCB
PERIOD COVERED
October 1, 1986 through September 30, 1987.
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Neuropeptide Secretion, Synthesis and Action in Neural, Endocrine and Immune Cells
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfiliatJon)
PI: Lee E. Eiden Pharmacologist LCB, NIMH
See Attached
COOPERATING UNITS (if any)
U. Strasbourg; D.C. V.A.; LDN, NICHHD; NIAAA; U. Innsbruck, UCSF; A. Einstein Coll
Med.; Merrell-Dow Res-. Inst.; Case Western Reserve; USUHS
LAB/BRANCH
Laboratory of Cell Biology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
6
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
n (a2) Interviews
□ (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We are examining the molecular mechanisms of neuropeptide secretion,
neuropeptide expression and biosynthesis and neuropeptide interactions with their
receptors in the developing and mature neuroendocrine system. We are attempting
to understand the structural features of peptides and proteins that confer
molecular specificity on these three processes. We hope to characterize and
develop pharmacological agents that mimick this specificity.
797
PHS 6040 (Rev. 1/84)
SPO SI4-«lt
ZOl MH 02386-01 LCB
Other Professional Personnel Engaged on Project:
D. Aunis Directeur de Recherche U. Strasbourg
M.F. Bader Charge de Recherche U. Strasbourg
D. Brenneman Pharmacologist LDN.NICHHD
M. Brownstein Chief LCB, NIMH
J. Dave Vi si ting Sci enti st NIAAA
J. Disbrow Guest Researcher LCB, NIMH
R. Eskay Chief, Sect, on Neurochem NIAAA
R. Fischer-Colbrie University Asst. U. Innsbruck
M. Grimes Fellow UCSF
CM. Hsu Biologist LCB, NIMH
A. lacangelo Microbiologist LCB, NIMH
J. Moskal Assoc. Professor A.Einstein Coll. Med.
J.M. Muller Guest Researcher LCB, NIMH
H. Okayama Visiting Scientist LCB, NIMH
D. Perrin Guest Researcher LCB, NIMH
R. Pruss Senior Scientist Merrell- Dow Res. Inst.
D. Rausch Guest Researcher LCB, NIMH
A. Rokaeus Visiting Fellow LCB, NIMH
R. Siegel Asst. Professor Dept. Pharmacology,
Case Western Reserve
P. Smith Asst . Professor Dept . Anatomy , USUHS
K. Timmers Assoc . Pro fessor Georgetown ,D . C . V . A.
J. Waschek Guest Researcher LCB, NIMH
Project Description:
The Unit of Molecular and Cellular Neurobiology, as a part
of the Laboratory of Cell Biology, investigates the molecular
mechanisms regulating biosynthesis and secretion of neuropeptide
hormones . Earlier, we have defined "stimulus-secretion-synthesis
c 0 u p 1 i n g " as the process by which secretagogues stimulate both
secretion and synthesis of peptide hormones in neuroendocrine
cells. Thus, cholinergic stimulation of chromaffin cells causes
a concommitant release of enkephalin peptides and an increase in
enkephalin synthesis as measured by an increase in enkephalin
peptides within the cell and a rise in cellular enkephalin mRNA
levels. Both these events are calcium-dependent, suggesting that
calcium is the common second messenger subserving the coupling of
enkephalin secretion and biosynthesis. In the past year, these
findings have been extended in several ways, Drs. Eiden, Dave
and Eskay have demonstrated that " st imul us- secret i on- synthes i s
coupling" occurs in other endocrine cell types. Cort i cotrophs of
the anterior pituitary employ cyclic AMP as the common second
messenger coupling secretion and synthesis of pro-
opiomelanocortin in response to corti cotropi n-rel eas i ng factor;
calcium playing only a permissive role. Calcium is the common
second messenger coupling secretion and synthesis of prolactin in
pituitary lactotrophs . Waschek and his co-workers have
demonstrated, using the calcium agonist barium, that distinct
calcium targets within the cell separately mediate the
stimulation of secretion and enhanced biosynthesis of enkephalin
and VIP in chromaffin cells, and prolactin in pituitary
798
ZOl MH 02386-01 LCB
lactotrophs . Hsu and co-workers have demonstrated that both
di hydropyridi ne-sensi ti ve and insensitive channels operate in
stimulus-secret ion-synthesis coupling. Dr. Perrin, in
collaboration with Dr. Smith, has developed an assay for
observing secretion from individual chromaffin cells
microscopically, and for injecting into single chromaffin cells
agents which can perturb or mimick the events leading to calcium-
dependent secretion. Dr. Perrin will attempt to determine the
epitopes of the fodrin molecule important in neurosecretion by
injection of fodrin peptide fragments into chromaffin cells, and
whether or not calmodulin participates in secretion by injection
of calmodulin antibodies and protein fragments.
Drs. Pruss, Eiden and Rokaeus have demonstrated that the
mRNA for the neuropeptide galanin is abundantly expressed in
bovine chromaffin cells and increased by phorbol esters (PMA).
Enkephalin mRNA on the other hand, is regulated much less
strongly, if at all, by PMA in the same cells that contain
galanin. In addition, treatment with PMA causes a blockade of
potassium-evoked stimulation of enkephalin mRNA . Drs. Eskay and
Eiden, and Drs. Pruss and Zamir, have used the chromaffin cell in
culture as a model to show that neurotensin, substance P, atrial
natriuretic hormone, and NPY-like immunoreactivity are
differentially affected by cyclic AMP, phorbol ester, and
calcium. Drs. Wascheck and Pruss have also demonstrated that
phorbol ester and forskolin act synergi st i cal 1 y to increase
endogenous VIP synthesis and exogenous VIP gene activation in
neuroblastoma cells. Using primary cultures of mouse spinal cord
as a model for the developing nervous system. Drs. Brenneman and
Foster of the NIHCD and Dr. Eiden have collaborated to show that
enkephalin and VIP, are biosynthesized at a high constitutive
level which is dependent on the maintenance of spontaneous
electrical activity (neuronal firing) in the cultures. We will
try to learn from this model if neuronal activity determines the
number of neurons expressing a given peptide in the developing
spinal cord. Further experiments will be directed towards the
role of calcium and specific neurotransmi tter-coupl ed receptor
activation in this process. Dr. Timmers has studied the
regulation of enkephalin and insulin secretion and mRNA levels by
second messengers and insulin secretagogues in rat insulinoma
(RIN) cells. Drs. Timmers and Rokaeus have shown that insulin
secretion is inhibited by galanin. Galanin levels are altered in
the pancreas of obese rats in which insulin secretion is
impaired. Direct inhibition of insulin secretion may be a part
of the role of galanin in vivo. Enkephalin mRNA levels are
stimulated synergi st i cal 1 y by phorbol esters and cyclic AMP in
RIN cells, whereas insulin mRNA appears to be expressed
const i tut ively. These data suggest that neuropeptide expression
and biosynthesis may be regulated in a developmentally and
t i ssue- s pec i f 1 c way by several receptor- 1 i nked second messenger
system s . This hypothesis may be useful in understanding the
ontogeny of neuropeptide diversity in the nervous system. Dr.
Dianne Rausch has developed methodology which will greatly aid in
studying the role of neurotransmitter and growth factor receptor
799
ZOl MH 02386-01 LCB
activiation in terminal differenation of neuroendocrine cells.
She has constructed retroviral vectors expressing src and ras
oncogenes, both of which cause high-efficiency infection and
differentiation of murine pheoc hromocytoma cells. These vectors
can now be used for genetic complementation of the
differentiating function of cyclic AMP, nerve growth factor and
epidermal growth factor. Based on previous reports that src
overexpression in developing neurons may delay the onset of
terminal differentiation, Drs . Rausch and Moskal have used a
temperature- sens i ti ve src retroviral vector to infect fetal CNS
neurons in culture. Infected cells have been maintained for
several passages in vitro. These vectors may be useful for
preparing clonal populations of developing neurons, in which the
signals that cause neuronal differentiation can be studied. Dr.
Rausch has identified, by Northern blot analysis, a messenger RNA
species specific to renal tissue which is homologous, but not
identical, to src which is devel opmental 1 y regulated in rat
cerebral cortex. She is currently screening a cDNA library
constructed by Dr. Brownstein from the rat cortex to isolate and
characterize this devel opmental 1 y regulated, sc r-homol ogous
messenger RNA species and the protein it encodes.
Chromogranin A, whose primary sequence was determined last
year by Ms. lacangelo and her co-workers is a marker for
developing chromaffin tissue in the autonomic nervous system, and
a secretory protein whose biosynthesis, unlike the other
neuropeptides described above, is not regulated by cyclic AMP,
protein kinase C or calcium, but rather by glucocorticoids.
Antibodies made against synthetic peptide fragments from the
chromogranin A sequence appear to recognize a protein of similar
size and isoelectric point in several metazoan and even a
protozoan animal. Its evolutionary persistence and stability
suggest that chromogranin A plays an important role in
neuroendocrine function or secretory cell structure. The
chromogranin protein structure, as deduced from the sequence of
its cDNA, has now been determined for rat, and partially for
human, as well as bovine chromogranin A (lacangelo et al., in
preparation; Grimes et al., in preparation). The hypothesis that
chromogranin A is a prohormone for biologically active peptides
is now being tested definitively by Dr. Perrin, by observing the
effect of synthetic peptides, conserved in the sequence of
chromogranin A from all three species, on the secretion of
hormones from a variety of neuroendocrine cells in culture. Dr.
Reiner Fischer-Colbrie, on leave from the University of Innsbruck
in Austria, has shown that chromogranin A expression in the
adrenal gland is under the control of the pituitary, while
enkephalin and neuropeptide Y expression are controlled by the
splanchnic innervation of the adrenal gland (in preparation).
The mRNA encoding chromogranin B, another acidic secretory
protein of the adrenal gland is not altered by either splanchnic
800
ZOl MH 02386-01 LCB
firing or hypophysec tomy , The adrenal medulla may contain at
least three classes of neurosecretory proteins: those regulated
by nerve traffic (enkephalin), those regulated by glucocorticoids
(chromograni n A) and those const i tuti vely expressed (neuropeptide
Y) . This work provides direct evidence for the concerted
influence of hormonal and neural factors in determining the
amount and type of each of several neuropeptides secreted by an
endocrine organ. We are currently examining the hypothesis that
humoral and neural factors together determine the basal secretory
activity and peptide phenotype of several endocrine organs
including the pancreas, pituitary gland and brain, using
chromogranin A synthesis and secretion as a prototype. In
addition, the gene encoding chromogranin A has been isolated and
will soon be characterized, allowing an investigation of the
sequences on the gene determing cell-specific expression and
glucocorticoid regulation
In collaboration with Dr. Tamir of Columbia University, the
discoverer of SBP, Mr. Disbrow has begun the biochemical
characterization of serotonin binding to SBP and fragments of the
protein derived by cleavage with V8 protease. Preliminary
experiments indicate that a serotonin-binding epitope of less
than 20,000 daltons can be generated from the 45,000 dalton
protein by protease treatment.
801
ZOl MH 02386-01 LCB
see
Proposed Course of Project :
The work described above will be followed to the endpoints
of identifying pairs of molecules whose interaction within or on
the cell is necessary for secretion, synthesis or action of
specific neuropeptides, and designing and testing peptide
fragments and analogues that mimick or block those
interactions. To this end, we are currently developing methods
for studying the behavior of individual neuropeptide secreting
and synthesizing cells and the effects of injection of purified
proteins, peptides and other factors into them.
Publications:
Brenneman, D.E. and Eiden, L.E.: Vasoactive intestinal
polypeptide and electrical activity influence neuronal survial.
Proc. Natl. Acad. Sci. USA 83: 1159-1162, 1986.
Dave, J.R., Eiden, L.E., Ka rani an, J. and Eskay, R.L.:
Ethanol exposure decreases pituitary corticotropi n-rel eas i ng
factor binding, adenylate cyclase activity, proopiomelanocortin
biosynthesis, and plasma B-endorphin levels in the rat.
Endocrinology 118: 280-286, 1986.
lacangelo. A., Affolter, H.U., Eiden, L.E., Herbert, E. and
Grimes, M.: Bovine chromogranin A sequence and distribution of
its messenger RNA in endocrine tissues. Nature 323: 82-86,
1986.
Eiden, L.E., Huttner, W.B., Mallet, J., O'Connor, D.T.,
Winkler, H. and Zanini, A.: A nomenclature proposal for the
chromogran i n/secretograni n proteins. Neurosci ence 21: 1019-
1023, 1987.
Dave, J., Eiden, L.E., Lozovsky, D., Waschek, J. and Eskay,
R.L.: Calcium- independent and calcium-dependent mechanisms
regulate CRF-stimul ated pro-opiomel anocorti n peptide secretion
and mRNA production. Endocrinology 120: 305-310, 1987.
802
ZOl MH 02386-01 LCB
Eiden, L.E.: Is chromogram'n a prohormone? Nature 325:
301, 1987.
Waschek, J., Dave, J.R., Eskay, R.L. and Eiden, L.E.:
Barium distinguishes calcium targets for synthesis and secretion
of peptides in neuroendocrine cells. Biochem. Biophys. Res.
Commun. 146: 495-501, 1987.
Eiden, L.E., lacangelo. A., Hsu, CM., Hotchkiss, A.J.,
Bader, M.F. and Aunis, D.: Chromogranin A synthesis and
secretion in chromaffin cells. J. Neurochem. 49: 65-74, 1987.
Waschek, J. and Eiden, L.E.: Calcium requirements for
barium stimulation of enkephalin and vasoactive intestinal
peptide biosynthesis in adrenomedul 1 ary chromaffin cells.
Neuropepti des , in press, 1987.
Bonnemann, C, Giraud, P., Eiden, L.E. and Meyer, O.K.:
Measurement of mRNA specific for preprochol ecystoki n i n in rat
caudatoputamen and areas projecting to it. Neurochem. Int . , in
press, 1987.
Eiden, L.E., Giraud, P., Hotchkiss, A.J. and Affolter,
H.U.: Regulation of enkephalin gene expression, prohormone
processing and secretion in bovine chromaffin cells. In:
Stefano, G.B. (Ed.): Handbook of Comparative Aspects of Opioid
and Related Neuropeptide Mechanisms v. 1. New York, CRC Press,
1986, pp. 27-36.
Ruth, J. A. and Eiden, L.E.: Enkephalins modulate
chronotropic responses and calcium flux in rat and guinea pig
atria. In: Stefano, G.B. (Ed..): Handbook of Comparative
Aspects of Opioid and Related Neuropeptide Mechanisms v. 2. New
York, CRC Press, 1986, pp. 91-102.
Dave, J.R., Eiden, L.E. and Eskay, R.L.: Differential
effect of various secretogogues on B-endorphin release and pro-
opiomelanocortin biosynthesis in rat anterior pituitary cells and
AtT-20 cells. In: Puett, D., Ahman, F., Black, S., Lopez, D.M.,
Melner, M.H., Scott, W.A., Whelan, W.J. (Eds.): Advances in gene
techno! oqy : Molecular biology of the endocrine system.
Proceedings of the Eighteenth Miami Winter Symposium v . 4 .
Cambridge, ICSU Short Reports, 1986, pp. 34-35.
O'Donohue, T.L., Chronwall, B.M., Pruss, R.M., Mezey, E.,
Kiss, J.Z., Eiden, L.E., Massari, V.J., Tessel, R.E., Pickel,
V.M., DiMaggio, D.A., Hotchkiss, A.J., Crowley, W.R. and
Zukowska-Gro jec , Z.: Neuropeptide Y and Peptide YY neuronal and
endocrine systems. Pepti des , in press, 1986.
Dave, J.R., Eiden, L.E., Lozovsky, D., Waschek, J. A. and
Eskay, R.L.: Differential role of calcium in stimulus-secretion-
synthesis coupling in lactotrophs and corti cotrophs of rat
anterior pituitary. Ann. N.Y. Acad. Sci. 493: 577-580, 1987.
803
ZOl MH 02386-01 LCB
Z.U1 I'ln u^oou-ui
Waschek, J. A., Pruss, R.M., Siegel, R.E., Eiden, L.E.,
Bader, M.F. and Aunis, D.: Regulation of enkephalin, VIP and
chromogran i n biosynthesis in actively secreting chromaffin
cells: multiple strategies for multiple peptides. Ann . N . Y .
Acad. Sci. 493: 308-323, 1987.
Grimes, M., lacangelo. A., Eiden, L.E,, Godfrey, B. and
Herbert, E.: Chromogranin A: the primary structure deduced from
cDNA clones reveals the presence of pairs of basic amino acids.
Ann. N.Y. Acad. Sci. 493: 351-378, 1987.
Eiden, L.E.: The cell biology of the peptidergic neuron.
In: Nemeroff, C, (Ed.): Peptides in Biological Psychiatry.
Baltimore, Johns Hopkins University Press, in press, 1987.
Dave, J.R., Eiden, L.E. and Eskay, R.L.: Elevation of
intracellular cyclic AMP by cort i cotroph i n- rel eas i ng factor links
secretion of beta-endor ph i n and biosynthesis of pro-
opiomelanocortin in cultured anterior pituitary and AtT-20
cells. Ann . N.Y. Acad . Sc i . , in press, 1987.
Beinfeld, M.C., Brick, P.L., Lowlett, A.C., Holt, I.L.,
Pruss, R.M., Moskal, J.R. and Eiden, L.E.: The regulation of VIP
synthesis in neuroblastoma and chromaffin cells. Ann . N.Y. Acad .
Sci. in press, 1987.
804
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZGl MH 02387-01 LCB
TITLE OF PROJECT (80 characters or less. Title rrjust tit on one line between the borders.)
Structural Analysis of the CD4/HIV Liqand/Receptor Dyad
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Lee E. Eiden Pharmacologist LCB, NIMH
Others:
Jeff Li f son
Pat Padgett
Pete Nara
Kou Hwang
Blair Eraser
Director of Immunology
Chemist
Biologist
Chemist
Chemist
Genelabs, Inc.
LMG, NINCDS
FCRF, NCI
Genelabs, Inc.
FDA
COOPERATING UNITS (if any)
Genelabs, Inc., Frederick Cancer Research Facility, FDA
LMG, NINCDS
LAB/BRANCH
Laboratory of Cell Biology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
1
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Peptide fragments of the CD4 antigen were synthesized and tested for anti-
viral activity. A derivatized 19 amino acid fragment of the molecule inhibtts"
HlV-mediated T-lymphocyte fusion and HTLV-IIIB infection of CEM cells, with an
ED50 of 10-100 >uM in the presence of approximately 25OTCID50 of the virus. The
peptide is ineffective to block T-cell infection by HTLV-I, or to block other
CD4-dependent cellular responses, e.g. the mixed lymphocyte reaction.
-80S
PHS 6040 (Rev. 1/84)
SPO «I4.BII
Project Description: ZOl MH 02387-01 LCB
The CD4 antigen appears to be the receptor for the human
immunodeficiency virus (HIV) in T-cell infection by this virus.
We decided to investigate the strucural requirements for this
interaction because of our interest in peptide ligand-receptor
interactions, and the presence of the CD4 antigen in both the
immune system, where it subserves c1 ass- 1 1- restri cted T-cell
helper function, and the central nervous system, where its
function is unknown. In addition, the structures of both the
gpl20 ligand and the CD4 receptor are known, and functional
assays for the ligand-receptor interaction exist, as do
antibodies against both ligand and receptor. We have synthesized
20-25 amino acid fragments of the CD4 molecule, and tested these
as competitive inhibitors of HIV infection and fusion of HIV-
positive cells with uninfected CD4+ cells. None of the purified
fragments were active to inhibit fusion of HTLV- 1 1 IB- i nf ected H9
lymphoma cells with VB CD4 positive indicator cells. A side
fraction of the synthesis of one of the peptides did inhibit
fusion completely at 125-250 ojM. Partial purification by
differential extraction increased the nominal activity to 50
ijM. Chemical derivatization of the inactive parent peptide
yielded preparations with nominal anti-viral activity of 60-120
-uM (complete blockade of fusion at these concentrations). The
original material synthesized is also active in a direct assay
for viral infection of CEM cells at a nominal concentration of
100 xiM (IC50 10 xiM). We hypothesize that the active peptide is a
side product of the original synthesis due to incomplete removal
of a protecting group during HF cleavage of the peptide from the
resin. The effect of the protecting group may be to force the
remainder of the peptide to assume a conformation, which the free
peptide does not, which is similar to the conformation of the
peptide segment in the native protein.
Significance to Biomedical Research: The development of
pan-specific anti-viral agents could result from further
modifications of the core sequences found so far which are of
modest potency but are fully efficacious to inhibit viral
infection in vi tro .
Proposed Course of Project: We intend to perform
experiments designed to validate our original hypothesis, that a
single continuous epitope of a protein receptor may act as an
antireceptor agent if sterically constrained in the appropriate
conformation. X-ray crysta 1 1 ogra ph i c and structure activity
studies as well as anti-viral assays in vivo in STLV- i nf ected
macaque monkeys are planned.
Pub! i cati ons : None
806
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02396-01 LCB
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Mechanical, Thermal and Optical Signs Of Excitation In the Nervous System
PRINCIPAL INVESTIGATOR (Ust other professional personnel tielow the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Ichiji Tasaki Chief, Unit of Neurobiology LCB, NIMH
Others: Paul M. Byrne Biomedical Eng. Technician LCB, NIMH
Michio Masumura Visiting Fellow LCB, NIMH
(appointed Jan. 1987)
COOPERATING UNITS (if any)
LAB/BRANCH
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
.Z^
PROFESSIONAL:
1.5
J.
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We continued and expanded our investigation of non-electrical signs of
excitation processes in the nervous system. Using isolated spinal cord prepara-
tions ot the bu I itrog and newborn rat, we found that afferent nerve impulses
arriving at the spinal cord evoke a rapid rise in the temperature of the cord.
A thorough examination of the observed temperature rise has indicated that trans-
mission of nerve impulses across the synapses at the terminals of the sensory
fibers is accompanied by generation of a considerable amount of heat in the
substantia gelatinosa. This discovery of the "thermal response" of the spinal
cord has given us a new, useful tool for studying the effects of various chemicals
on synaptic transmission. We also examined excitation processes in the bullfrog
retina by using our thermal detectors and piezoelectric sensors. We found that
the photoreceptors in the dark-adapted retina are capable of releasing thermal
energy which is more than one million times as large as the energy of the light
pulse used for stimulation. Furthermore, we found it possible to analyze the
processes of synaptic transmission in the retina by taking its mechanical
responses as an index.
This is a continuation of Project #Z01 MH 00981 LNP.
807
PHS 6040 (Rev. 1/84)
SPO ai4.SIB
Prelect Description: ZOl MH 02396-01 LCB
Objectives : The objective of the present research is to
elucidate the function of the nervous system by examining non-
electrical manifestations of excitation processes. The major
portion of our present-day knowledge of the function of the
nervous system is derived from experimental findings obtained by
measuring changes in electric potentials and currents in various
parts of the system. The high sensitivity and the rapidity of
response of various devices designed to record electrical events
had led us to heavily rely on the results of analyses of the
electrical signs of excitation processes. In recent years,
however, it has become possible to record optical, mechanical and
thermal changes in the nervous system with a reasonably high time
resolution. During the past years, the sensitivity and the
response- time of the thermal detectors we have constructed are
greatly improved. Consequently, we expect that our investigation
of functions of the nervous system by use of our optical, thermal
and mechanical devices continues to unravel new phenomena in the
nervous system.
Methods : Piezoelectric sensors are employed for measuring
rapid changes in the tension or pressure in the nervous system.
Bifurcated light guides are used for detecting changes in the
turbidity. Thin sheets of pol yv i nyl i dene f 1 uor i de--synthet i c
pyroelectric materi al --are employed to construct heat sensors
with a high sensitivity and a short response time. Quite
recently, we have constructed a sensitive heat sensor which has a
very small effective surface.
Major Findings:
( 1 ) Detection of heat production associated with synaptic
transmission.
Using bullfrog spinal cord preparations, we found that
electric stimulation of the dorsal roots evokes a rapid rise in
the rate of heat production by the cord. Immersion of the cord
in a salt solution containing a low calcium and high magnesium
ion concentration was found to suppress the observed "thermal
response" (i.e. the transient rise of in heat production evoked
by stimulation). Strong electric shocks applied to the ventral
roots did not evoke any thermal response. The observed amplitude
of the thermal response was enhanced considerably when the spinal
cord was hemisected horizontally and the cut surface of the
dorsal half-cord was brought in contact with the heat sensor.
This and other tests have clearly indicated that the observed
heat production is associated with synaptic transmission at the
afferent fiber terminals. During the past three months, we have
carried out a quantitative analysis of this newly discovered
phenomenon. A study of the effects of various
neuropharmacological agents on the synaptic heat is now in
progress. In a preliminary experiment, we have demonstrated
synaptic heat in the spinal cord of the new-born rat.
808
,„, ,. ZOl MH 02396-01 LCB
(2 ) Heat generated by bullfrog photoreceptors.
Last year, we found that the photoreceptors in the bullfrog
retina respond to brief light pulses with a rapid generation of
heat. This year, we analyzed properties of the thermal responses
of the photoreceptor cells in detail by using greatly improved
heat sensors. We found that, at the level of light intensity
which delivers roughly one photon per rod, the energy released by
the photoreceptors (in the form of heat) is more than one million
times as large as the energy absorbed by the receptors. The
significance of this finding is not altogether clear at present.
(3 ) Study of spread of excitation processes in the retina.
By taking mechanical responses of the isolated bullfrog
retina as an index, the sequence of spread of excitation
processes from the photoreceptor cells to the ganglion cells was
analyzed. The time course of the force developed by the retina
was explained by comparing the sequence of force development with
that of the el ectro-reti nogram. We came to the conclusion that
the nerve cells in the retina swell during depolarization and
shrink during hyperpol ar i zat ion .
Significance to Biomedical Research:
Our knowledge of the function of the vertebrate nervous
system is at present quite limited. There are inconsistencies in
the present-day interpretation of the effects of drugs on the
spinal cord. Studies of non-electrical manifestations of
excitation processes are expected to lead us to a better
understanding of the normal, as well as abnormal, function of the
nervous system.
Proposed Course of Project:
We have just started investigating the effects of various
chemical agents on the bullfrog spinal cord by taking heat
production as an index. We are planning to apply our technique
of recording non-electric signs of excitation processes to the
vertebrate cerebrum.
Publ i ca t i ons :
Tasaki, I., and Byrne, P.M.: Heat production associated
with synaptic transmission in the bullfrog spinal cord. Brai n
Res. 407: 386-389, 1987.
Tasaki, I.: On the mechanism of hypersensitivity in nerve
fibers and cells. In Chal azoni ti s , N., and Gola, M. (Eds.):
Inacti viation of Hypersensitive Neurons. New York, Alan R. Liss
Inc., 19S7, pp. 511-315.
Tasaki, I., and Byrne, P.M.: Rapid mechanical changes in
the amphibian retina evoked by brief light pulses. Biochem.
809
ZOl MH 02396-01 LCB
Biophys. Res. Commun. 143 (1): 93-97, 1987.
Tasaki, I., Byrne, P.M., and Masumura, M.: Detection of
thermal responses of the retina by use of pol yvi nyl i dene fluoride
multilayer detector. Japan . J. Physiol . (in press), 1987.
810
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00881-31 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. We rrtust tit on one Ime tietween f/ie borders.)
Intermediary Energy Metabolism in Mammalian Brain
PRINCIPAL INVESTIGATOR (List ottter professiortal persor\net betow ttie Principal Irtvestigator) (Name, title, laboratory, artd institute affiliation)
P.I.: Elaine E. Kaufman Research Chemist LCM, NIMH
Others: Thomas Nelson Medical Officer (Research) LCM, NIMH
COOPERATING UNITS (it any)
None
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.7
PROFESSIONAL:
1.2
OTHER:
1.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues S (c) Neither
n (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed ttte space provided.)
This report will describe work carried out in this laboratory which resulted in
the discovery and characterization of a mammalian mitochondrial transhydrogenase ,
an enzyme capable of transferring hydrogens from specific hydroxyacids to equally
specific ketoacids. This work suggests new catabolic pathways for
L-g-hydroxybutyrate , y-hydroxybutyrate and g-ketoglutarate and may explain the
origin of the elevated levels of certain hydroxyacids found in patients with
specific organic acidemias.
811
PHS 6040 (Rev. 1/84)
CPO gi4>»it
Z01 MH 00881-31 LCM
Project Description:
Objectives:
The objectives of this study has been the isolation, characterization and
purification of a mitochondrial hydroxyacid-oxoacld transhydrogenase found in
brain, liver and kidney. The role of this transhydrogenase in the metabolism of
such key metabolic intermediates as L-g-hydroxybutyrate, and a-hydroxyglutarate
and Y-hydroxybutyrate is also of interest.
Methods:
The mitochondrial transhydrogenase described in this report has been purified
using the following techniques: 1) differential centrifugation, 2) salt
fractionation, and 3) column chromatography. The products of the reaction have
been identified using gas-liquid chromatography either alone or in combination
with mass spectroscopy, paper and thin layer chromatography. Subcellular
localization and quantification of one of the enzymes has been confirmed by
antibody titration. Enzyme kinetics have, in general, been carried out using a
spectrophometric method.
A mitochondrial hydroxyacid-oxoacid transhydrogenase was discovered when
experiments with an antibody to the purified cytosolic 7-hydroxybutyrate
dehydrogenase indicated that there was a second enzyme which could catalyze the
oxidation of the hydroxy-acid, T-hydroxybutyrate. This new enzyme was located
in the mitochondria fraction isolated from brain, liver, and kidney and,
moreover, did not require added NAD* or NADH. The oxidation of the hydroxyacid
did, however, have an absolute requirement for a-ketoglutarate. In contrast to
the well known a-ketoglutarate requiring dioxygenases the reaction catalyzed by
this enzyme did not require molecular oxygen and resulted in the formation of
a-hydroxyglutarate .
We later found that several other oxoacids, pyruvate, oxolactate and
a-ketoadipate could substitute for a-ketoglutarate but that a-ketoglutarate is
the preferred substrate. The hydroxyacids which we have so far found to be
substrates for this enzyme are L-6-hydroxybutyrate, Y-hydroxybutyrate and
D-a-hydroxyglutarate .
Inasmuch as no added cofactor is required by this enzyme, we assume that the
enzyme contains a tightly bound cofactor, possibly NAD"^, NADH, which is oxidized
and reduced on the protein. Proof of this must await purification of sufficient
quantities of the protein to identify the cofactor.
In summary, a new enzyme has been isolated which can catalyze transhydrogenation
reactions involving specific hydroxyacids and equally specific oxoacids. This
is the first report of a mammalian enzyme capable of oxidizing
L-6-hydroxybutyrate .
Significance to Biomedical Research and to the Program of the Institute:
A completely new enzyme which can catalyze the transfer of hydrogens from
hydroxyacids to oxoacids has been isolated. The discovery of this enzyme may
812
ZOl MH 00881-31 LCM
provide new clues to the origin of specific hydroxyacids such as the
2-hydroxyglutaric acid found in the inherited metabolic aciduria, glutaric
aciduria type II, a condition which leads to both peripheral and central nervous
system disorders.
Proposed Course:
Aspects of this project which deal specifically with the metabolism and mode of
action of Y-hydroxybutyrate- have been completed and are being prepared for
publication as is the work described above on the mitochondrial
transhydrogenase .
A new initiative which will involve an investigation of some of the metabolic
interrelationships of glia and neurons, in particular those for which GABA and
glutamate are the predominate neurotransmitter, will be started. It is
anticipated that these studies will utilize freshly isolated tissue, cells in
culture as well as brain dialysis techniques.
Publications:
Kaufman, E. and Nelson, T. : Evidence for the participation of a cytosolic
NADP'^-dependent oxidoreductase in the catabolism of Y-hydroxybutyrate i_n vivo.
J. Neurochem. 48: 1935-19^11, 1987.
813
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00882-20 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
riTLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Studies on Regional Cerebral Circulation and Metabolism
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
P.I.: Louis Sokoloff Chief, Lab. Cerebral Metabolism LCM, NIMH
Others: Charles Kennedy
Thomas Nelson
Carolyn B. Smith
Gerald A. Dienel
Nancy Cruz
Nancy Eng
Guest Researcher
Medical Officer (Research)
Research Chemist
Senior Staff Fellow
Biologist
Chemist
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
COOPERATING UNITS (if any)
Theoretical Statistics & Mathematics Branch, NIMH (C.S. Patlak & K.D.
NINCDS, NIH (I. Kopin); NIDA, ARC, Baltimore, Maryland (L. Porrino) .
Pettigrew) :
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
9.50
PROFESSIONAL:
6.00
OTHER:
3.50
CHECK APPROPRIATE BOX(ES)
U (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues B (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The deoxyglucose method for the quantitative determination of rates of local
glucose consumption in the discrete functional and structural components of the
^^^^ii^ of conscious or anesthetized laboratory animals was developed in this
laboratory over 10 years ago. In this method [^^C] deoxyglucose is employed as a
t?^^cer for glucose flux through the hexokinase step; the product,
[ C]deoxyglucose-6-phosphate, is measured by quantitative autoradiography. The
method continues to be used to study alterations in local energy metabolism in a
variety of physiological, pharmacological and a limited number of pathological
states. Its suitability to a wider range of pathologic conditions is being
extended and special time constraints which may be present in the method's
adaptation for use in human subjects with [ ^^C] f luorodeoxyglucose and PET have
been examined.
815
PHS 6040 (Rev. 1/84)
GPO si4-aie
ZOl MH 00882-20 LCM
OTHER INVESTIGATORS (CONTINUED)
Kathleen Schmidt
Victor Ho
Giovanni Lucignani
There^e M. Jay
Kentaro Mori
Quang Vo
Ernesta Palombo
Hajime Nakanifhi
Project Description:
Computer System? Analyst
Guest Researcher
(Hughes Scholar)
Guest Researcher
Visiting Fellow
Visiting Fellow
Computer Programmer
Visiting Fellow
Visiting Fellow
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
LCM, NIMH
The deoxyglucose method, both in its original form and in its adaptation for use
in human subjects, has been widely used by investigators throughout the world
for over a decade. It has also been employed by members of this laboratory in
the study of a variety of physiological conditions as reported previously and as
given below. In the interests of extending the method's general applicability,
the Laboratory has refined the model on which the method is based. This
revision takes into account new information on the intracellular sites of
phosphatase activity, and thereby defines more accurately the late time course
when radioactive label is lost from the tissue. The new model provides the
basis for understanding the time limits after administration of the labeled
deoxyglucose during which valid measurements can be made, and to correct for
processes that become significant with the long scan times required when PET is
employed. Also many of the experiments planned over a year ago to permit the
deoxyglucose method to be extended for use in a wide range of pathophysiologic
states have been successfully accomplished, and work done in response to
criticisms by others has been concluded. These diverse studies related to the
deoxyglucose method are separately described below.
I. APPLICATIONS OF THE DEOXYGLUCOSE METHOD
A, Dr. Linda Porrino (NIDA) and Dr. Ernesta Palombo, in collaboration with
Drs. Irwin Kopin and Krystof Bankiewicz of NINCDS, have extended their
studies with the deoxyglucose method applied to the Parkinson's
syndrome induced by l-methyl-4-phenyl-l ,2, 3,6-tetrahydropyridine (MPTP).
Objectives:
1) To measure glucose utilization in the neural pathways involved in the
production of abnormal motor function in the monkey with
hemiparkinsonism and to map the circuits involved in the therapeutic
administration of L-DOPA.
2) To study the acute effects of MPTP on glucose utilization in monkeys
and their prevention by pretreatment with MAO-inhibitor drugs.
816
ZOl MH 00882-20 LCM
Major Findlngg;
In the animals with hemiparkinsonism decreases in glucose utilization
were found in the external segment of the globus pallidus ipsilateral to
the side of neuro- toxin administration while decreases were found
bilaterally in the substantia nigra and in the subthalamic nuclei.
Treatment with L-DOPA restored normal rates of glucose utilization in
the subthalamic nucleus and in the lateral substantia nigra while
resulting in rates above normal in the substantia nigra reticulata and
in the internal segment of the globus pallidus. The findings in
hemi parkinsonian monkeys in the visual system and pathways concerned
with eye movements have been incorporated in a manuscript now in press.
Acute administration of MPTP resulted in marked increases in glucose
utilization in the substantia nigra pars compacta and portions of the
ventral tegmental area, along with severe metabolic depression in
virtually all other structures of the brain, Pretreatment with the
MAO-inhibitor , pargyline, which protects against the long term toxicity
in the nigra, also prevents the acute increases in the nigra and ventral
tegmental area.
Dr. Therese Jay completed her studies of local cerebral blood flow in the
mouse. While these left unexplained the reason for the relatively high rate
of metabolism of the fasciculus retroflexus in the normal mouse, they served
to provide the methodology for local cerebral blood flow in this species.
The report of this work is now in press.
Drs. Kentaro Mori, Hajime Nakanishi, and Charles Kennedy, in collaboration
with Dr. Kenneth Kellar of the Department of Pharmacology of Georgetown
University School of Medicine, are completing the study of the local
metabolic effects of phencyclidine, the agent now prominent because of its
mind-altering effect? and abuse among young people. Detailed analysis of
the autoradiographs has lagged because of limited availability of personnel
and computer times, but it is under high priority examination.
Objectives:
To correlate the known psychological and behavioral effect? of phencyclidine
with the effects on local cerebral glucose utilization in the rat over a
wide range of doses.
Major Findings;
Selective reductions in the rates of metabolism were found in primary
sensory pathways, such as auditory and visual pathways, while enormous
increases were observed in limbic structures, such as the amygdala, hippo-
campus, cingulate gyrus, etc. The work is in progress, but it already
demonstrates that limbic seizure activity may be a major consequence of PCP
use and the basis of the violent and aggressive behavior a??ociated with
the use of this drug.
«17
ZOl MH 00882-20 LCM
D. Drs. Therese Jay and Charles Kennedy, in collaboration with Dr. Robert
Abratns of the Department of Obstetric? and and Gynecology at the University
of Florida in Gainesville, concluded their study of local metabolic rate of
the brain during rapid-eye-movement sleep. The result? showed diffuse
increase? in local cerebral glucose utilization throughout the brain during
REM sleep. The manuscript reporting this work has been submitted for
publication.
E. Dr. Linda Porrino concluded her evaluation of the effect? of cocaine on
cerebral metaboli?m, a project initiated jointly with Dr. Floyd R. Domer in
the previou? year.
II. EXTENSION OF THE DEOXYGLUCOSE MODEL TO A 5-PARAMETER MODEL
The deoxygluco?e method assumes that products of deoxyglucose phosphorylation
are trapped in the tissues for the duration of the experimental period. In its
application no detectable loss is found during the first ^5 minutes, a small
loss occurs at 60 minutes, and progressively greater losses occur at 90 and 120
minute? following an intravenou? pul?e of [^ ^C]deoxygluco?e. The loss, when it
occurs, is presumably due to the action of glucose-6-phosphatase. Fishman and
Karnovsky (J. Neurochem. 46: 371, 1986) have explained the lag in the appearance
of the phosphatase activity by finding that the hydrolysis i? rate-limited by
the diffu?ion of deoxygluco?e-6-phosphate from the cytosol, where it is formed,
across the endoplasmic reticular membrane to the site where
gluco?e-6-pho?phatase resides. The original deoxyglucose model was, therefore,
revised to include this new information, and an equation was derived to describe
the kinetic behavior of labeled deoxyglucose and deoxyglucose-6-phosphate in the
ti??ue in the pre?ence of the?e con?traints. The new equation contains 5 rate
constants, including one for the diffusion of deoxyglucose-6-pho?phate across
the endoplasmic reticular membrane and another for its hydrolysis by
glucose-6-phosphatase.
Drs. Giovanni Lucignani, Kentaro Mori, Therese Jay, Erne?ta Palombo, Thoma?
Nel?on and M?. Kathleen Schmidt carried out the necessary experiment? to obtain
estimates of the values of these rate con?tant?. The?e experiments provided the
time courses of arterial plasma and tissue concentrations of ^ '^C over a period
from 2.5-120 minutes. The new equation was fit to the measured data by a
non-linear least-squares routine to obtain the best fitting rate con?tant?. The
data were also analyzed by the graphical evaluation technique which provides an
estimate of the time when loss of product begins to take place. These studies
have been presented af an international meeting, and a manuscript on this work
i? in preparation.
III. ADAPTATION OF THE DEOXYGLUCOSE METHOD FOR USE IN PATHOPHYSIOLOGICAL
CONDITIONS
The original deoxyglucose method was designed for use in laboratory animals
under physiologic conditions. When normal physiological limit? are exceeded, a?
may occur during ?tatus epilepticus, ischemia, or s-evere hypoglycemia, the
lumped constant of the operational equation is altered. In order to make
818
ZOl MH 00882-20 LCM
possible the application of the deoxyglucose method to such pathophysiological
conditions it is planned to develop a procedure to obtain value? for the local
lumped constant. Through the use of [^ ^C]methylglucose it is now possible to
measure local glucose concentration in the brain. This, in turn, makes possible
an estimate of the ratio of the distribution volumes for glucose and
deoxyglucose in local brain regions and, therefore, of the lumped constant
itself. The experiments require the preparation of animals in which various,
specified plasma concentrations of glucose over a wide range can be induced and
maintained for long periods. An equation and programmed infusion schedule for
this purpose has now been refined and tested with success by Dr. Kentaro Mori.
The complex experiments in which [^Hjdeoxyglucose or [^ ^C]methylgluco?e and
glucose concentrations in local regions of brain will be measured simultaneously
in the same animal under steady state conditions have begun. Others
participating in these experiments are Drs. Gerald Dienel, Thomas Nelson,
Therese Jay, Ernesta Palombo, Charles Kennedy, Carolyn Smith, and Ms. Nancy
Cruz.
Objectives:
To make possible the determination of the local lumped constant in the brains of
animals under pathophysiological conditions. This determination of glucose
utilization in all local subdivisions of brain could be reliably measured in
pathophysiological states.
Major Findings:
The large number of complex experiments involving the creation of a steady state
at specified plasma glucose concentrations and, simultaneously, a steady state
for plasma deoxyglucose or methylglucose, have been completed. These
experiments, terminated with a procedure for instantaneous freezing of the
brain, and followed by analyses of plasma and tissue for their concentrations of
glucose, deoxyglucose or methylglucose, have permitted the calculation of a
family of curves describing distribution volumes over a wide range of plasma
glucose concentrations. These fundamental studies are preliminary to the
conduct of a planned group of experiments involving double label isotopes for
the autoradiographic measurement of brain concentrations of glucose and
deoxyglucose in the same animal. Information from the combined groups of
experiments will make possible the calculation of the local lumped constant in
such conditions as ischemia, sustained seizures, and cerebrovascular disorders.
IV. WORK IN RESPONSE TO PAPERS CRITICAL OF THE DEOXYGLUCOSE METHOD
The laboratory has largely completed the time-consuming projects of responding
to criticisms of the deoxyglucose method a year ago. The claims of Huang and
Veech that the activity of glucose-6-phosphatase in brain is very active (J.
Biol. Chem. 257: 11358-11363, 1982) were shown to be without foundation and due
to inadequate purification procedures. It remained for Dr. Gerald Dienel to
identify the major sources of contaminating compounds in the brain glucose
fraction of Huang and Veech. He has shown these to consist of at least 7 amino
acids in addition to glucosamine. Furthermore, Dr. Dienel showed that the
819
ZOl MH 00882-20 LCM
glucose purification procedure? employed by Huang and Veech not only failed to
purify the radioactive glucose but actually provided an environment for
detritiation of the labeled glucose and for side reactions that converted
glucose and contaminants to other ^ ^C labeled compounds. The improperly
purified glucose fraction was responsible for the erroneous conclusions of Huang
and Veech. One more paper by G. Dienel describing these studies is close to
final form for submission and publication.
Significance to Biomedical Research and to the Program of the Institute:
The deoxyglucose method has made it possible for the first time to measure the
rates of glucose utilization simultaneously in all functional and structural
components of the central nervous system of conscious, behaving animals and now
also in man. Because the method was developed in our Laboratory, it has been
our responsibility to survey its applicability to the various types of
conditions in which it might be applicable. The program has, therefore, been
somewhat heterogeneous covering a wide range of physiological, pharmacological,
pathological, and altered behavioral states. The method and its wide-ranging
usefulness have now been more or less established, and it is used extensively
throughout the world in neuroanatomical , neurophysiological,
neuropharmacological , psychiatric, neurological, and neurosurgical research.
Its wide acceptance is directly related to the results of studies in this
project.
Proposed Course:
Application? of the deoxyglucose method to problem? of neurophysiology,
neuropharmacology, neurology, and psychiatry will be continued. A project has
been initiated and will be continued to adapt the method for use in
neuropathological condition? such as stroke, status epilepticus, etc. Effort?
will be made to improve the quantitative re?olution of the method to the ?ingle
cell and subcellular level?. Immunocytochemical technique? will be introduced
with the aid of Dr. Bernard Dri?coll to correlate local cerebral rate? of
gluco?e utilization with local level? of neuropeptides and the host of putative
neurotransmitter? and neuromodulators. A cell culture facility has been
established in the Laboratory by Dr. B. Driscoll to allow studies of cellular
mechanism? of carbohydrate transport across cell membranes which are necessary
to define the rates of glucose utilization in neuronal and glial cellular
components of the cerebral tissue.
Publications:
Sokoloff, L. : Basic principles in the imaging of rates of biochemical processes
in vivo. In Hayaishi, 0. (Eds.): Biomedical Imaging — From Anatomy to
Physiology and Biochemi?try . Tokyo, Japan, Academic Press, Japan, Inc., 1986,
pp. 183-217.
Nelson, T., Dienel, G., and Sokoloff, L. : Gluco?e-6-pho?phata?e activity in
brain. Science 234: 1128-1129, 1986.
820
ZOl MH 00882-20 LCM
Sokoloff, L. and Porrlno, L. : Some fundamental con?i deration? in the
application of the deoxyglucope method to pharmacological studies. In
Krieglstein, J. (Ed.): Pharmacology of Cerebral Ischemia. Amsterdam, Elsevier
Science Publishers B.V., 1986, pp. 65-76.
Sokoloff, L. : Mapping cerebral functional activity with radioactive
deoxyglucose. In Adelman, G. (Ed.): Encyclopedia of Neuroscience. Cambridge,
MA, Birkhauser Boston, Inc., 1987, pp. 604-609.
Namba, H., Lucignani, G., Nehlig, A., Patlak, C, Pettigrew, K., Kennedy, C,
and Sokoloff, L.: Effects of insulin on hexose transport across blood-brain
barrier in normoglycemia. Amer. J. Physiol. Endocrinol. & Metab. 252
(Endocrinol. Metab. 15): E299-E303, 1987.
Porrino, L.J., Burns, R.S., Crane, A.M., Palombo, E., Kopin, I.J., and Sokoloff,
L.: Changes in local cerebral glucose utilization associated with parkinson's
syndrome induced by 1-Methyl-4-Phenyl-l ,2, 3, 6-Tetrahydropyridine (MPTP) in the
primate. Life Sci. 40: 1657-1664, 1987.
Sokoloff, L. : Book Review. Radionuclide Imaging of the Brain. (Vol 1,
Contemporary Issues in Nuclear Imaging. Edited by B. Leonard Holman. New York:
Churchill Livingstone, 1985, 232 pg . ) . J. Neurosci. Res. 17: 199, 1987.
Lucignani, G., Namba, H., Nehlig, A., Porrino, L.J., Kennedy, C, and Sokoloff,
L. : Effects of insulin on local cerebral glucose utilization in the rat.
J. Cereb. Blood Flow & Metab. 7: 309-31^*, 1987.
Kadekaro, M., Vance, W. H., Terrell, M. L., Gary, Jr., H., Eisenberg, H.M., and
Sokoloff, L.: Effects of antidromic stimulation of the ventral root on glucose
utilization in the ventral horn of the spinal cord in the rat. Proc. Natl.
Acad. Sci. USA 84: 5492-5495, 1987.
Porrino, L.J., Burns, R.S., Crane, A.M., Palombo, E., Kopin, I.J., and Sokoloff,
L.: Local cerebral metabolic effects of L-DOPA therapy in
l-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine-induced parkinsonism in monkeys.
Proc. Natl. Acad. Sci. USA 84: 5995-5997, 1987.
Sokoloff, L.: Foreword, In Wood, J.H. (Ed.). Cerebral Blood Flow: Physiologic
and Clinical Aspects. McGraw-Hill, 1987, (in press).
Ito, M., Kadekaro, M., and Sokoloff, L. : Local glucose utilization of the brain
and pineal gland during stimulation of the cervical sympathetic trunk.
J. Pineal Res, (in press) 1987.
Ho, V.W., Porrino, L.J., Crane, A.M., Kopin, I.J., and Sokoloff, L. :
Alterations in local cerebral utilization in the oculomotor system of
MPTP-induced Parkinsonian monkeys. Ann. Neurol, (in press) 1987.
Sokoloff, L., Kennedy, C, and Smith, C. B. : The [ ^ ^c jdeoxyglucose method for
measurement of local cerebral glucose utilization. Neuromethods Vol. 15.
Humana Press, 1.987, (in press).
821
ZOl MH 00882-20 LCM
Sokoloff, L. : Circulation and energy metabolism of the brain. Bagic Neurochem.
Mth Ed. . Raven Pre??, 1987, (in press),
Sokoloff, L. : Basic principles in imaging of regional cerebral metabolic rates
with radioisotopes. (Proceedings of the NATO ASI Meeting in L'Aquila, Italy,
June, 1986) (in press).
Jay, T.M., Lucignani, G., Crane, A.M., Jehle, J., and Sokoloff, L. : Measurement
of local cerebral blood flow with [ ^ ^C jiodoantipyrine in the mouse. J. Cereb.
Blood Flow & Metab. (in press) 1987.
Abrams, R.M., Hutchison, A. A,, Jay, T.M., Sokoloff, L., and Kennedy, C: Local
cerebral glucose utilization nonselectively elevated in rapid eye movement sleep
of the fetus. Brain Res, (in press) 1987.
Palombo, E., Porrino, L.J., Krzysztof, S., Bankiewicz, K.S., Kopin, I.J., and
Sokoloff, L. : Comparison of acute and chronic effects of MPTP on local cerebral
glucose utilization in monkeys. (International Symposium on Neurotoxicology ,
Turin, Italy, May 5-7, 1987) (in press) 1987.
Jay, T.M., Abrams, R.M., Hutchison, A. A., Kennedy, C, Schmidt, K., and
Sokoloff, L.: Variations du debit sanguin et raetabolisme cerebral au cours du
sommeil. Cereb. Circ. & Metab. (in press) 1987.
Sokoloff, L. : Foreword to Proceedings of the Eric K. Fernstrom Symposium,
Neural Regulation of Brain Circulation. (Proceedings of the Eric K. Fernstrom
Symposium, Lund, Sweden, June 21-23, 1985), (in press) 1987.
822
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00887-10 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.)
The Extended Visual System of the Macaque Monkey
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
P.I.: Charles Kennedy Guest Researcher LCM, NIMH
Others: Louis Sokoloff
Chief
LCM, NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland
TOTAL MAN-YEARS:
2.0
PROFESSIONAL:
1.25
0.75
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Although the project began with its focus on mapping the visually responsive
cortical areas, the experiments also permit an analysis of the sensory-motor
system. Normal control monkeys with their visual pathways intact responded to
the visual cues by pressing a lever with one hand. This involved an asymmetrical
pattern of local glucose utilization in brain involving a wide expanse of cortical
and sub-cortical structures. An analysis of the pattern of asymmetry provides new
information with respect to the localization sensory-motor function. The data
obtained to date indicate that a much larger portion of brain regions are uni-
laterally activated on unimanual activity than has been appreciated previously.
823
PHS 6040 (Rev. 1/84)
GPO SI4-aiB
ZOl MH 00887-10 LCM
Project Description;
Objective:
To map all regions of monkey brain (motor and sensory) which are involved in
the performance of a task involving the use of one hand.
Methods Employed:
Normal controls involved in continuous, unimanual lever pressing in response to
visual cues were studied with the deoxyglucose method and provided an
opportunity to study the localization of motor activity. To the extent that
motor activity involving one arm and hand is accompanied by neuronal discharges
in the contralateral hemisphere, a side to side comparison of metabolic rates in
homologous regions provides a measure of the functional participation of the
regions in the motor behavior. During such behavior there is, of course, a
concomitant stimulation of a number of sensory modalities, eg. touch, pressure
and joint position. The metabolic studies of the unimanually active monkeys
therefore permitted a mapping of sensory pathways. The large task of making a
detailed side to side comparison of the large number of potentially
activated structures in the animals was begun only recently.
Major Findings:
In the study of local regions responsive to sensorimotor stimulation by
unimanual lever pressing the analysis has indicated that the metabolically
activated regions are widely distributed in both cortical and sub-cortical
structures. Some structures, which might be expected to have large
right-left differences in their metabolic rates on the basis of others'
neurophysiologic observations, proved to have relatively small differences,
eg. the globus pallidus. Conversely, surprisingly large differences were
found in several cortical regions generally not regarded as being active
during motor activity. Among these was the inferior bank of the superior
parietal lobule. Lesion and behavioral-electrophysiological experiments
indicate that many independent functional entities coexist in their
representation in the superior parietal lobule. The deoxyglucose method as
applied here appears to have localized with great precision the part of this
diverse cortical region which contributes to visually directed
movements of one arm and hand. Metabolic evidence of the participation of
several other regions is demonstrated in these studies. The analysis of the
data is continuing.
Significance to Biomedical Research and to Program of the Institute:
These studies add new information on the localization of functional activity in
brain.
Publications
None
824
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00889-08 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 Characters or less. Title must lit on one line tietween the borders.)
A Method for the Determination of Local Rates of Protein Synthesis in Brain
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation)
P.I.: Carolyn B. Smith Research Chemist LCM, NIMH
Others: Louis Sokoloff
Christina Eintrei
Charles Kennedy
Mortimer Mishkin
Richard Nakamura
Chief
Visiting Fellow
Guest Researcher
Chief
Guest Researcher
LCM, NIMH
LCM, NIMH
LCM, NIMH
LN, NIMH
LN, NIMH
COOPERATING UNITS (H any)
Department of Neurosurgery, Univ. of Texas (M. Kadekaro) ; Dept. of Obstetrics &
Gynecology, Univ. of Florida (R. Abrams); Department of Neurosurgery, SUNY
(D. Dow- Edwards)
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.8
PROFESSIONAL:
1.6
1.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues H] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A method has been developed for the estimation of local rates of protein synthesis
in brain in vivo. The method is based on the use of L-[^ ^CJleucine as a tracer
for the incorporation of leucine into protein. Six kinetic models for the
behavior of leucine in brain have been designed. By mathematical analysis of the
kinetics of exchange of the amino acid between plasma and the tissue pooKs) and
its incorporation into protein, equations have been derived for each model that
define the rate of amino acid incorporation into protein in terms of the time
course of plasma-specific activity, final tissue concentration of '^C, and
experimentally determined kinetic constants. Tissue concentrations of ^^C are
determined by quantitative autoradiography. Experiments have been carried out to
test the validity of the various models and to determine the kinetic constants to
be used in the operational equation.
Studies of the effects of normal development, sleep, anesthesia, electrical
stimulation and hypothyroidism on cerebral protein synthesis have been undertaken
in order to examine the potential usefulness of the leucine method.
825
PHS 6040 (Rev. 1/84)
CPO SI4-9I*
Z01 MH 00889-08 LCM
Project Degcrlption;
Major Objectives:
The overall objectives of this research project are:
1 . To develop a method for the measurement of local rates of protein synthesis
in brain.
2. To test the usefulness of the method in the study of neurobiological
problems.
A method is being developed for the estimation of local rates of protein
synthesis in brain ui vivo. This method is similar to the [ ^ ^C jdeoxyglucose
method in that it is based on enzyme kinetic principles as applied to the
measurement of reaction rates i_n vivo with labeled tracers as substrates. The
two requirements for this type of method are: 1) label must be primarily in the
form of labeled product (labeled protein) at the end of the experiment, and 2) a
model for the behavior of the precursor in brain in order to calculate the
integrated specific activity of the precursor at the site of the reaction in
terms of measurable variables. In order to satisfy the first requirement,
[l-^ ^CJL-leucine has been chosen as the labeled tracer. The only metabolic
pathway for leucine, apart from protein synthesis, entails a transmination
followed by a decarboxylation. Therefore, in the metabolism of [ 1-^ ^C Jleucine
the label is transiently transferred to a-ketoisocaproic acid and ultimately to
^^CQ2 which is then diluted in the large CO2 pool in brain produced by
carbohydrate metabolism. There are, therefore, no residual radioactive products
other than labeled protein. In order to meet the second requirement we have
designed kinetic models for the behavior of leucine in brain. All of these
models are based on the following assumptions and requirements:
1 ) Homogeneous tissue compartments
2) No isotope effect
3) No release of labeled leucine from labeled protein during the
60-minute experimental period
M) Complete loss of label from metabolic degradation of leucine
5) Steady state for unlabeled leucine
We have developed six such models, and by mathematical analysis we have derived
operational equations for all of them. The models are of progressively
increasing complexity as regards the number of compartments and the
interrelationships among them. Regardless of the degree of complexity of the
model used, the operational equation has the same general form. The numerator
is composed of the total amount of radioactivity in the tissue at the end of the
experiment minus term(s) for the lag between the precursor pool and the plasma.
Optimal design of the experimental procedure allows us to reduce this expression
to the total radioactivity in the tissue divided by the integrated plasma
specific activity. The terms in the numerator for the free leucine pools can be
eliminated by fixing and washing tissue sections. The expression for the lag in
the denominator can be made very small by administering a pulse of [^^cjleucine
826
ZOl MH 00889-08 LCM
and allowing 60 minutes, estimated to be more than 10 half-lives of the
precursor pool in white matter, for clearance of the free pool.
The specific aims pursued during this fiscal year were:
1. To determine the relative contribution of plasma leucine to the precursor
pool for protein synthesis, and
2, To apply the [^^c]leucine method to the study of development and other
neurobiological problems.
Methods Employed:
1. The determination of the relative contribution of plasma leucine to the
precursor pool for protein synthesis.
The experiment consists of the determination of the specific activity of brain
leucyl-tRNA and plasma leucine in a rat in a steady state for both labeled and
unlabeled leucine in the plasma. If all of the leucine is derived from the
plasma, the specific activity of the leucyl-tRNA will eventually reach that of
the plasma. If there is a significant contribution of leucine from protein
degradation, the ratio of the specific activities of leucyl-tRNA and plasma
leucine will be a measure of the fractional contribution from plasma. A
programmed intravenous infusion schedule for [^HJleucine was designed that would
produce and maintain a constant concentration of [Sfijleucine in the arterial
plasma. This input function was obtained from a Laplace tansform of the
relationship between a pulse input of [SHJIeucine and the multiexponential
output. The controlled infusion was achieved by means of a programmable
infusion pump. Rats were maintained under a steady state for both labeled and
unlabeled leucine for at least 30 minutes at which time they were guillotined
and the brains and livers were rapidly removed. Brain and liver tRNA was
separated and purified by differential centrifugation, and acid and phenol
extraction techniques. Purified tRNA was deacylated at pH 10 and the amino
acids were separated from the RNA by ethanol precipitation. The specific
activity of the leucine in this amino acid fraction and the specific activity of
the plasma samples were determined as follows: The amino acids were derivatized
with [''^CJdansyl chloride. The dansylated neutral amino acids were separated
from other products of the dansylation reaction by thin-layer chromatography.
Dansyl leucine was separated from other dansylated neutral amino acids by HPLC
with a CI 8 column. The dansyl leucine peak was collected and counted for both
^'^C and 3h. The ratio of 3h to ^ ^C is a measure of the specific activity of the
leucine .
2. Studies of neurobiological problems.
Local rates of cerebral protein synthesis were determined with the
r^_l'<c]leucine method (Smith et al . , J. Neurosci. _4: 2489-2496, 1984).
827
ZOl MH 00889-08 LCM
Major Findings:
1 . The determination of the relative contribution of plasma leucine to the
precursor pool for protein synthesis.
Results obtained in 6 experiments show that in brain 50-60? of the leucine pool
which serves as the precursor pool for protein synthesis is derived from the
plasma whereas in liver about kO% of the tRNA leucine pool is derived from the
plasma. These results indicate that protein turnover will have to be taken into
consideration in future studies of protein synthesis. This is the first time
that the relative contribution of the plasma leucine to the tRNA amino acid pool
has been determined in either brain or liver _in vivo. Because these results are
of Interest in and of themselves they will be reported separately.
2. Studies of neuroblological problems.
In order to examine the potential usefulness of the leucine method, studies of
neuroblological problems are being pursued with the assumption that the relative
contribution of plasma leucine to the precursor pool for protein synthesis does
not change with the experimental conditions. We have determined local rates of
protein synthesis in normal, conscious male rats with Dr. Charles Kennedy. The
course of normal development in Rhesus monkeys is also being examined. In
addition, studies of slow wave sleep in Rhesus monkeys have been undertaken.
Dr. Elntrei is Investigating the effects of anesthesia on local rates of protein
synthesis in rats. Experiments have been carried out on animals under either
light barbiturate anesthesia or ketamlne anesthesia. The autoradiographic
results of these experiments are now being analyzed. Studies have been carried
out in collaboration with Dr. Kadekaro at the University of Texas on the effects
of repetitive stimulation of the sciatic nerve on local rates of protein
synthesis in the spinal cord and the dorsal root ganglia. The autoradiographic
results of these experiments are bein'g analyzed. Studies of the effects of
thyroidectomy in Infant rats on cerebral protein synthesis have been carried out
with Dr. Dow-Edwards at SUNY. The results of these experiments are being
analyzed. A collaboration effort with Dr. Robert Abrams at the University of
Florida to study the developmental time course of protein synthesis in fetal
sheep brain has been Initiated. Cerebral protein synthesis rates measured in
two fetal sheep at 130 days gestation are exceedingly high (range 15-25moles
leucine/g/min) .
Significance to Biomedical Research and Program of the Institute:
Protein synthesis is probably the most important biochemical process underlying
the development, maturation, plasticity, maintenance, and long-term regulation
of the nature and degree of functional activity of the nervous system. The
structural, functional, and metabolic properties of the tissues largely reflect
the role of structural and enzymatic proteins. Peptides that are considered to
be neurotransmitters are in some, and possibly all, cases derived from the
cleavage of large parent protein molecules. Many hormones within and outside
the nervous system are proteins. It is, therefore, certain that changes in
protein synthesis can and do alter function and that some mental and
neurological dysfunctions reflect disturbances in this vital biochemical process.
828
ZOl MH 00889-08 LCM
This research is directed at determining the rates of protein synthesis in
specific regions of the central nervous system with an ultimate resolution down
to the cellular level. This provides for the first time the opportunity to
study at the individual structural or anatomical level the changes in protein
synthesis that may be the causes, consequences, or correlates of normal
conditions, such as maturation, plasticity, differentiation, sleep, learning and
memory, behavioral patterns, etc., or pathological conditions, such as hormonal
disorders, aging, regeneration in response to injury, convulsive disorders,
coma, etc.
Proposed Course:
Additional experiments will be carried out in order to determine by an
independent analytical method the relative contribution of plasma leucine to the
brain t-RNA pool. The Laboratory has just purchased a new Beckman amino acid
analyzer which has the capability of detecting picomolar levels of amino acids.
With the assistance of Gladys Deibler we are setting up this instrument to
separate and detect the tRNA pool amino acids derived from rat brains in a
steady state for both labeled and unlabeled leucine.
Applications of the ['' '^Cjleucine method to studies of the effects of anesthesia
on nerve stimulation, sleep, development, and hypothyroidism will be continued.
Studies of the effects of testosterone on regeneration will be carried out in
collaboration with Dr. Amy Yu, City University of New York Medical School.
Publications;
Holcomb, H.H., Links, J., Smith, -C, and Wong, D. : Positron emission
tomography: Measuring the metabolic and neurochemical characteristics of the
living human nervous system. In Andreasen, N.C. (Ed.): Brain Imaging:
Implications for Psychiatry. Washington, D.C., Amer. Psychiatric Press, 1987,
(in press) .
829
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00903-10 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on arte line between the borders.)
Purification and Identification of Brain Proteinases and their Cleavage Products
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Gladys E. Deibler Research Chemist LCM, NIMH
Others:
Marian W. Kies
Audrey Stone
Chemist
Guest Researcher
LCM, NIMH
LDMI, NICHHD
COOPERATING UNITS (if any)
Multiple Sclerosis Research Center; Dept. of Neurology, Georgetown University
(J. Richert)
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.5
PROFESSIONAL:
1.5
1.0
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our exhaustive review of the many papers on brain proteinases led us to choose
calcium activated neutral proteinases (CANP I, II and III) as -the brain enzymes
that we will investigate. CANP I is located in the neurons, CANP II is located in
the glia cells, and CANP III has not been associated with a particular structure.
Presently, we are developing the specific analytical methods needed to extract,
purify, characterize and assay the CANF's.
Our previous investigation of human myelin basic protein (HBP) led to the
identification and characterization of a new form of HBP with a molecular weight
of 17.2 kDa and highly purified HBP-component 1, the unmodified 18.5 kDa protein.
Highly purified HBP-component 1 was cleaved with thrombin and the resulting two
peptides (1-97 & 98-170) were purified and characterized. HBP-component 1, 17.2
kDa HBP and the two thrombic peptides (residues 1-97 & 98-170) were used in
collaboration with Dr. John Richert, Dept. of Neurology, Georgetown University.
Forty myelin basic protein-reactive T cell clones were isolated from a multiple
sclerosis patient and used to identify human T cell recognition sites on the HBP
molecule. At least three sites have been identified: One in the N-terminal half
of the molecule (residues 1-97), one in the C-terminal (residues 98-170), and one
which spans residues 97-98.
In collaboration with Dr. Audrey Stone, we have completed the investigation on the
role of phosphorylation on the conformational adaptability of bovine myelin basic
protein (MBP). The limited digestion of MBP-components 2+3 yielded a mono-
phosphorylated component which was phosphorylated only at threonine 97. From the
circular dichroism of this homogeneous mono-phosphorylated MBP, we determined that
the g-structure of this protein was increased by 7%, when compared with
MBP-component 1 . The single phosphorylation on threonine 97 produced a change in
the macrostructure of the protein involving about 12 amino acid residues.
PHS 6040 (Rev. 1/84)
GPO SI4-91B
Z01 MH 00903-10 LCM
Project Description;
Objectives:
The completion of the determination of the T cell active sites of human myelin
basic protein.
The isolation and purification of the calcium activated neutral proteinases and a
study of their bond specificity with known CNS proteins and/or peptides as
substrates.
Methods Employed:
Ion-exchange, affinity and size-exclusion chromatography, FPLC, HPLC, amino acid
analysis and polyacrylamide slab gel electrophoresis were used in all our previous
studies and will be used in our new project. A mini slab SDS polyacrylamide
electrophoresis method and the Bradford Protein Assay have been developed for our
new project. HPLC procedures are being investigated for the identification of
digestion products of CANP.
Methods and programs for the identification and quantitation of methionine
sulfoxide, methionine sulfone, N^N^'dimethylarginine and monomethylarginine are
being developed for our new 7300 Beckman amino acid analyzer.
Major Findings:
Our discovery that thrombin will not cleave the bond between arginine-98 and
threonine-97, when the latter is phosphorylated, made it possible for us to obtain
a homogeneous mono-phosphorylated MBP-component 3. The analysis of the CD spectra
of this component, and components 1 and 2 indicates that the phosphorylation of
threonine-97 promoted a 7% increase in the g-structure of the polypeptide chain.
The isolation of T cell clones reactive to HBP from a patient with multiple
sclerosis proves the relevance of HBP as a potential autoimmune target in human
demyelinating disease. Analysis of reactive T cell clones revealed at least three
different antigenic determinants: one in the N-terminal (1-97) half of the
molecule, one in the C-terminal (98-170) and one which spans residues 97-98. More
clones were reactive to the C-terminal half of the molecule. All clones were
reactive to the 17.2 kDa HBP. This means that the deletion of Exon 5 (residues
106-116) was not a part of an antigenic site. This is the first successful
cloning of BP-reactive cells specific to different regions of the molecule.
Significance to Biomedical Research and the Program of the Institute:
The forty myelin basic protein (HBP)-reactive clones will be useful for both the
further delineation of the human T cell recognition sites on HBP and the
generation of anticlonotypic monoclonal antibodies which might be used in the
determination of the active site in a demyelinating disease.
Phosphorylation of threonine-97 has been shown to increase the ordered
macrostructure of bovine myelin basic protein. These data may contribute to
832
ZOl MH 00903-10 LCM
understanding the role of phosphorylation of MBP in the development and
stabilization of the myelin sheath.
Proposed Course:
The continued collaboration with Dr. John Richert to determine all of the HBP
reactive T cell sites.
The extraction, purification and characterization of CANP I, II, III and the
determination of their cleavage sites in brain proteins.
Publications;
Kira, J., Bacon, M.L., Martenson, R.E., Deibler, G.E., Kies, M.W., and Alvord,
E.G., Jr.: Experimental allergic encephalomyelitis in rabbits. A major
encephalitogenic determinant within residues 1-44 of myelin basic protein. J.
Neuroimmmunol. 12: 183-193, 1986.
Alvord, E.G., Jr., Hruby , S., Martenson, R.E., Deibler, G.E., and Law, M.J.:
Evidence for specific polypeptide chain folding in myelin basic protein from
reactions between fragments of the protein and monoclonal antibodies. J.
Neurochem. 4?: 754-771, 1986.
Deibler, G.E., Krutzsch, H.C., and Kies, M.W.: A new form of myelin basic protein
found in human central myelin. J. Neurochem. 47: 1219-1225, 1986.
Richert, J.R., Rueben-Burnside, C.A., Deibler, G.E., and Kies, M.W. : Peptide
specificities of myelin basic protein-reactive human T cell clones. Neurology,
in press, 1987.
833
PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
ZOl MH 02216-04 LCM
TITLE OF PROJECT (80 characters or less, rule must lit on one line tietween We tmrders.)
Metabolic Mapping of the Brain during Rewarding Self-Stimulation
PRINCIPAL INVESTIGATOR (List other prolessional personnel tielow the Principal Investigator) (Name, title, laboratory, and institute altiliation)
P.I.: Linda J. Porrino Guest Researcher LCM, NIMH
Others: Louis Sokoloff Chief LCM, NIMH
COOPERATING UNITS (H any)
None
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.00
PROFESSIONAL:
1.5
OTHER:
0.5
CHECK APPROPRIATE B0X{ES)
D (a) Human subjects D (b) Human tissues ^ (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The deoxyglucose method is being used to study alterations in local cerebral
glucose utilization resulting from rewarding electrical brain self-stimulation
to discrete brain sites as well as resulting from the administration of drugs of
abuse. By mapping metabolic activity in rats under these conditions, information
can be obtained about those areas of the brain involved in motivation and
re in for c emen t .
835
PHS 6040 (Rev. 1/84)
GPO •<4-»l(
ZOl MH 02216-04
Project Description:
Objectives:
The goal of the present project is to map those regions of the rat brain
activated during positive reinforcement processes. Both rewarding electrical
brain stimulation and the administration of psychostimulants known to produce
rewarding effects are being studied.
Methods Employed:
The 2-[^ ^Cjdeoxyglucose method affords a novel and unique opportunity to map
functional neural pathways simultaneously in all anatomical components of the
central nervous system. This method, therefore, allows the identification of
complex neural circuits that are functionally active during various behavioral
and pharmacological manipulations.
The goal of the present project is to map those regions of the rat brain
activated during positive reinforcement processes. Both rewarding electrical
brain stimulation and the administration of psychostimulants known to produce
rewarding effects are being studied.
Major Findings:
The results of previous experiments in this series indicate an involvement of
the nucleus accumbens, medial thalamus and prefrontal cortex in reinforcement.
These data, however, are confounded to a degree by differences in the motor
behavior of the compared groups - i.e. lever-pressing vs. sitting quietly. In
order to eliminate the motor component and to distinguish better between motor
and reinforcement effects, the following experiment was conducted. The standard
protocol for the 2-DG method was applied to four groups: 1) intracranial
self-stimulation (ICSS) prior to 2-DG in stimulation chamber, 2) experimenter
administered stimulation prior to 2-DG in stimulation chamber, 3) no stimulation
prior to 2-DG in same chamber, and 4) ICSS prior to 2-DG in home cage. All
animals received no stimulation for the 2 sessions just prior to and during the
2-DG procedure. Since the animals in all groups were stimulated during the
measurement of glucose utilization, the only difference between the groups was
their stimulation history.
Preliminary analysis of these data indicates that in the large majority of brain
regions, rates of glucose utilization were identical, a similarity that can be
considered a reflection of the similarity in the behavioral states of the
animals in all groups. There were, however, differences in a small number of
structures, the prefrontal cortex and nucleus accumbens, in which rates of
energy metabolism were higher in the ICSS group tested in the chamber in which
they had had ICSS experience than rates of rats in other groups. These
differences cannot be attributed to stimulation effects, as groups 2 and 4 also
received similar amounts of electrical stimulation, either ICSS or
experimenter-administered. Although these results are highly preliminary, they
suggest that these areas are important for the expression of conditioned
positive reinforcement.
836
ZOl MH 0221 6-OM
Proposed Course:
The application of the 2-deoxyglucose method to studies of reinforced behavior
will be extended to include behavior reinforced by other reinforcers other than
drugs and electrical stimulation. The effects of the administration of other
drugs of abuse that are considered highly rewarding such as phencyclidines is in
progress .
Publications:
Porrino, L.J.: Using the quantitative 2-[ ^ ^C jdeoxyglucose method for metabolic
mapping of the brain during reinforced behavior. In Dahlstrom, A. (Ed.):
(Proceedings of the VI International Catecholamine Symposium, Jerusalem, Israel,
June 14-19, 1987), (in press).
837
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 02217-04 LCM
TITLE OF PROJECT (80 characters or less. Title must fit on one line tietween the borders.)
Plasticity in the Developing Monkey Visual System
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Carolyn B. Smith Research Chemist LCM, NIMH
Others: Louis Sokoloff
Karen D. Pettigrew
Susan Herdman
Chief
Res. Math. Statistician
Guest Researcher
LCM, NIMH
TSMB, NIMH
LCM, NIMH
COOPERATING UNITS (if any)
Department of Neurology, Johns Hopkins Medical School, Baltimore, MD (R. Tusa)
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.8
PROFESSIONAL
0.5
0.3
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) interviews
n (b) Human tissues B (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The postnatal development of the central visual pathways depends on the quality of
the Visual environment. During the critical period in the primate visual system
environmental manipulation can modify the physiological properties of visual
cortical cells. The purpose of this project is to study the underlying
biochemical events that imbue the nervous system with the property of plasticity.
Protein synthesis is a biochemical process which is involved in bringing about
changes in morphology, adjustments in growth rates, and remodeling and maintenance
of structures. We have, therefore, used the [^ ^CJleucine method to study the
relationships between local plastic changes which occur in the developing monkey
visual system and local rates of protein synthesis.
839
PHS 6040 (Rev. 1/84)
SPO BI4.9II
ZOl MH 02217-01^ LCM
Project Description:
Objectives:
The purpose of this project is to study the biochemical events associated
with plasticity. The developing rhesus monkey visual system has been chosen
as a model system because the physiological and anatomical responses to
deprivation have been so well described by others. We have focussed
initially on the process of protein synthesis because it is a requirement
for growth and development and because changes in morphology and rates of
growth and remodeling and even maintenance of existing structures should be
reflected in changes in rates of protein synthesis.
The postnatal development of the central visual pathways depends on the
quality of the visual environment. During the critical period alterations
in the conditions of visual input can modify the physiological properties of
visual cortical cells. If a monkey is monocularly deprived during the
first few weeks of life there is a reorganization of the striate cortex such
that the ocular dominance columns representing the functioning eye extend
beyond their boundaries and broaden at the expense of the adjacent columns
representing the deprived eye. Eventually, most of the striate cortex may
be incorporated into a monocular visual system that serves only the deprived
eye. The organization of the lateral geniculates (dLGN), the locus of the
cell bodies of the terminals in striate cortex, remains normal.
Our specific aims for this fiscal year were:
1 . To determine the effects of acute monocular occlusion on local rates of
protein synthesis in the laminae of the dLGN in 25 and 50 day old rhesus monkeys.
These animals will serve as controls for our chronic monocular deprivation
groups. The addition of these 2 groups of animals will make it possible to test
for the statistical significance of changes with chronic deprivation.
2. To determine the effects of chronic binocular deprivation of local rates of
protein synthesis in 25 day old monkeys. These experiments were designed to
examine whether the changes found in local rates of protein synthesis with
chronic monocular deprivation are the causes or the consequences of the
reorganization which takes place in the striate cortex.
3) To examine the effects of our reverse-suture procedure on eye movements.
During the course of the experiments in which monkeys were reverse-sutured it
was noted that a nystagmus develops during the second 25 days. In collaboration
with Dr. R. Tusa, Johns Hopkins Medical School, eye movements are being recorded
in these animals.
Methods Employed:
Local rates of cerebral protein synthesis were determined with the
[l-'''^c]leucine method (Smith et al., 198iJ).
840
ZOl MH 02217-04 LCM
Unilateral or bilateral tarsorrhaphies were performed under ketamine anesthesia
on newborn and 25 day old rhesus monkeys. In some animals a reverse-suture
paradigm was used, i.e., unilateral lid suture was performed at birth and at 25
days the sutured eye was opened and the other eye was closed.
Eye movements were recorded with scleral search coils that were implanted at the
time of the reverse-suture.
Major Findings:
In our early studies the effects of acute and chronic monocular deprivation in
the newborn rhesus monkey on rates of protein synthesis in the laminae of
the dLGN were examined. The results showed that in newborn monkeys acute
monocular deprivation produced no differential changes in rates of protein
synthesis in any of the dLGN laminae. Chronic monocular deprivation (from
0-25 days) resulted in decreases of about ^5% in the rates of protein
synthesis in the laminae innervated by the deprived eye, whereas in
geniculate laminae innervated by the functioning eye rates of protein
synthesis were normal as compared with monkeys with binocular vision. In
order to test the statistical significance of these effects we have
completed another control group of animals, i.e. age-matched monkeys with
one eye occluded acutely. The rates of protein synthesis determined in the
six laminae of left and right LGN in acutely and chronically monocular occluded
monkeys were analyzed by Dr. Pettigrew with a 3 factor analysis of variance
with repeated measures. The results show a highly significant (p <.001)
interaction of group (acute/chronic) and condition (deprived/nondeprived) . All
other interactions and all main effects were not significant. This analysis
shows that chronic monocular occlusion results in a significantly different
pattern of protein synthesis in the LGN as compared with acute monocular
occlusion. Rates of protein synthesis are decreased in the deprived eye laminae
of the LGN and unchanged in the nondeprived eye laminae. These effects on
protein synthesis occur when the deprivation is begun at a point in the critical
period when there is considerable overlap of the representation from the two
eyes in layer IV of striate cortex. These results suggest that the
underdevelopment of the deprived columns in striate cortex may be the result of
inadequate growth and/or maintenance of axon terminals with consequent default
of synaptic connections to the normally maintained terminals of the functional
pathway.
In experiments in which monocular deprivation is carried out later in the
critical period (from 25-50 days) when there is less overlap in striate
cortex, the primary effect is on the nondeprived laminae of the dLGN. In
these experiments the nondeprived laminae have increased rates of protein
synthesis in comparison to normal, age-matched controls with binocular
vision, and the deprived laminae are unchanged. In order to test the
statistical significance of these results we have completed an additional
control group, i.e. age-matched monkeys with one eye occluded acutely.
These experiments have been carried out and are currently being analyzed. The
initial results indicate that there is no significant difference in rates
of protein synthesis in the laminae of the dLGN between the 50 day old
animals with binocular vision and those with acute monocular occlusion.
Thus, the reorganization that occurs as a result of the monocular
841
ZOl MH 02217-OiJ LCM
deprivation from 25-50 days may require a more active process than the
default that was seen when monocular deprivation was begun at birth.
In order to determine if the changes in protein synthesis are the causes or
the consequences of the reorganization of striate cortex, we have studied
the effects of binocular deprivation. Physiological experiments have shown
that binocular deprivation during the critical period has no effect on the
organization of striate cortex. Rates of protein synthesis were determined
in the 6 laminae of the dLGN in 10 25-day old monkeys with normal binocular
vision and 8 25-day old binocularly deprived animals. The results were
analyzed by Dr. Pettigrew with a 2-f actor analysis of variance with repeated
measures. The results show no significant interaction between groups and
laminae, no significant main effect of group and a significant effect of
laminae (P<.001), i.e. laminae 2 has a lower rate of protein synthesis than
the other 5 laminae. The results of this analysis show that there is no
significant effect of chronic binocular deprivation from 0-25 days on the
rates of protein synthesis in the dLGN. The results of these experiments
were presented at the 1987 meeting of the International Symposium of
Cerebral Blood Flow and Metabolism.
During the course of the experiments in which monkeys were reverse-sutured it
was noted that a nystagmus develops during the second 25 days. In collaboration
with Dr. R. Tusa, Johns Hopkins Medical School, eye movements are being recorded
in these animals. In one experiment eye movements were normal on Day 25 at the
time of the reverse-suturing. Eye movements became progressively abnormal
during the ensuing 25 days and resembled somewhat the nystagmus associated with
congenital blindness in humans. A second animal is being studied under these
conditions. Eye coils were implanted in both eyes and the progressive
development of the nystagmus is being followed. The abnormal movements are
conjugate and did not decrease in intensity when both eyes were opened. These
studies indicate that this is a good experimental model of congenital nystagmus.
A manuscript of these results is in preparation.
Significance to Biomedical Research and the Program of the Institute;
Plasticity, the capacity of the nervous system to respond to changes in the
environment, is one of the most fundamental properties of nervous tissue.
Learning, a form of plasticity, is a process of intense interest to
neuroscientists the world over. In an attempt to study some of the biochemical
processes underlying plastic changes, we have embarked on this study of the
developing monkey visual system about which the physiology and anatomy are well
known. Studies with the [^^Cjleucine method for local rates of protein
synthesis and the [^ ^Cjdeoxyglucose method for local rates of glucose
utilization are directed at first a description of some of the biochemical
events which occur and then a determination of the regulation of these events.
The understanding of these events may provide some insight into the unique
properties of the critical period which make it so responsive to environmental
manipulation. In addition, this research may have some direct implications on
the clinical management of children with congenital cataracts and strabismic
amblyopia.
842
Z01 MH 02217-04 LCM
Proposed Course;
The recent experiments on acute monocular occlusion will be analyzed, the
statistical analyses will be carried out and manuscripts will be prepared.
Experiments on the congenital nystagmus model (in collaboration with Dr. Tusa)
will be continued. Control experiments such as binocular deprivation will be
carried out. Strabismus produced at birth by surgical section of ocular muscles
in conjunction with monocular deprivation will be examined for its ability to
produce the abnormal eye movements.
Publications;
None
843
DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 02220-04 LCM
TITLE OF PROJECT (80 characters or less. Title mt/s( tit on one line tjetween the borders.)
Regional Biochemical Changes in the Normal Aging Brain
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
P.I.: Carolyn B. Smith Research Chemist LCM, NIMH
Others ;
Louis Sokoloff
Ernesta Palombo
Chief
Visiting Fellow
LCM, NIMH
LCM, NIMH
COOPERATING UNITS (if any)
Department of Physiology-Anatomy, University of California, Berkeley, CA
(M. C. Diamond)
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.5
PROFESSIONAL;
0.4
OTHER:
0.1
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
n (a2) Interviews
□ (b) Human tissues H (c) Neitiier
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies have been carried out on the effects of aging on cerebral protein
synthesis and glucose utilization in rats. With the application of local methods
developed in this Laboratory, discrete regions of the brain can be examined in
normal conscious animals. The regional changes in glucose utilization indicate
that entire sensory pathways are affected by the aging process. The fact that
similar changes are found in the same pathways with respect to protein synthesis
suggests that some of these changes reflect an adaptation of the nervous system to
a chronic lack of input. Our findings of age-dependent decreases in glucose
utilization in the striatum have been followed up with studies of the effects of
aging on the metabolic responsiveness to the dopaminergic agonist apomorphine.
845
PHS 6040 (Rev. 1/84)
CPO B1 4.9 1 •
Z01 MH 02220-OM LCM
Project Description:
Objectives:
The overall purpose of these studies is to examine the effects of normal aging
in rats on rates of regional metabolic processes in the brain. In our previous
studies, decreases in rates of glucose utilization with age were found in the
brain as a whole. On a local level senescent decreases in glucose utilization
were found with the most profound effects in the components of the primary
auditory and visual pathways. These were effects similar to those seen
following acute sensory deprivation of these systems. These results raised the
question of whether or not some of the central nervous consequences of normal
aging are due to sensory deprivation due to sense-organ degenerative changes,
inasmuch as there is known to be some retinal and inner ear degenerative change
with age. With only a few exceptions, the rates of glucose utilization in
structures of the limbic and motor systems remained unchanged with age. The
exceptions were several regions of white matter and the caudate-putamen.
Measurements of energy metabolism do not differentiate between the immediate
functional demands of cerebral structures and the longer term maintenance
processes within the nervous system. Long term effects that are related to
changes in morphology, structural maintenance, and remodeling in the nervous
system are more likely reflected in biosynthetic biochemical processes, such as
protein synthesis. In another study the effects of aging on local rates of
protein synthesis in brain were examined by means of the quantitative
autoradiographic [ ^ '^C Jleucine method. The results show that aging is associated
with significant decreases in rates of protein synthesis in the brain as a whole,
as well as in several specific brain regions. Brain regions involved in visual
and auditory function were selectively affected, perhaps due to a chronic lack
of sensory input. Several regions involved in motor function and two areas in
the limbic system had significantly decreased rates of protein synthesis in the
old rats. Notably, there was a significant age-related decrease in protein
synthesis in the locus coeruleus which contains the cell bodies of origin of the
major ascending noradrenergic innervation of the cortex.
There were two major objectives of the ongoing work during this fiscal year: 1)
To study the functional consequences of senescent changes in the nigrostriatal
dopaminergic system by determining the effects of normal aging on the metabolic
responsiveness of dopamine-receptor activation of systemically-administered
apomorphine, and 2) To examine the effects of environmental enrichment in adult
rats on resting local rates of cerebral glucose utilization.
Methods Employed:
Studies of the effects of age on the metabolic responsiveness to apomorphine:
In this study Fisher 3^14 male rats were obtained from the colony maintained by
the National Institute on Aging. Three age groups were studied: young adults,
4-6 months of age; middle-aged rats, 14-16 months of age; and old rats, 23-25
months of age. The ['' '^C ]deoxyglucose method (Sokoloff et al., 1977) was used to
determine local rates of cerebral glucose utilization. Rats were administered
with apomorphine at 0.5 mg/kg, 1.5 mg/kg or 5.0 mg/kg or normal saline vehicle
846
ZOl MH 02220-Oil LCM
10 minutes before the administration of [ ^ ^CJdeoxyglucose. Behavioral and
physiological responses were monitored throughout the study.
Major Findings:
Effects of age on metabolic responsiveness to apomorphine:
The results of our pilot study showed significant dose-dependent effects of
apomorphine in 6 of the 14 brain regions examined in the young rats. In the
lateral habenula and anterior cingulate cortex the effect of apomorphine was to
decrease the rate of glucose utilization, whereas in the subthalamic nucleus,
inferior olivary nucleus, substantia nigra (pars compacta), and substantia nigra
(pars reticulata), apomorphine stimulated glucose utilization. Age-dependent
changes in responsiveness to apomorphine were found in the subthalamic nucleus,
substantia nigra (pars reticulata), and inferior olivary nucleus. In the
subthalamic nucleus the stimulation of glucose utilization by apomorphine was
decreased in the old rats at all doses, including those that elicited maximal
responses. In the inferior olivary nucleus and the substantia nigra (pars
reticulata), the dose-response curves were markedly depressed in the aged group.
No significant effect of apomorphine on the rate of glucose utilization was
found in the caudate-putamen as a whole, but a significant stimulation of
glucose utilization was found only in the ventral portion in the young animals.
The significant age-dependent decreases in responsiveness to apomorphine found
in the subthalamic nucleus, substantia nigra (reticulata), and inferior olivary
nucleus may reflect the functional consequences of the reported loss of dopamine
receptors in the caudate-putamen with aging. Because the results of this pilot
study were promising, a complete study has been undertaken.
Studies have been completed on 72 rats. The integrated plasma specific activity
of deoxyglucose has been calculated for each of the animals. The
autoradiographs of brain sections have been prepared. The resulting 216 films
are being analyzed with the aid of our image processing system. Rates of
glucose utilization are being determined in 42 brain structures and in the
brain, as a whole. When all the results have been tabulated, a dose-response
curve for the effects of apomorphine on local rates of cerebral glucose
utilization will be constructed for each age group, and each brain structure
examined.
Effects of Environmental Enrichment on Local Rates of Cerebral Glucose
Utilization:
The work of Rosensweig, Diamond and others has shown that environmental
enrichment can result in histological changes in the cerebral cortex of adult
rats. As environmental enrichment may be the converse of sensory deprivation,
we have examined the effects of enrichment on local rates of cerebral glucose
utilization. In our studies, results from four "enriched" rats were compared
with results from six "standard" rats. Brain regions examined included eight
cortical areas, nine limbic areas, ten other subcortical areas, two regions of
white matter and the reticular formation. Of the 30 brain regions examined,
there were no significant changes in 28 of these. There were no significant
increases in any of the regions examined in the "enriched" animals. In both
frontal and parietal cortex, rates of glucose utilization were decreased
847
Z01 MH 02220-OiJ LCM
increages in any of the regions examined in the "enriched" animals. In both
frontal and parietal cortex, rates of glucose utilization were decreased
(p .05) by 135S in the "enriched" rats. Whether or not these effects are random
events, or are actually due to the experimental conditions, can only be
determined by a second series of experiments. This work was presented at the
November 1986 Annual Meeting of the Society of Neuroscience.
Significance to Biomedical Research and the Program of the Institute:
Insofar as aging is rapidly becoming a problem of increasing social significance,
this research, which is focused on senescent changes in the ability of the brain
to function, may be of considerable importance to the medical community.
Furthermore, our results indicate that some of the changes that occur with age
may be the consequence of a decreased functional activity. Confirmation of this
possibility, and further understanding of the basic biochemical processes
underlying plastic changes in the nervous system of either an involutional or
developmental nature, may be useful in trying to prevent and/or reverse such
senescent changes.
Proposed Course:
Completed experiments on the effects of aging on the metabolic responsiveness to
apomorphine are currently being analyzed. When the analysis is completed a
manuscript of this work will be prepared.
Publications :
None
848
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 02308-02 LCM
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.;
Growth and Development of Dopaminergic Neurons
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Bernard F. Driscoll
Research Biologist
LCM, NIMH
COOPERATING UNITS (It any)
None
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
NIMH, Bethesda, Maryland 20892
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20E
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
1.0
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues (xl (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Development of central nervous system pathways involves the action of soluble
factors on target cells and interactions between various cell types. One of the
best defined CNS pathways is the nigrostrlatal pathway. To examine events
involved in the formation of this pathway, development of dopaminergic neurons
from the embryonic rat mesencephalon was examined in dissociated cell cultures.
Cells were grown in serum-free medium or serum-containing medium in the presence
or absence of the basement membrane component, laminin. Mesencephalic cells were
obtained from embryonic day 13 to embryonic day 15 rat brains; cells from other
regions were obtained from embryonic or postnatal rat brains at times when
subsequent in vitro development was shown to be optimal. Development of the
dopaminergic neurons in the mesencephalic cultures was determined by uptake of
exogenous, labelled dopamine.
Mesencephalic dopaminergic neurons grown alone developed vigorously in culture for
9 days. After this time development ceased. In contrast, if mesencephalic
neurons were grown in the presence of cells from other brain regions, the cultures
continued to develop to day 20 or day 27. Glial cells from these other brain
regions appeard to be important for the continued development of the mesencephalic
neurons. There also appeared to be a later stage of development or cell survival
that was dependent on the presence of specific target cells in the culture.
Striatal neurons are the normal iri vivo target for most of the mesencephalic
dopaminergic neurons. Cells from the striatum provided optimum i_n vitro
conditions for development of the dopaminergic neurons.
849
PHS 6040 (Rev. 1/84)
SPO X4-S
Z01 MH 02308-02 LCM
Project Description:
Objective:
To determine the factors Involved In development of central nervous system
pathways by observing survival and development of neurons In dissociated cell
cultures.
Methods;
Cells were prepared from specific regions of embryonic day (ED) 13 to postnatal
day 8 rat brains. Five specific regions were used; the ability of cells from
any particular region to grow i^ vitro is dependent on the time in embryonic
development when they are removed. Therefore, cells must be obtained from
embryos or neonates of various ages.
The cells were cultured in vitro in either serum containing medium (SCM-10?
fetal calf serum) or in serum-free medium (SFM). Use of SFM allows for greater
control over factors to which the cells are exposed. On the other hand,
cultures in SCM are morphologically different than those in SFM; this provides
additional conditions under which to examine the cells' development.
We are interested in assessing the development of the mesencephalic dopaminergic
neurons, and in particular the effect of target (striatal) neurons on this
development. The degree of development was determined by quantitating the level
of dopamine uptake by the reuptake system which is present in all dopaminergic
neurons. The uptake of ^H-labelled dopamine was measured by liquid
scintillation counting or (for qualitative morphologic studies) by
autoradiography. To identify and quantitate the cell types present,
representative cultures were fixed, permeabilized and exposed to antibodies
which recognize various cell-type specific antigens, particularly antigens
present in neurons as astroglia. After exposure to a second antibody containing
a fluorochrome, the cultures were examined by fluorescence microscopy.
Major Findings:
One reason for culturing cells in SFM is to control the proliferation of
astroglia. In SCM, these cells, if unchecked by mitotic inhibitors, will
completely overgrow the neurons. In SFM, the proliferation of astroglia is
inhibited presumably due to the lack of growth factors normally present in fetal
calf serum. Surprisingly, when the SFM cultures were examined for the presence
of astroglia using an antibody to the glial-specific antigen glial fibrillary
acidic-protein (GFAP), glia were found to be present in almost all cultures at
d-9. More important was the observation that the amount of GFAP was increased
in cultures at d-20 and even more so by d-27. We do not know whether the
increase in GFAP represents astroglial proliferation or process development.
Regardless, while astroglia do not overgrow these cultures, they do represent an
unexpectedly large component of the cultures grown in SFM.
Like all neurons in the CNS, the mesencephalic dopaminergic neurons can grow in
vitro but to do so they must be removed from brains at a narrow, defined time of
embryonic development. Dopaminergic neurons from embryonic day 13 (EDI 3) brains
850
Z01 MH 02308-02 LCM
grow vigorously in low cell numbers and are exquisitely sensitive to the
basement membrane component laminin. EDI 4 cells grow slightly less vigorously
and require higher cell numbers to develop. They have a moderate to good
response to laminin. EDI 5 cells grow poorly unless cultured in high cell
numbers and have moderate sensitivity to laminin. By EDI 6, mesencephalic
dopaminergic neurons fail to grow even in the presence of laminin. Other types
of neurons in the EDI 5 brain grow vigorously in culture but these cultures
contain no demonstrable dopaminergic neurons.
Mesencephalic dopaminergic neurons examined in our experiments were taken from
EDI 3 to EDI 5 brains. To assess the effect of target neurons on this
development, the dopaminergic neurons were cultured in the presence of neurons
from the striatum (ED16-17), the cortex (ED16), the hippocampus (ED19) and the
cerebellum (postnatal days 5-8). At the times noted, cells from these regions
grew well i_n vitro in SFM, which was used in most of the experiments.
Mesencephalic dopaminergic neurons developed extremely well when cultured alone.
However, some time after d-9 in vitro, these cells show a decline as assessed
morphologically and by the dopamine uptake assay. If they are cultured in the
presence of cells from other regions, this decline does not occur and in many
cases their development increases to d-20 or d-27 ini vitro. Dopaminergic
neurons cultured with striatal cells display excellent development; however,
dopaminergic neurons cultured with cells from other regions also develop well
but not as consistently as when striatal cells are in cultures. As mentioned
previously, most of these cultures contain glial cells in unexpectedly large
amounts. These astroglial cells could be masking any specific effects that
target neurons might have on development.
The current working hypothesis is that the mesencephalic dopaminergic neurons
can survive and develop when cultured alone for at least 9 days. They must
receive all necessary factors from endogenous mesencephalic cells until this
time. After day 9 the dopaminergic neurons depend on the presence of cells
outside the mesencephalon for continued development. These cells could be glial
cells but this has not yet been proven. As has been demonstrated in other
studies, it is possible that the dopaminergic neurons produce (specific?)
factors that induce the glial cells to in turn secrete factors critical for the
development of the dopaminergic neurons. Finally, long term survival could be
dependent on the presence of target neurons in culture.
Proposed Course;
Current experiments are designed to define the role of astroglial cells or
factors produced by these cells in the survival and development of the
dopaminergic neurons in cultures. For instance, since the mesencephalic
dopaminergic neurons do not continue to develop when cultured alone, the
mesencephalic astroglia (which are present in these cultures along with the
neurons) must be qualitatively different than the glia from other brain regions.
Also, the simple physical presence of glial cells in culture appears to be
insufficient to induce neuronal development. Factors secreted by the glial
cells are probably of critical importance.
851
ZOl MH 02308-02 LCM
Another area of interest is the communication between the neurons and the glia
that leads to glial enhancement of neuronal development. A deficit in this
communication may be the reason that mesencephalic astroglia do not support the
development of the mesencephalic neurons: the glia do not respond to the
signals produced by the neurons and do not produce the requisite factors. If
glial soluble factors can be identified and are sufficient to allow neuronal
development, the presence of glial cells can be diminished or eliminated from
cultures and the effect of target neurons on the development and terminal
survival of dopaminergic neurons can be determined.
Concurrent with the above studies, we have developed techniques for growing
predominantly glial or neuronal cultures from various brain regions. These
cultures will be used in collaboration with other investigators in the lab to
examine CNS energy metabolism with emphasis on the kinetics of glucose
metabolism including studies involving control of glucose transport.
Significance to Biomedical Research and the Program of the Institute:
Neurological disorders, whether accompanied by obvious pathologic changes or
more subtle, as yet undetectd, changes are all due to malfunctioning CNS cells.
Knowledge about the mechanisms by which these cells develop into organized
pathways and are maintained over time is of obvious importance. Studying the
subtle signals involved in brain development i_n vivo has proven to be difficult
due to the compactness, complexity and relative inaccessibility of the brain.
Removing the developing cells from this environment and growing them in a more
controllable environment in vitro allows ready access to the cells for
observation and manipulation. There is no doubt that increased knowledge about
the normal development and maintenance of these pathways will provide
information useful for the detection of abnormal development or maintenance that
leads to mental disorders.
There are limitations to i_n vitro studies and these observations must always be
related to in vivo (or in the case of humans - clinical) observations. A
judicious blending of data from these two areas will eventually provide a
solution to the puzzle of devastating neurological diseases.
Publications:
Levenbook, I., Elisberg, B., and Driscoll, B. : Rhesus diploid rabies vaccine
(adsorbed): neurological safety in guinea pigs and Lewis rats. Vaccine 'J:
225-227, 1986.
8^2
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02307-02 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must lit on one /me befween the borders.)
Role of Proteinases in Production and Control of Neuropeptides
PRINCIPAL INVESTIGATOR (List other pratess/ona/ personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliation)
P.I.: Marian W. Kies Chemist LCM, NIMH
Others: Gladys E. Deibler
Research Chemist
LCM, NIMH
COOPERATING UNITS (il any)
None
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Developmental Neurochemistry
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
U (a) Human subjects
D (a1) Minors
D (a2) Interviews
B (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has temporarily been postponed due to the prolonged absence of the
Principal Investigator on account of illness. It is planned that the research
will resume the beginning of FY 1988.
853
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GPO 914-Sie
DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00507-05 LCM
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Clinical Brain Imaging
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfiliation)
Robert M. Cohen, M.D., Ph.D., Chief, CBI, LCM, NIMH
COOPERATING UNITS (if any)
Clinical Neuroscience Branch, NIMH (D. Pickar) ; Clinical Center, NIH (C. Channing
and R. Carson); Child Psychiatry Branch, NIMH (A. Zametkin)
LAB/BRANCH
Laboratory of Cerebral Metabolism
Section on Clinical Brain Imaging
INSTITUTE AND LOCATION
National Institute of Mental Health
9000 Rockville Pike. Bethesda, MP 20892
TOTAL MAN-YEARS:
8.5
PROFESSIONAL:
4.5
4.0
CHECK APPROPRIATE BOX(ES)
© (a) Human subjects
D (al) Minors
B; (a2) Interviews
O (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The major areas of effort in this project have been (1) to develop new
tracers or other approaches for the study of neurotransmitter function in
normal and abonormal physiology; and (2) to apply what tracer methodologies we
have available to the study of neuropsychiatric disorders. To these ends, the
following achievements are notable. A series of studies of [18F]-cyclofoxy
have been completed in baboons that delineate its usefulness as a measure of
opiate receptor avidity. An application for its use in humans has been
submitted to the Food and Drug Administration. We found that we could
successfully apply PET measurement of glucose metabolism to determine
biological determinants of attention as we observed for what we believe to be
the first time a direct relationship between the metabolic activity of a brain
region, the mid-prefrontal cortex and quantitative measures of the accuracy of
ongoing performance of auditory discrimination in normals. In patients with
schizophrenia, even those who performed as well as normals, the metabolic rate
in the middle prefrontal cortex was found to be significantly lower than
normal and unrelated to performance. Furthermore, preliminary analysis
suggests that medicated patients with schizophrenia demonstrate a similar
relationship between the middle prefrontal cortex and performance as normal
controls. The findings point to a role of the mid-prefrontal cortex and its
dopamine neurotransmitter pathway input in sustained attention and to
dysfunction of this region and of its dopamine modulation in some patients
with schizophrenia.
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ZOl MH 00507-05 LCM
OTHER PROFESSIONAL PERSONNEL
Thomas Nordahl , M.D., Ph.D., Medical Staff Fellow, LCM, NIMH
Michael Gross, M.D., Medical Staff Fellow, LCM, NIMH
Steven M. Larson, K.D., Chief, NM, CC, NIH
David Pickar, M.D., NSB, NIMH
Robert M. Post, M.D., Chief, BPB, NIMH
Daniel R. Weinberger, M.D., ETB, St. Elizabeths Hospital
William Semple, Ph.D., Psychologist, LCM, NIMH
John Cappelletti, Computer Programmer Analyst, LCM, NIMH
A. Catherine King, Psychology Technician, LCM, NIMH
Mary S. Dowl ing-Zimmerman, Psychologist, LCM, NIMH
Michael Channing, NM, CC
Dale Kiesewetter, NM, CC
Alan Zametkin, M.D., CPB, NIMH
MAJOR FINDINGS
Method Development:
In the last annual report, we summarized our early findings with a new
selective opiate receptor dependent tracer [18F]cyclofoxy. We have now
completed a series of studies demonstrating the hoped for displacement of
cyclofoxy binding with the selective opiate receptor antagonist naloxone in
baboons. This data is currently being analyzed by two different compartment
models to determine a best fit for the data. Furthermore, the quality control
issues for the reliable and safe production of cyclofoxy for administration to
humans have been settled. An application for the use of cyclofoxy in normals
and neuropsychiatric patients has been submitted to the Food and Drug
Administration.
Patient Studies with PET:
Early studies by several groups including the intramural NIMH had
indicated that either while receiving electric shock or while resting,
patients with schizophrenia when compared to normal controls appeared to have
relatively lower levels of frontal cortex to posterior cortex brain activity
(metabolism), i.e. be hypofrontal . Moreover, the "normally" observed
relatively greater metabolism in the frontal cortex, a region somewhat
specialized for higher cognitive function, compared to the more posterior
cortical regions of the brain that may be more closely associated with primary
processing, suggested to investigators that the hyperfrontal pattern of
metabolism represented an important dimension of normality, and that the
observation of "hypofrontal ity" in schizophrenia was a reflection of the
"executive" function pathophysiology of schizophrenia.
We were concerned, however, by several problems with the approach. First,
hypofrontal ity failed to be observed in some studies. Second, the finding,
even in the positive studies, was frequently dependent upon idiosyncratic
definitions and selective statistical analysis. Third, affectively disordered
patients, even while not substantially depressed, appeared to be more
hypofrontal than patients with schizophrenia. Moreover, we felt that the
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ZOl MH 00507-05 LCM
normal or abnormal functioning of the frontal cortex should in many instances
be evaluated in the context of a specific behavioral requirement which is
difficult to establish in subjects at rest or who are receiving "meaningless
electric shock".
Nevertheless, we attempted to assess the importance of the hypofrontality
concept by (1) employing analogous measures of hypofrontality as those used in
prior investigations (2) measuring the associations between clinical symptoms
and hypofrontality and (3) observing if the chronic administration of
neuroleptics, the treatment of choice in schizophrenia, would substantially
effect this index. This approach was not fruitful in establishing the
significance of the findings of hypofrontality in schizophrenia.
To remedy the above problems, we elected to study frontal cortical
function in schizophrenia in the context of a specific executive function,
maintenance of directed attention. A continuous performance test (CPT) based
on auditory discrimination was chosen because CRTs have consistently been
reported to demonstrate deficits in the maintenance of directed attention in
schizophrenia and are presumed to be associated more directly to genetic
errors than the overt symptomatology of schizophrenia. Furthermore, as the
frontal cortex is quite large in man and consists of a number of functionally
somewhat independent entities, we elected to do a detailed anatomical
examination of this area and the rest of the brain. This meant that we needed
to study a large number of normals both in the resting state and while
performing CPT so as to allow for a statistical analysis that would reveal
replicable results while retaining some degree of power.
We found that we could successfully apply the FDG-PET methodology to
determine biological determinants of attention, i.e. those anatomical
structures that may contribute to the reception and modulation of sensory
stimuli. We observed metabolic rate differences in the middle prefrontal,
cingulate and superior posterior parietal cortices of normals performing
auditory discrimination compared to resting subjects or subjects receiving
electric shocks (Cohen, et al_., 1987). Furthermore, a direct relationship was
observed between metabolTc rates in the middle prefrontal cortex and the
accuracy of a normal subject's auditory discrimination. We believe that this
is the first time that the metabolic activity of a brain region has been
specifically linked to quantitative measures of the accuracy of ongoing
performance in normals.
In patients with schizophrenia, even those who performed as well as
normals, the metabolic rate in the middle prefrontal cortex was found to be
significantly lower than normal and unrelated to performance (Cohen, et a^. ,
1987). Furthermore, we have partially completed our analysis of the data of 8
patients who were receiving neuroleptics and find that the medicated patients
with schizophrenia demonstrate a clear association between brain activity in
the mi d-pref rental cortex and performance (Cohen, et aj^. , in press/. Tne
findings point to a role of the mid-prefrontal cortex and its dopamine
neurotransmitter pathway input in sustained attention and to dysfunction of
this region and of its dopamine modulation in some patients with schizophrenia.
857
ZOl MH 00507-05 LCM
SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE
The central tenet of the Laboratory is the belief that our increasing
knowledge of the molecular genetics, biochemistry and cytology of the central
nervous system will fall short of allowing us a complete understanding of
normal and abnormal behavior regardless of the degree of sophistication that
genetic probes and postmortem analyses achieve without studies of the
functioning system. Advances in the understanding of the functioning system
are required if we are to delineate the pathophysiology of psychiatric
illnesses such as schizophrenia. If for no other reason such work should
facilitate a reduction in the heterogeneity of patient samples in research
studies of psychiatric disorders by ensuring that patients who have the
syndrome of schizophrenia also share the same pathophysiology. This may be of
particular importance to genetic studies.
Furthermore, were a single gene found to be principally responsible for
the genetic determination of schizophrenia prior to the elucidation of the
pathophysiology, schizophrenia researchers would still need information about
pathophysiology to tackle the difficult problems of determining the mechanisms
responsible for the development of the phenotype and to improve treatment
strategies.
At the NIMH-IRP program we have made a sustained effort to use positron
emission tomography (PET) to help elucidate the pathophysiology of psychiatric
disorders. We have made this commitment because we believe that PET, one of a
number of new brain imaging technologies with its unsurpassed ability to
precisely localize and quantify in the human brain tracers used for the study
of physiological processes, offers the greatest promise for delineating the
pathophysiology of schizophrenia.
In our most recent PET studies we chose to examine the functional
localization of the ability to perform continuous auditory discrimination
because consistent deficits in continuous performance had been reportea in
schizophrenia and in subjects at increased risk for schizophrenia and because
sustained attention is also fundamental to the development and execution of
all "goal-directed" behavior. These defects may lie closer to the primary
defects presumed to be associated with genetic errors than the overt
symptomatology of schizophrenia (fully expressed phenotype). We were able to
observe dysfunction of the middle prefrontal with respect to sustainea
attention in schizophrenia. Although the importance of this dysfunction for
understanding the pathophysiology of schizophrenia remains to be delineated,
preliminary evidence of the dopamine dependence of this function in
schizophrenia as evidenced by change in prefrontal cortex function with
neuroleptic treatment suggests that detailed studies of anatomic and
neurotransmitter pathways involved in attention are warranted.
PROPOSED COURSE:
The majority of subjects that have participated in our protocols to date
were scanned on the ECAT II scanner. The resolution of the ECAT II scanner
was 1.8 cm. and the number of slices that could be obtained was at best 7.
Furthermore, the attenuation corrections were calculated and could not be
858
ZOl MH 00507-05 LCM
determined empirically, thus limiting the brain structures that could be
accurately examined. The Scanditronix scanner now in use for this protocol
has a 5 mm. resolution, can gather 28 slices from a single scan, and an
empirically derived attenuation correction can be made. Thus, work with the
Scanditronix should allow us to replicate our previous findings and search for
additional biological determinants of attention and abnormalities in
schizophrenia. The use of other populations, e.g., neuropsychiatric
disorders, including adult attention deficit disorder (ADD) and the
examination of patients while on medications for their disorders (e.g. while
ADD patients are on stimulants) should help with the search for additional
biological determinants of sustained attention and the delineation of the
specificity of these findings for schizophrenia.
Although the success that we have had in pursuing an understanding of the
brain metabolic map of sustained attention and a component of its
neurotransmitter dependence is gratifying, it is very likely that the study of
other cognitive processes will be required to define the pathophysiology of an
illness as complex as schizophrenia. Just as no one phenomenological variable
such as hallucinations is sufficient to delineate schizophrenia, we will
probably need to evaluate brain activity with respect to a number of cognitive
tasks as well as with respect to the other major component of behavior,
emotion if we are to arrive at an understanding of complex neuropsychiatric
disorders such as schizophrenia. Therefore, we will also be pursuing
biological determinants of other components of cognition and emotion.
PUBLICATIONS:
Kiesewetter, W.C, Eckelman, W.C, Cohen, R.M., Finn, R.D., and Larson, S.M.:
Syntheses and D2 receptor affinities of derivatives of spiperone containing
aliphatic halogens. Appl . Radiat. Isotopes 37: 1181-88, 1986.
Ostrowski, N.L., Burke, T.R., Jr., Rice, K.C., Pert, A., and Pert, C.B.: The
pattern of ^H-cyclofoxy retention in rat brain after in vivo injection
corresponds to the in vitro opiate receptor distribution. Brain Res. 402:
275-286, 1987.
Post, R.M., DeLisi, L.E., Holcomb, H.H., Udhe, T.W., Cohen, R.M., and
Buchsbaum, M.S.: Glucose utilization in the temporal cortex of affectively
ill patients: Positron emission tomography. Biol. Psychiatry 22: 545-553,
1987.
Cohen, R.M., Semple, W.E., Gross, M,, Nordahl , T.E., DeLisi, L.E., Holcomb,
H.H., King, A.C., Morihisa, J.M., and Pickar, D.: Dysfunction in a prefrontal
substrate of sustained attention in schizophrenia. Life Sci. 40: 2031-2039,
1987.
Cohen, R.M., Semple, W.E., Gross, M., Nordahl, T.E.: From syndrome to
illness: Delineating the pathophysiology of schizophrenia with positron
emission tomography. Schizophrenia Bull ., in press.
859
ZOl MH 00507-05 LCM
Ostrowski, N.L., Hill, C.B., and Pert, A.: Autoradiographic visualization of
sex differences in the pattern and density of opiate receptors in hamster
hypothalamus. Brain Res. , in press, 1987.
Ostrowski, N.L., Pert, C.B., and Pert, A.: Visualization of opiate receptors
in vivo. In France Leslie's (Ed.): Receptor Localization: Ligand
Autoradiography. New York, Alan Liss, Inc., 1987 in press.
860
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02296-02 LCM
PERIOD COVERED
October 1, 1986 through September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must tit on one line iMtween ttie tmrders.)
In Vivo Tomographic Imaging of Dopaminergic Systems and their Turnover
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, latxiratory, and institute aftillation)
C. C. Chiueh Pharmacologist LCM NIMH
COOPERATING UNITS (if any)
Nuclear Medicine, CC, NIH (S. Larson); Office of the Director, IRP, NINCDS
(I. Kopin) ; Dept. of Nuclear Medicine, McMaster Univ. Medical Centre, Hamilton,
Ontario, Canada (G. Firnau)
LAB/BRANCH
Laboratory of Cerebral Metabolism
SECTION
Section on Clinical Brain Imaging
INSTITUTE AND LOCATION
National Institute of Mental Health, 9000 Rockville Pike Bethesda, }m 20892
TOTAL MAN-YEARS:
3.0
PROFESSIONAL:
2.5
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues H (c) Neither
n (a1) Minors
n (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have established a neurochemical basis for the use of purified
[F-18]-labe1ed L-6-F-dopa as a presynaptic imaging ligand for brain
dopaminergic neurons. A potential clinical use of this PET imaging ligand for
brain dopamine in determining degrees of brain damage in parkinsonian patients
was demonstrated in this study by using the MPTP-induced primate model of
parkinsonism. Despite a high background activity due to contamination, we
were able to image striatal dopaminergic neurons in living monkeys by using
the Scanitronix PET scanner at the NIH Clinical Center. The striatal
[F-18]-labeled dopaminergic activity was diminished in the monkeys that
received a partially purified [F-18]-labeled 6-F-dopa and indicates a need to
purify further this PET imaging ligand prior to the application of this
6-F-dopa/PET procedure in humans. By using animal models of parkinsonism, we
have also evaluated a new SPECT imaging ligand for D2 dopamine receptors,
[I-123]-labeled IBZM (3-iodobenzylamide derivative). It has been shown that
IBZM specifically binds to D2 dopamine receptors in the brain and is
displaceable by both agonists and antagonists of the D2 dopamine receptor.
The in vivo imaging of the D2 dopamine receptors in the caudate nucleus,
nucl?iJs-iHumbens, olfactory tubercle and the kidney were obtained within
thirty minutes following the administration of the radioactively labeled
IBZM A denervation induced decrease in dopaminergic fibers and increase in
D2 dopamine receptors in the basal ganglia of experimental parkinsonian
animals have been imaged by using these pre- and post-synaptic imaging
liqands Thus, this pre-clinical study has demonstrated potential clinical
uses of these imaging ligands for studying in vivo the dopaminergic activities
in patients with neuropsychiatric or Parkinson disorders.
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ZOl MH 02296-02 LCM
Other Professional Personnel Engaged on the Research Project:
R.M.
D.
P.
W.
I.
T.
J.J.
H.
Cohen
Doudet
Douillet
Singhaniyom
Namura
Bruecke
Chen
Miyake
I.J.
Y.F.
T.
Kopin
Tsai
Kondo
R.
S.M.
Finn
Larson
T.S.
J.L.
K.L.
D.
Chen
Sun
Kirk
Furlano
H.F.
Kung
G.
Firnau
Section Chief
Visiting Fellow
Visiting Fellow
Visiting Fellow
Guest Researcher
Guest Researcher
Visiting Fellow
Visiting Fellow
Director
Visiting Fellow
Visiting Fellow
Chemist
Director
Pharmacologist
Pharmacologist
Section Chief
Chemist
Professor
Professor
LCM
LCM
LCM
LCM
LCM
LCM
LCM
LCM
IRP OD
NI
NI
NM CC
NM CC
OB
LC
LC
NIMH
NIMH
NIMH
NIMH
NIMH
NIMH
NIMH
NIMH
NINCDS
NINCDS
NINCDS
NIH
NIH
FDA
FDA
NIADDK
NIADDK
Dept. of Nuclear Medicine
Univ. of Pennsylvania
Dept. of Nuclear Medicine
McMaster University
Medical Centre
Hamilton, Ontario, Canada
862
ZOl MH 02296-02 LCM
Project Description:
Objectives:
This research project continues the brain imaging project begun several
years ago in the Laboratory of Clinical Science (see Chiueh et al., 1984; ZOl
MH 02241-01-LCS).
The first goal of this project is to develop an ideal positron emitting
pre-synaptic ligand (either carbon-11 or fluorine-18) for in vivo imaging of
brain dopaminergic systems and to provide an index of the functional turnover
rate of dopamine by positron emission tomographic (PET) scanning procedures.
The second goal is to develop and evaluate gamma emitting post- synaptic
ligands (iodine-123) for imaging of D2 and Dl dopamine receptors in the brain
by single photon emission tomographic (SPECT) procedures.
Such tomographic brain imaging procedures may prove to be useful for
determining brain damage in Parkinson's disease, for visualizing regeneration
of striatal dopamine and for evaluation of up and down regulation of
dopaminergic activities and receptors in neuropsychiatric disorders.
Methods Employed:
A. Synthesis of Fluorine-18 labeled L-6-fluoro-dihydroxyphenyl alanine and
iodine-123 labeled 3-iodobenzylamine:
Flourine-18 labeled L-6-F-dopa is synthesized by Dr. R . Finn using the
procedure of Firnau et al . (1984) and Adams et al^. (1986). The L-6-F-dopa is
separated from 5-F- and/or 2-F-species by using HPLC procedures. [F-18]
isotope (2 hr. half-life) is being generated and produced by using the newly
installed cycltron in the NIH Nuclear Medicine Department. Carbon-14 labeled
6-F-dopa is enzymatically synthesized by Furlano and Kirk (1987).
Iodine-123 or iodine-125 labeled 3-iodobenzylamine (specific activity:
greater than 1000 Ci/mmol) is produced and purified by Kung et al^. (1986).
B. Animals:
Adult rhesus monkeys (Macaca mulatta) and/or baboons of both sexes (5-8
kg) were used. These animals were housed individually in primate quarters on
a 12-hr light/dark cycle. Purina monkey chow, water, juice, and fresh fruit
were given ad lib. L-dopa therapy (Sinemet 100/10, q.i.d.) was given to
severly affected parkinsonian monkeys. Full and hemiparkinsonism is induced
in monkeys after an intravenous or intra-arterial administration of a dopamine
neurotoxin, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), respectively.
During the PET procedure, animals were anesthetized with pentobarbital
sodium (35 mg/kg, i.v.). A head mold was developed and designed to hold the
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ZOl MH 02296-02 LCM
head steady in the PET scanner. Acute arterial and venous catheters were
placed to monitor blood pressure, for blood sampling, and for injection of
tracers.
Rats (200 g) or mice (25 g) were pretreated with peripheral decarboxylase
inhibitor (MK-486, 25 to 75 mg/kg i.p., 30-60 min.). Carbon-14 labeled L-Dopa
(sp. act. 10.9 mCi/mmole) was administered through a venous catheter placed in
the tail vein. Animals were sacrificed at various times after the treatment
for autoradiographic procedures and/or neurochemical assays.
C. Neurochemical Procedures:
The metabolites of 6-[F-18]-dopa in plasma and/or brain samples, i.e.,
6-F-dopamine, 6-F-dihydroxyphenyl acetic acid, 6-F-homovanillic acid and
0-methylated 6-F-dopa were separated and quantified by HPLC procedures. A
semi -preparatory HPLC system was used for an isolation of radioactively
labeled 6-F-dopa.
D. PET Imaging of Brain Dopamine Neurons:
The brain dopamine imaging procedure described by Garnett et ^. (1983)
was used. Briefly, following an intravenous injection of 1.5 to 2 mCi
purified L-6-[F-18]-dopa, anesthetized monkeys were examined in a 10 mm
horizontal brain slice along the orbitomeatal plane in the Scanditronix PET
scanner for 2 to 4 hours. At various intervals, blood samples were drawn and
assayed for 3-0-methyl-6-F-dopa, the major metabolite of 6-F-dopa. Some
animals were sacrificed at the end of the experiment. Disc-shaped punches of
tissue from the caudate nucleus and the putamen were assayed for dopamine and
its biosynthetic enzymes in order to correlate the brain imaging data with the
endogenous dopaminergic activities.
E. Autoradiographic Imaging of Brain Dopamine and its Receptors in Small
Experimental Animals:
Some of the experimental designs of future clinical brain imaging
procedures are being conducted in small experimental animals following an
intravenous administration of L-3-[C-14]-dopa, L-2-[C-14]-6-F-dopa and
[I-125]=IBZM. Mice or unilaterally-lesioned rats were pretreated with
antipsychotic agents (haloperidol ) , MAO inhibitor (deprenyl), dopamine uptake
blocker (amfonelic acid), L-dopa decarboxylase inhibitor (NSD-1015), and major
tranquilizer (reserpine) in order to manipulate dopaminergic activities in the
brain. Animals were sacrificed at various intervals after the administration
of imaging ligand in order to simulate clinical brain imaging procedures for
investigating the turnover rate of dopaminergic neurons. The brain was
quickly dissected and frozen in -20 C isopentane. Brains were cut frozen into
30 urn thick sections which were mounted on gel-coated slides. Sets of serial
sections through the striatum, hypothalamus and mid-brain were processed for
autoradiographic demonstration of brain dopaminergic systems using LKB
ultrofilm. The autoradiographic imaging was quantified by uising a
computerized densitometer and external radioactivity standards.
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ZOl MH 02296-02 LCM
F. Imaging of D2 Dopamine Receptors by SPECT Procedures:
Radioactive iodo-amphetamine has been used in SPECT procedures for in
vivo imaging of blood flow of the human brain. This clinical procedure is
modified for the current preclinical study using subhuman primates.
Significance to Biomedical Research:
The MPTP-induced primate model of parkinsonism was employed in the
present preclinical study to evaluate these newly developed PET and/or SPECT
imaging tracers for assessment of brain dopaminergic functions in the living
organism. The current preclinical results showed clearly that [F-18]-6-F-dopa
and [I-123]-IBZM are excellent dopamine imaging ligands and have potential
uses as a diagnosic tool in the clinic for not only identifying parkinsonian
patients in the subclinical stage, but also for elucidating dopaminergic
mechanisms of neuropsychiatric disorders. Further development could lead to
the procedure's use in the investigation of dopamine turnover rate and D2
dopamine receptor density in studying neuropsychiatric disease.
Proposed Course:
A. Preclinical Studies:
The 6-F-dopa/PET procedures including the generation of [F-18] gas by a
cyclotron, the fluorination of L-dopa, and a partial purification of
[F-18]-labeled 6F-dopa, and scanning of living monkeys have been successfully
tried at the NIH medical center in living monkeys. Before we apply the brain
imaging procedure to human subjects, vigorous HPLC purification and additional
toxicological and pharmacological evaluations have to be performed in order to
meet the safety guidelines stipulated by the Food and Drug Administration. So
far, the rate limiting step of this project is the isolation and purification
of [F-18]-labeled L-6-F-dopa.
1. Visualization of Degree of Damage to Brain Dopamine in MPTP-induced
Hemi-parkinsonism by the 6-F-dopa/PET Procedures.
The MPTP-induced primate model of parkinsonism is being used extensively
for research in dopaminergic functions. These parkinsonian monkeys require
L-dopa therapy and sometimes tube- feeding in order to sustain their life.
Recently, a hemi-parkinsoian monkey model has been developed. A post-mortem
neurochemical assay revealed that the damage to brain dopamine of these
monkeys was limited to the infusion side, contralateral to the side of motor
dysfunction. Since these monkeys suffered parkinsonism only in one side of
their body, the animals still were able to care and feed themselves.
Furthermore, each animal can serve as its own paired control in testing these
brain imaging ligands designed for PET and SPECT studies.
Triplicate PET scans will be performed on these hemi-parkinsonian monkeys
in order to obtain quantitative and statistical information on the performance
of the Scanditronix PET scanner. The in vivo PET data will be verified at the
865
ZOl MH 02296-02 LCM
end of the experiment by performing ex vivo neurochemical measurement of
dopamine, its synthetic enzymes, and metabolites in each animal and
histological confirmation of cell death or survival. The correlation between
the in vivo PET activity and the degree of parkinsonism may provide standards
for Tuture clinical diagnosis of parkinsonian patients even in the subclinical
stage.
Major Findings:
This research project on the use of L-6-F-dopa for PET imaging of brain
dopamine has been conducted at NIMH since 1981. In collaboration with Dr.
Irwin Kopin (NINCDS), Dr. Kenneth Kirk (NIADDK), Dr. C. Robert Creveling
(NIADDK), Dr. Ronald Finn (NIH, Nuclear Medicine), and Dr. Gunter Firnau
(McMaster University Medical Centre), we have established a neurochemical
basis for the use of purified L-6-F-dopa as a presynaptic imaging ligand for
brain dopamine and demonstrated potential clinical applications of the
6-F-dopa/PET imaging procedure in collaboration with the McMaster and the NIH
nuclear medicine PET imaging groups.
The MPTP-induced brain damage to the basal ganglia in three parkinsonian
monkeys was visualized by using purified fluorine-18 labeled L-6-F-dopa and
the McMaster PET scanner (Chiueh et al^. , 1986). This PET procedure is being
adapted and tested at the Department of Nuclear Medicine of the NIH Clinical
Center. In the control monkeys, the fluorine-18 activity in the striatum of
the brain increased by three- fold over the background activity seen in the
non-dopaminergic brain regions following the administration of a partially
purified 6-F-dopa (specific activity: 100-150 mCi/mmol; 70 to 80 chemical
purity; prepared by Dr. R. Finn). The major contaminant of the 6-F-dopa was
identified by our quality control procedure as 2-F-dopa which was not
decarboxylated to l^orm 6-F-dopamine but was rather 0-methylated to
3-0-methoxy-2-F-dopa and thus interfered with the 6-F-dopa imaging of brain
dopamine neurons as indicated from the results of our autoradiographic studies
in small experimental animals. This partially purified [F-18]-labeled
6-F-dopa was not able to image in vivo brain transplants in parkinsonian
monkeys. These results will be reported at the Xth International Congress of
Pharmacology (August 23-28, 1987 Sydney, Australia).
It is currently believed that MPTP exerts its toxic effects through its
metabolite MPP"*". We reported that intranigral injection of MPP* caused a
dose-dependent depletion of striatal dopamine in rats. In the present study,
we used radioactive pre- and post-synaptic ligands in addition to [Ca-45] to
visualize autoradiographically MPP"^ effects on dopaminergic systems.
Autoradiographic procedures were performed two weeks after unilateral
lesioning of the median forebrain bundle following intravenous administration
of [C-14]-L-dopa or [I-123]-IBZM. In controls, the in vivo presynaptic
[C-14]-L-dopa imaging revealed dopamine-rich areas, such as the caudate
nucleus, the nucleus accumbens, and the median eminence. Dopamine fiber
imaging completely disappeared in the striatum of the unilaterally
nigrallesioned rats. A denervation-induced dopamine receptor supersensitivity
was visualized by using the in vivo [I-125]-IBZM binding and ex vivo
866
ZOl MH 02296-02 LCM
autoradiographic procedures (in collaboration with Dr. Kung of the University
of Pennsylvania). D2 dopamine receptors as seen by the [I-125]-IBZM binding
increased by 50 in the denervated side of basal ganglia of
unilateral-lesioned rats and in hemi-parkinsonian monkeys. The [I-125]-IBZM
binding to brain receptors was found to be highly specific to 02 dopamine
receptor sites and was displaced by 02 agonists and antagonists. Furthermore,
the in vivo imaging of 02 dopamine receptors in the brain were obtained within
thirty minutes following the administration of [I-125]-IBZM and also yielded a
four to one ratio of specific to nonspecific activity. Our report to the
Vlllth Catecholamine Symposium (June 14-19, 1987, Jerusalem, Israel) indicates
that IBZM is a new SPECT imaging ligand of 02 dopamine receptor when labeled
with the short half-life isotope 1-123.
2. Turnover of Brain Dopaminergic Systems Following Administration of
Antidepressants, Antipsychotics, Tranquilizers, and Antiparkinsonian
Drugs:
Efforts will be focused on the PET procedure for measuring the in vivo
turnover rate of dopamine in the mesolimbic and mesocortical systems. The
previously published procedures of measuring turnover rate of brain dopamine
required at least 5-6 animals per group at 3 different time points. The
present PET procedure by using a purified [F-18]-6-F-dopa ( 98 purity) can
offer a complete time curve in each animal in vivo following
neurophamarcological manipulations.
a. Dopamine receptor antagonists: Antipsychotics
b. Dopamine uptake blockers and dopamine releasing agents:
amphetamine and cocaine
c. Electrical activation of the dopaminergic systems:
e.g., electrical convulsion shock treatment
3. In vivo SPECT Imaging of 02 Dopamine Receptors by [I-123]-IBZM:
The newly developed 02 dopamine receptor imaging ligand will be tested in
a clinical SPECT imaging camera by using a hemi-parkinsonian monkey model.
Our in vitro autoradiographic study has demonstrated a 25-50 increase in the
binding sites of 02 dopamine receptors in the caudate nucleus and putamen of
the MPTP-lesioned parkinsonian monkeys. This SPECT procedure will use
[I-123]-labeled IBZM with a 13-hour half-life. This SPECT imaging ligand will
be synthesized and provided by Dr. H. Kung of the University of Pennsylvania
in this preclinical trial.
B. Clinical Studies:
After passing the preclinical toxicological evaluations, these pre- and
post-synaptic dopaminergic ligands will be administered to normal volunteers,
parkinsonian patients, bipolar manic-depressive patients, schizophrenic
patients, and other patients suffering from disorders which may involve brain
dopamine. These clinical projects will be conducted following established NIH
guidelines and with the collaboration of different clinical branches of NIH
and NIMH.
867
ZOl MH 02296-02 LCM
Publications:
Namura, I., Douillet, P., Sun, C.J., Cohen R.M and Chiueh, C.C.: MPP*
(l-methyl-4-phenylpyricline) is a neurotoxin to dopamine-, norepinephrine-, and
sertonin-containg neurons. Eur. J. Pharmacol . 136: 31-37, 1987.
Chiueh, C.C: Dopamine in the Extrapyramidal Motor Function: A Study Based
upon the MPTP-induced Primate Model of Parkinsonism. In Joseph, J. (Ed.):
Central Determinants of Aqed-re1ated Declines in Motor Function. New York,
Annals New York Academy of Science, 1987, in press.
868
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 00931-14 LGCB
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) ' '
Characteristics and Regulation of S-Adenosylhomocystein Hydrolase
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
LGCB NIMH
P.I. P. S. Backlund, Jr. Senior Staff Fellow
R. R. Aksamit
G. L. Cantoni
Research Chemist
Chief, Laboratory of General
and Comparative Biochemistry
LGCB NIMH
LGCB NIMH
COOPERATING UNITS (if any)
Department of Biochemistry, Toyama Medical and Pharmaceutical University,
Toyama, Japan
LAB/BRANCH
Laboratory of General and Comparative Biochemistry
SECTION
Section on Proteins
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
3.5
PROFESSIONAL
1.5
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (a1) Minors
D (a2) Interviews
(b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceedthe space provided.)
S-adenosyl homocysteine hydrolase plays a critical role in regulating AdoMet-
dependent methylations in eukaryotlc cells by regulating the ratio of
Ad oMe t/ Ad oH cy . Several approaches are being used to determine the structure
and function of this enzjrme.
1) Structure Determination: The enzyme has been purified from rat liver, and
cloned from a rat liver cDNA library. Ihe amino acid sequence was determined
and a putative NAD binding site identified. The rat liver enzyme has been
expressed in E. coli and site-directed mutagenesis is in progress to determine
the function of specific amino acid residues. Conformational changes for
active and inactive forirs of the enzyme have been examined by fluorescence,
and circular dichroism.
2) Ligand Binding and Kinetic Properties: The role of NAD, nucleotide, and
cAMP binding in regulating the catalytic activity has been studied, and
photoaf f inity ligands are being used to label the binding sites. A large
nunber of adenosine and adenosylhomocysteine analogs have been examined for
their ability to function as inhibitors and/or substrates of the enzyme.
3) Biological Effects of Inhibitors: In vivo these adenosine analogs can
form very potent and specific inhibitors of transmethylation reactions, and
these inhibitors have a wide range of biological activities, including
antiviral activity against several RNA and DNA viruses , inhibition of
leukocyte chemotaxis, and stimulation of cell differentiation.
869
PHS 6040 (Rev. 1/B4)
CPO SI4-S1I
ZOl MH 00931-14 LGCB
Other Investigators:
J. Kasir Visiting Fellow LGCB NIMH
T. Gomi Visiting Fellow LGCB NIMH
T. Caryk Chemist LGCB NIMH
M. Fujioka Toyama Medical & Pharmaceutical
University, Toyama, Japan
Project Description:
As is well known, S-adenosylmethionine (AdoMet) is a key intermediate in
biological transmethylation and transalkylation reactions. There are hundreds
of reactions, each catalyzed by a specific enzyme, that utilize AdoMet as a
substrate. It is obvious that the utilization of AdoMet in biological systems
must be under regulatory controls, but at the present time little is known about
the nature of these controls. It has been established that S-adenosylhomocysteine
(AdoHcy), one of the products of transmethylation reactions that utilize AdoMet
as methyl donor, is a competitive inhibitor of most reactions in which AdoMet
participates. From the result of work in this and other laboratories, it has
been proposed that the intracellular ratio of AdoMet/ AdoHcy must be of key
importance in the regulation of biological alkylation reactions, and that this
ratio plays a role in determining the hierarchy of biological methylation reac-
tions. In eukaryotes, AdoHcy is metabolized through a single metabolic pathway
by S-adenosylhomocysteine hydrolase (AdoHcyase), an enzyme which catalyzes the
reversible hydrolysis of AdoHcy to adenosine and homocysteine. Because of the
central role of AdoHcyase in the metabolism of AdoHcy and in maintaining the
ratio of AdoMet/ AdoHcy , this enzyme has been under intensive study in this and
other laboratories.
S-Adenosylhomocysteine hydrolase has been purified from a variety of sources.
Previous work has shown that the mammalian enzyme consists of structurally iden-
tical subunits, contains four mols of tightly bound NAD/mol of enzyme, and also
binds cAMP and adenosine. The chemistry of the catalytic reaction is fairly well
understood, but very little is known about the structure of the enzyme and its
relation to function. Our studies are directed towards 1) the elucidation of the
primary structure of the hydrolase by molecular cloning of its cDNA and by
inference, its secondary and tertiary structure, 2) the determination of the
specific polypeptide sequences that are involved in its binding, catalytic,
and regulatory sites, 3) characterization of the conformational changes that
accompany activation and binding of substrates and cofactors, and 4) crystalliza-
tion of the enzyme to provide an absolute three-dimensional structure by X-ray
diffraction.
The enzyme was purified to homogeneity, and the amino acid sequence of
several cyanogen bromide and tryptic peptides were determined. The purified
enzyme was also used to produce antibodies against the hydrolase, and the
antibodies were used to screen a Xgtll cDNA library from rat liver to clone
and sequence the rat liver enzyme. The cloned sequence was used to determine
the amino acid sequence of the protein, and a putative NAD binding region was
identified by the homology of the amino acid sequence with other NAD binding
proteins. The cloned hydolase was also expressed in E. coli, at an induced level
reaching approximately 10% of the bacterial proteins. Site directed mutagenesis
870
ZOl MH 00931-14 LGCB
is also being attempted, in order to examine structure/function relationships for
different regions of the enzyme, such as the NAD and adenosine binding sites.
The cloned cDNA sequence is also being used to examine the level of mRNA expres-
sion in different cell types, and in other species. In addition, the genomic
organization of the hydrolase is also being examined.
The hydrolase has binding affinities for nucleosides and we are investigating
the possible role of ATP, adenosine, cAMP, and the tightly bound NAD's in the
regulation of the enzyme. We have shown that the enzyme is inactivated by
Mg*"^, ATP, and KCl with the loss of four molecules of NAD, and it can be
reactivated upon incubation of the enzyme with NAD. When NAD is bound to
the enzyme, little cAMP binds, while more cAMP binds to the enzyme lacking NAD
suggesting that the cAMP may bind to the NAD site. Fluorescence, circular
dichroism, differential ultraviolet spectroscopy studies, and photoaf f inity
labeling have been used to monitor changes in conformation of the enzyme upon
inactivation and reactivation. The emission and excitation spectra of inacti-
vated enzyme, for example showed a loss in tryptophan fluorescence intensity
which appears to be restored upon reactivation. ATP, adenosine, and cAMP have
binding affinities for the enzyme and it is not clear how they fit in catalysis
and/or regulation. Various affinity reagents have been used to label the
hydrolase, and the labeled fragments will be isolated and sequenced to determine
the amino acid residues that comprise the active site and/or binding clefts in
the protein. The amino acid sequence determined from the cloned cDNA sequence
will help to determine the regions of the protein modified by these affinity
reagents. Modification of the amino acids at these sites by site directed muta-
genesis, will provide independent data on the role of these amino acid residues
in catalysis.
While the biochemical mechanisms of transmethylation reactions have been
elucidated many years ago, largely as a result of the studies by Canton! and his
collaborators at NIH, the correlation between many methylation reactions and
cellular functions remains obscure. For instance, the significance of the
methylation of a variety of informational macromolecules, such as proteins and
nucleic acids (DNA, ribosomal- , messenger-, viral and tRNA, etc.), or of complex
polysaccharides, or even simpler compounds such as guanido acetic acid, nicotina-
mide, etc., is not immediately obvious and is the subject of much debate. A
role for DNA methylation in gene expression has been suggested by observations
from several laboratories. We have shown that both 3-deaza-Ado and 3-deaza-Ari
can stimulate cell differentiation in a number of cell lines, including the
differentiation of myoblasts to form myotubes, and the differentiation of myloid
cells to synthesize globin. It is possible that 3-deaza-Ado may cause
differentiation of these cells by inhibiting DNA methylation. However, since
3-deaza-Ado also inhibits a number of other methylation reactions, further work,
will be required to identify the reactions involved. In collaboration with Dr.
Razin, we have proposed a novel mechanism for the transient demethylation of DNA
during differentiation where 5-methylcytosine is replaced enzymatically by
cytosine, by a mechanism destinct from conventional excision-repair (see ZOl MH
02321-2 LGCB).
Since AdoHcyase is the only enzyme known to metabolize AdoHcy in eukaryotes,
inhibition of this enzyme by analogs can be used to alter the ratio of AdoMet/
AdoHcy in the cell. We decided some years ago to take advantage of this fact
871
ZOl MH 00931-14 LGCB
and initiated a long range experimental project designed to study in depth the
properties of AdoHcyase, and then to develop a series of specific inhibitors of
this enzyme. As a result of these studies on the properties of AdoHcyase, we
have established that the use of specific inhibitors makes it possible to alter
the intracellular levels of AdoHcy and/or to accumulate intracellular ly congeners
of AdoHcy of the general formula S-purinylhomocysteine (PurHcy). By using these
inhibitors, it is possible to modulate the AdoMet/ AdoHcy and/or AdoMet/PurHcy
ratio in different cellular systems , and to examine the consequences of these
changes on cellular functions.
Our studies, confirmed and extended in other laboratories, have identified
several inhibitors of AdoHcyase. These compounds have a variety of biological
effects and may have important clinical applications, and explain some of the
mechanisms of action of some clinically important compounds. Irreversible
inhibitors of AdoHcyase include the compounds 9-6-D-arabinof uranosyladenine
(Ara-A), 3-deaza-9-6-Darabinof uranosyladenine (3-deaza-Ara-A) , and 2-chloro-
adenosine. Ara-A has been used by others in chemotherapy for cancer patients.
3-Deaza-Ara-A and 2-chloroadenosine might be expected to have clinical effects
similar to Ara-A, since they produce similar inhibition of AdoHcyase. Of the
many reversible inhibitors tested, two compounds have been extensively studied
in this laboratory as prototype compounds of this group; 3-deazaadenosine
(3-deaza-Ado) and 3-deazaaristeromycin (3-deaza-Ari) . 3-Deaza-Ado is a potent
competitive inhibitor of AdoHcyase with Y.^ of 5-8 yM, and as a substrate has
a Kj^ value about equivalent to the natural substrate, adenosine. In contrast
to 3-deaza-Ado, 3-deaza-Ari is not a substrate for AdoHcyase, but it is a very
potent competitive inhibitor, with Kj^ of 2.0 nM for the hamster liver enzyme.
Neither compound is a substrate for either adenosine kinase or adenosine
deaminase.
The capacity of AdoHcyase to synthesize AdoHcy analogs in vivo, as has been
shown with 3-deaza-Ado, demonstrates the exciting possibility of synthesizing
potent and specific methylation inhibitors intracellularly . Comparison of the
biological effects of 3-deaza-Ado and 3-deaza-Ari has made it possible to
attribute some of the differences in specificity to the finding that 3-deaza-
AdoHcy is a more potent and specific inhibitor of some transmethylation reactions
than AdoHcy. We have found that macrophage chemotaxis is specifically inhibited
by the intracellular formation of 3-deaza-AdoHcy , brought about by treatment of
the cells with 3-deaza-Ado, while chemotaxis is unaffected by accumulation of
AdoHcy by treatment with 3-deaza-Ari (see ZOl MH 00942-06). We have further
shown that inhibition of chemotaxis by 3-deaza-Ado is correlated with inhibition
of the synthesis of specific proteins which are not inhibited by 3-deaza-Ari.
The inhibition of synthesis of specific proteins was attributed to an inhibition
of mRNA synthesis. Both 3-deaza-Ado and 3-deaza-Ari were used to inhibit mRNA
methylation, and the methylation of adenosine on the N-6 position was very
sensitive to inhibition, while methylation of the guanosine in the mRNA cap was
only slightly inhibited. In contrast, mRNA synthesis was greatly inhibited with
3-deaza-Ado and only partially inhibited with 3-deaza-Ari. Both 3-deaza-Ado and
3-deaza-Ari also inhibit the replication of various RNA and DNA viruses. The
sensitivity of various viruses to these two drugs is different, and it seems
probable the some of the antiviral effects can be attributed to an inhibition
of viral mRNA synthesis or methylation. The specific reaction(s) involved in
inhibition of RNA synthesis has not been identified, and the effect of both
872
ZOl MH 00931-14 LGCB
compounds on viral RNA methylation may be useful for examining the role of these
reactions in the synthesis and processing of different classes of viral RNA.
We have shown that the cytostatic effect of 3-deaza-Ari for RAW264 cells,
can be reversed by micromolar concentrations of homocysteine. Since AdoHcy is
the only cellular source of homocysteine, we concluded that cells incubated with
3-deaza-Ari cannot recycle methyltetrahydrof olate and regenerate tetrahydrof olate
for use in d^^ novo synthesis of purines and pyrimidines. This condition is
similar to the situation with vitamin Bjl2 deficiency, which inactivates methionine
synthase, and causes methyltetrahydrof olate to accumulate. In addition, it would
be expected that cells incubated with 3-deaza-Ari would contain less cystathionine,
an amino acid without a known function that is found in high concentration in
the brain. These findings could have clinical significance in situations where
AdoHcyase is inhibited such as the administration of Ara-A and patients with
adenosine deaminase deficiency.
In a series of recent studies in Europe and in this country, it has been
found that AdoMet, given parenterally to depressed patients produced rapid and
remarkable improvement in the clinical picture. These studies indicate that
AdoMet has approximately the same antidepressant activity as the standard
tricyclics, such as imipramine, amitryptiline, etc. It is noteworthy, however,
that administration of Adol>fet is not accompanied by any toxic side effects, and
thus, this mode of therapy may represent a considerable improvement over the
therapeutic regimens currently in use. The mechanism of action of Adoffet in
depressive illness is unknown. It should be pointed out, however, that the
dose of AdoMet found to be effective in the management of clinical depression
(200-400 mg/i.v./day) is very small compared to the daily flow of methionine
through AdoMet. Human adults synthesize and metabolize about 20 millimoles of
AdoMet/day, or 20-40 times the dose used in clinical trials.
Significance to Biomedical Research and the Program of the Institute:
Studies of the AdoHcyase and its inhibitors are important to understanding
the regulation and function of biochemical transmethylations, and have possible
clinical applications in the development of specific inhibitors for certain
methylation reactions. Since AdoMet dependent methylation reactions are involved
in the synthesis of so many compounds, including DNA, RNA, proteins, lipids,
and neurotransmitters, the regulation of these reactions can alter many cell
functions. Inhibitors of methylation reactions have been shown to affect cell
differentiation, leukocyte chemotaxis, and virus replication. The possible
clinical applications could be in the development of compounds for use in
chemotherapy, immunosuppression, and antiviral drugs. Because of the important
role of methylation in neurotransmitter synthesis, these compounds could have
important effects on brain function as well.
Proposed Course of Research:
The cloned cDNA sequence will be used for site directed mutagenesis in
order to characterize the role of specific amino acids in the enzyme catalysis.
The genomic organization of the hydrolase gene is also being investigated.
Studies on several inhibitors will continue in order to determine specific
mechanisms of inhibition, and to determine correlations between inhibition of
873
ZOl MH 00931-14 LGCB
specific reactions and the physiological effects of these compounds. Much of
the work will focus on methylation reactions involved in leukocyte chemotaxis,
and on the role of DNA methylation in gene expression. The mechanism for how
the pattern of DNA methylation is changed during differentiation will continue
to be examined. In addition, the role of different RNA methylations on RNA
metabolism and protein synthesis will also be examined.
Publications:
de la Haba, G. , Agostini, S., Bozzi, A., Merta, A., Unson, C. and Cantoni, G.L. :
S-Adenosylhomocysteinase: Mechanism of reversible and irreversible inactivation
by ATP, cAMP and 2' -deoxyadenosine. Biochemistry, 16 8337-8342, 1986.
I
Backlund, P.S. Jr., Carotti, D. and Cantoni, G.L. : Effects of the S-adenosyl-
homocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on
RNA methylation and synthesis. Eur. J. Biochem. 160 : 245-251, 1986.
Ogawa, H. , Gomi , T. , Mueckler, M.M. , Rijioka, M. , Backlund, P.S. Jr., Aksamit,
R.R. , Unson, C.G. , and Cantoni, G.L. : Amino acid sequence of S-adenosyl-L-
homocysteine hydrolase from rat liver as derived from the cDNA sequence. Proc.
Natl. Acad. Sci. USA. 84: 719-723, 1987.
Gahl, W.A., Finkelstein, J.D., Mullen, K.D. , Bernardini, I., Martin, J . J . ,
Backlund, P., Ishak , K.G. , Hoofnagle, J.H. , and Mudd, S.H. : Hepatic methionine
adenosyltransf erase deficiency in a 31-year-old man. Am. J. Hum. Genet. 40:
39-49, 1987.
874
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 00936-23 LGCB
TITLE OF PROJECT ^80 chartctera or Jau. 7W* must tit on om line belwttn tha borders.)
Homocystinuria: Methionine Metabolism in Mammals
PRINCIPAL INVESTIGATOR (Lift other proteasionel personnel behw the Phncipal Investigator.) (Name, title, laboratory, and institute attillation)
S.H. Mudd Chief, Section on Alkaloid Biosynthesis LGCB NIMH
COOPERATING UNITS f/f«nw William Gahl Human Genetics Branch Child Health and Human Dev
James Finkelstein, Va Hospital and George Washington Univ., Washington, D.C.
Alfred Tangerman, Dept of Medicine, St. Radboud Univ. Hospital., Nijmegen,
The Netherlands
LAB/BRANCH
Laboratory of General and Comparative Biochemistry
SECTION
Section on Alkaloid Biosynthesis
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.1
PROFESSIONAL:
0.1
OTHER:
0
CHECK APPROPRIATE BOX<ES)
El (a) Human subjects D (b) Human tissues D (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies of a 31-year-old man with proven partial deficiency of hepatic
methionine adenosyl transferase (MAT) activity have been contlTiued. Identified
abnormal metabolites in his bodily fluids, urine, and/or breath include:
L-methionine-d-sulf oxide , 4-methylthio-2-oxo-butyrate , 3-methylthiopropionate,
dimethylsulfide, and a mixed disulfide CH^S-SX, the structure of v^^iich is
still under investigation. Balance studies" have permitted calculation of the
fluxes of methyl- and sulfur-containing compounds and shown that: In spite of
the deficient activity of hepatic MAT, the patient forms a normal amount of the
product of this enzyme, S-adenosy Methionine (SAM). In spite of the normal rate of
fbrmation of SAM, the patient does not convert methionine sulfur to sulfate at
a normal rate. In spite of his high body load of methionine, the patient conserves
methionine by N§-metAyltetrahydrofolate-dependent metAylation of homocysteine.
The later observations are explained in terms of the regulatory effects of
SAM on cystathionine synthase and methylenetetrahydrofolate reductase. In the
presence of 20- to 50-fold elevations of methionine, the transamination pathway
metabolizes about 20% of the normal methionine intake of this patient, although
the transamination pathway is clearly not sufficiently active to prevent the
accumulation of methionine.
875
PHS 6040 (Rev. 1/84) '"•° •'«-•••
Project No. ZOl MH 00936-24 LGCB
Studies of a 31-year-old man with documented partial (93% decrease) deficiency
of hepatic methionine adenosyltransferase (MAT) have been continued to answer
the questions posed in last year's annual report. MAT activity has been assayed
in erythrocytes and cultured skin fibroblasts and found to be normal. The
implication is that extra-hepatic form(s) of MAT are under genetic regulation
different from that governing the chief hepatic form. Lean body mass has been
measured by determination of total body potassium in a whole body counting
chamber, and found to be 63.8 kg, or 87% of total body weight. In nonnal persons
this would be reflected by excretion of 14.7 mmol creatinine/day. The patient
excreted 13.9 +/- 0.4, very close to the predicted value. His liver is normal
on histological examination and by a variety of function tests.
The following sulfur-containing compounds have now been identified in
urine, breath, and/or bodily fluids of the patient in abnormal amounts:
(a) L-Methionine-d-sulf oxide. The presence of one diastereoisomer, only, implies
the existence of a here-to-fore undescribed enzyme in humans forming this
sulfoxide.
(b) 4-Methylthio-2-oxobutyrate, the immediate product of methionine
transamination.
(c) 3-Methylthiopropionate, formed by oxidative decarboxylation of the above
keto acid.
(d) Dimethylsulf ide, present in 17-fold normal concentrations in the breath of
the patient, and accounting for the peculiar breath odor v\*iich was his
chief complaint.
(e) A mixed disulfide, CH3S-SX, which is a quantitatively important urinary
excretion product at 2.1 mmDl/day. The structure of this hitherto undescribed
compound is under investigation.
The patient was placed upon several diets, each with a different methionine
intake. Excretions of methyl- and sulfur-containirq compounds were measured,
both in a steady-state during intake of a diet normal for this individual,
and during methionine restriction and supplementation. The results obtained
enabled us to construct methyl and sulfur balances, and to arrive at several
important and novel conclusions:
(a) In spite of his proven deficiency in hepatic MAT activity, the patient
forms the product of this reaction, S-adenosylmethionine (SAM) , at
a virtually normal rate of 18 mmol/day. Presumably this is achieved
by virtue of the build-up of methionine proximal to the block driving
flux through any residual activity of hepatic high Kjj, MAT. The normal
extrahepatic MAT also must contribute, but the extent of this contribution
is uncertain.
(b) In spite of the normal rate at vrfiich SAM is formed, the patient does
not convert methionine sulfur to sulfate at a normal rate on his usual
diet, and does not respond normally to changes in methionine intake with
commensurate changes in sulfate output.
(c) In spite of his extremely high body-load of methionine, the patient
carries out a high rate of N^-methyltetrahydrofolate-dependent methylation
of homocysteine (8.8 mmol/day). He thus reconverts homocysteine to
methionine and conserves the latter amino acid under circumstances in
which a normal human would not do so.
876
project No. ZOl MH 00936-24 LGCB
(d) The apparently anomalous conclusions outlined in (c) and (d) , above,
can be reconciled and explained in terms of a lack of the regulatory
effects of SAM upon cystathionine synthase and methylenetetrahydrofolate
reductase. These effects, until now demonstrated only ui vitro, each
tend to enhance the proportion of available homocysteine v^iich, under
conditions of methionine excess, is converted to cystathionine for
eventual degradation rather than beirq conserved by methylation to
reform methionine.
(e) The transamination pathway metabolizes at least 20% of the methionine
degraded by this patient. This is achieved in the presence of 20-50 fold
elevations of methionine above normal. The transamination pathway is clearly
not active enough to prevent such accumulations.
Publications:
1. Mudd, S.H.: Homocystinuria, In J.B. W/ngaarden and L.H. Smith (Editors),
Cecil Textbook of Medicine, 18th edition, W.B. Saunders, Philadelphia, 1987,
in press
2. Mudd, S.H. , Levy, H.L., Skovby, F.: Disorders of transsulfuration. In
Scriver, C.R. , Beaudet, A.L. , Sly, W.S. , Valle, D. (Editors). The Metabolic
Basis of Inherited Disease, 6th edition, McGraw Hill Book Co., N.Y. 1987,
in press
3. Gahl, W.A. , Bernardini, I., Finkelstein, J.D. , Tangerman, A., Martin,
J.J., Blom, H.J., Mullen, K.D. , and Mudd, S.H. Transsulfuration is an adult
with hepatic methionine adenosyltransf erase deficiency, submitted J. Clin.
Invest. 5/11/87
877
DEPARTMENT OF HEAI.TH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00940-06 LGCB
PERIOO COVERED
October 1, 1986 to September '^0, T
TITLE OF PROJECT (80 etmncmt or Im*. TTtto miM monontitnt bttwtn tfM bordft.)
Methionine Biosynthesis in Higher P^ar.^
PRINCIPAL INVESTIGATOR (LM othtf pntttionl fmnonnti btlow (ft* Principal Invtttiguor.) (Nam; Vtlt, laOontory, and insutula altiUation)
A.H. Datko .Biologist -r ^^
^ "^ LGCB NIMH
S.H. Mudd Chief, Section on Alkaloid Biosynthesis
LGCB NIMH
COOPERATING UNITS (» any)
R. Aksamit, LGCB NIMH
LAS/BRANCH
Laboratory of General and Comparative Biochemistry
SECTION
Section on Alkaloid Biosynthesis
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAL:
1.7
CHECK APPROPRMTE eOX(ES)
D (a) Human subjects
U (a1) Minors
D (a2) Interviews
D (b) HufTian tissues 9 (c) Neither
SUMMARY OF WORK (Utt uandani unmiuead typa.Donol axcaad Iha apaea prwktad.)
Investigation of the pathways for biosynthesis of phosphatidylcholine
has been extended to two plants in addition to the Lemna previously
studied, and to rat liver and hepatoma cells. Especial attention was
focussed upon the role of phosphoethanolamine derivatives by use of
methods v*iich had demonstrated the participation of such derivatives
in Lemna. Surprisingly, each of the plant tissues uses different steps to
carry out the methylation reactions involved, although methylation of
phosphoethanolamine appears to be the committing step common to eacTT
plant. Choline, or a metabolite thereof, markedly down-regulates entry
of methyl groups into- the network of methylated ethanolamine derivatives
in each of the three tissues.
Rat liver uses a pathway Oiich is again different in which all three
methylations take place at the phosphatidyl-base level. In hepatoma cells
it was demonstrated that entry of methyl grbups into this pathway is"^
regulated also by the availability of choline.
879
SPO tl4.*ll
Project No. ZOl MH 00940-06 LGCB
Project Description:
During the past year we have continued our investigations of the biosynthesis
of phospholipids. Our earlier studies in this area, reported in previous
annual reports, had demonstrated that Lemna utilizes a pathway for formation
of phosphatidylcholine different from that previously accepted for both plants
ar>d animals. To assess the biological generality of this new pathway, ve
have now studied two additional higher plants, and reexamined liver using
our new techniques. Surprisingly, the pathways utilized for this major
biosynthetic process appear to be different in each of the four systans
examined.
The plant systems have each been studied by a number of techniques. These
include:
(a) Measurement of the rates of transfer of radioactively labeled
methyl groups originating in methionine to methylated ethanolamine derivatives
of three classes: the free bases, methylethanolamine (MEA) , dimethylethanolamine
(DMEA) , and the trimethyl derivative, choline; the phosphate esters of
these bases, i.e. phospho-bases (abbreviated as P-MEA, P-DMEA, and P-choline,
respectively); and the phosphatidyl derivatives of the same bases, i.e.
phosphatidyl-bases (abbreviated as phtd-MEA, phtd-DMEA, and phtd-choline) .
In these studies Lemna, or tissue-cultured cells of soybean or carrot
were exposed continuously under unperturbed growth conditions to a tracer
dose of methionine labeled with tritium in the methyl group. Exposure
times were short, from 0.5 to 15 minutes in various experiments. The
plant materials were harvested and rinsed rapidly, and homogenized at
-60 degrees. Each of the methylated ethanolamine derivatives mentioned above
was purified and the amount of tritium in each determined.
(b) Assay in cell-free extracts of enzyme activities catalyzing the following
reactions:
(1) Transfer of methyl groups from S-adenosylmethionine to phospho-
ethanolamine (P-EA) , P-MEA, or P-DMEA.
(2) Transfer of methyl groups from S-adenosylmethionine to phosf*iatidyl-
ethanolamine (phtd-EA) , phtd-MEA, phtd-DMEA.
(3) Transfer of the cytidyl moiety of CTP to P-MEA, P-DMEA, or P-choline,
forming the respective CDP-bases.
(c) Experiments similar to those described under (a), but utilizing plant
material v*iich had been grown for at least several doublings in a high external
concentration of choline. These studies were aimed at investigating possible
regulatory effects of exogenous choline.
The major results with the respective plants, and our interpretations of these
results are as follows:
(a) Lemna. This plant rapidly incorporates methyl groups into P-MEA, P-DMEA,
and P-choline. Radioactive methyls appear in phosphatidyl-tases only after a
marked initial lag. For example at earliest times, about 100 times as much
radioactivity has entered the phospho-bases as the phtd-bases. Radioactivity
eventually accumulates almost entirely in phtd-choline, but the partially
methylated phosphatidyl derivatives, phtd-MEA and phtd-DMEA, never acquire more
than trivial amounts of radioactivity. The free bases at short times are
880
Project No. ZOl MH 00940-06 LGCB
virtually unlabeled. Enzyme activities methylating P-EA, P-MEA, and P-DMEA
were each demonstrated. Microsomes could methylate phtd-MEA and phtd-DMEA,
but not phtd-EA. Cyt idyl-trans f erases active with P-MEA, P-DMEA, and
P-choline were demonstrated. These facts suggest that the major pathway is:
P-EA — > P-MEA —> P-choline > CDP-choline — > phtd-choline
Minor pathways may occur via cytidyl transfers to P-MEA and P-DMEA, with
subsequent formation of the phosphatidyl compounds, and their methylation
to phtd-choline.
(b) Soybean. These cells rapidly incorporate methyl groups into P-MEA,
but differ from Lemna in failing to incorporate detectable radioactivity
into either P-DMEA or P-choline. Radioactivity appears rapidly also in
phtd-MEA, phtd-DMEA, and phtd-choline, but not in any of the free bases.
An enzyme methylating P-EA was demonstrated, but no activity was detected
for methylation of P-MEA or P-DMEA. Microsomes were similar to those of
Lemna, catalyzing methylation of phtd-MEA and phtd-DMEA, but not phtd-EA.
Cytidyl transferase activities were similar to those of Lemna. These facts
suggest that the major pathway is:
P-EA — > P-MEA > CDP-MEA > phtd-MEA > phtd-DMEA >• phtd-choline
(c) Carrot. These cells resemble Lemna (and differ from soybean) in
that they incorporate methyls rapidly into each of the three phospho-bases,
P-MEA, P-DMEA, and P-choline. Conversely, carrot cells differ from Lemna
(and resemble soybean) in that they incorporate methyls rapidly into
each of the three phosphat idyl-bases, phtd-MEA, phtd-DMEA, and phtd-choline.
Activities for methylation of P-EA, P-MEA, P-DMEA, phtd-MEA, and phtd-DMEA
were demonstrated, but, again, none for methylation of phtd-EA. Cytidyl
transferase activities were similar to those of both Lemna and soybean.
These facts suggest that in carrot significant methylation occurs at
both the phospho-base and the phtd-base levels. Quantitative interpretation
is more difficult with these cells, but to emphasize the third possibility
for a major pathway we tentatively suggest the following:
P-EA— > P-MEA — >-P-DMEA > CDP-DMEA > phtd-DMEA >- phtd-choline
There are probably more or less significant cytidyl transfers to both
P-MEA and P-choline also.
(d) With each tissue, growth in exogenous choline down- regulated entry of
methyls into the total network of methylated ethanolamine derivatives. Such
down-regulation amounted with Lemna to a 93% decrease; with soybean, to a 77%
decrease; and with carrot, to a 99% decrease. The locus of this down-regulation
may be placed with some confidence at the step v*iich initially introduces
methyl groups into this network. Methylation of P-EA, viiiich we postulate is the
committing step ccmman to the three tissues examined would appear to be the
best candidate for this regulation.
881
Project No. ZOl MH 00940-06 LGCB
We have recently extended these studies to the synthesis of phtd-choline
by rat liver and by hepatoma cells grown in tissue culture. Although it
is generally accepted that mammals synthesize phtd-choline by successive
methylations at the phosphat idyl-base level, a review of the evidence revealed
that in the early experiments by Bremer and coworkers and Gibson and coworkers
vi^ich are regarded as establishing the mammalian pathway, no attempt was
made to directly detect the partially methylated P-bases after short incubations
with methionine labeled in the methyl group. Further, in later experiments
published in 1973, Salerno and Heeler (Biochim Biophys Acta 326, 325, 1973)
reported that after intraportal injection of [H^C] methionine the specific
activity of P-choline in rat liver rose rapidly to a maximum at 30 seconds
after injection and then declined to a basal equilibrium before phtd-choline
had readied one-half of its maximum value. These authors interpreted their
results "to indicate the direct methylation of P-EA to P-choline". These obser-
vations have not been confirmed or refuted by subsequent publications. In
addition, the possible regulation of phtd-choline synthesis by choline had not
been settled for mammalian systems. For these reasons we decided to apply the
methods which had successfully settled the analogous questions in plant tissues.
To study rat kiver in as unperturbed a state as possible, two experiments
were carried out: First, an intraperitoneal injection of [H^C] methionine with
sacrifice of the animal 10 minutes after injection. Separate control experiments
showed that, after intraperitoneal injection, total radioactivity in the liver
increased almost linearly with time for at least 20 minutes. During the
10 minute experiment the liver was therefore exposed continuously to
[ H-jC] methionine. Second, an intraportal injection over 30 seconds with
immediate sacrifice of the animal. After the 10 minute experiment, 31%
of the radioactivity in liver was converted to phtd-choline, 3% was recovered
in phtd-DMEA, and 0.7% in phtd-MEA. No radioactivity (<0.02%) was detected
in P-MEA or P-DMEA, with 0.05% in P-choline. After the 30 second experiment,
1.9% of total hepatic radioactivity was recovered in phtd-choline, 1.5% in
phtd-DMEA, and 0.42% in phtd-MEA. No radioactivity (<0.004%) was detected in
P-bases. These results provide strong support for the conclusion that in rat
liver the pathway is:
phtd-EA > phtd-MEA > phtd-DMEA ^ phtd-choline
With hepatoma cells, the relative proportion of methionine methyl converted
to phtd-choline was much less, about 0.3% in 24-hour experiments. Again, no
indication that P-bases participate as intermediates in the methylation process
was obtained. However, it was possible to find conditions for these cells under
vi^ich their growth was limited by external choline. After several days of exposure
to such conditions, the rate of entry of methionine methyls into the phtd-bases
was increased about 3- fold, demonstrating a considerable degree of regulation
of the methylation pathway by choline, or a metabolite thereof.
882
Project No. ZOl MH 00940-06 LGCB
Publications:
Mudd, S.H. , Datko, A.H.: Phosphoethanolamine Bases as Intermediates in
Phosphatidylcholine Synthesis by Lemna. Plant Physiol. , 82, 126-135 (1986)
883
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 00942-06 LGCB
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less Title must lit on one line between the borders.)
Biochemical Reactions in Mammalian Cell Chemotaxis
PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I. R. R. Aksamit Research Chemist LGCB NIMH
P. S. Backlund, Jr. Senior Staff Fellow
G. L. Cantoni
Chief, Laboratory of General
and Comparative Biochemistry
LGCB NIMH
LGCB NIMH
COOPERATING UNITS (if any)
Office of Biologies, FDA; Molecular Pathophysiology Section, NIDDJ;
Department of Biochemistry and Pharmacology, University of Glasgow, Scotland
LAB/BRANCH
Laboratory of General and Comparative Biochemistry
SECTION
Section on Proteins
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1
PROFESSIONAL:
0.5
0.5
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
D (a1) Minors
D (a2) Intervievi/s
(b) Human tissues D (c) Neither
SUMM/KRY Of WOBK (Use standard unreduced type. Do not exceed the space provided ) . ..... , , _ , „^^h«^,^
Chemotaxis by the RAW264 mouse macrophage cell line was inhibited by 3-deazaadeno-
sine but not by 3-deazaaristeromycin. A search for biochemical reactions inhibited
by3-deazaadenosine but not by 3-deazaaristeromycln has revealed that only one reac-
tion, the synthesis of a small number of proteins identified after separation by
two-dimensional polyacrylamide gel electrophoresis, has the necessary Inhibitor
specificity for involvement in the 3-deazaadenoslne-sensitive step of chemotaxis.
A study with several adenosine analogs showed a correlation between inhibition of
chemotaxis and inhibition of the synthesis of a common subset of proteins. These
analogs also inhibited the synthesis of polyadenylated mRM, leading us to postulate
that incubation of cells with 3-deazaadenosine inhibits methylation reaction(s)
required for the formation of functional mRNA coding for one or more proteins
required for chemotaxis.
Experiments to identify attractant-specif ic proteins have been limited because
chemically defined attractants for RAW264 cells have not been available. This
problem has been overcome by the isolation of a stable cell hybrid from a fusion
between human leukocytes and a thioguanine-resistant RAW264 cell line. The hybrid
expressed functional genes for chemotaxis to fMet-leu-phe, a commercially available
synthetic attractant. Binding of fMet-leu-phe to hybrid cell membranes indicated
that the binding constant was 2 nM and each cell had an average of 1200 receptors.
In addition to chemotactic receptors, one or more guanine nucleotide binding
proteins are required for chemotaxis by RAW264 and the hybrid cells. This conclu-_
sion is based on the observation that chemotaxis of either RAW26i( or hybrid cells is
inhibited upon incubation of the cells with either cholera toxin or pertussis toxin.
In all cases entry of the toxin is required and there is a correlation between
toxin-catalyzed ADP-ribosylation of a guanine nucleotide binding protemand the
inhibition of chemotaxis. Although both cholera toxin and pertussis toxin affect
CAMP levels elevated cAMP levels per se do not inhibit chemotaxis. By immunochemi-
^Sr-and electrophoretic techniques, the pertussis toxin substrate involved in
chemotaxis has been identif i,ed-a^-Ni:^-a J?rg^gijl-.that_is also found in brairu
PHS 6040 (Rev 1/84)
GPO 914-SIB
Z01 MH 00942-06 LGCB
Other Investigators:
A. Spiegel Chief, Molecular Pathophysiology NIDDK
Section
G. Milligan Assistant Professor, Univ. of Glasgow
T.M. Caryk Chemist LGCB NIMH
L. Harvath Research Microbiologist DEEP FDA
The important discovery in this laboratory that chemotaxis by a macrophage cell
line is specifically inhibited by 3-deaza-AdoHcy has allowed us to assess the
significance of certain biochemical reactions in macrophage chemotaxis. Our
conclusion was based on the finding that RAW254 chemotaxis is inhibited by
3-deazaadenosine but not by 3-deazaaristeromycin, and a search was initiated for
a biochemical reaction that also showed this inhibitor specificity.
The synthesis of phosphatidylcholine by methylation of phosphatidylethanolamine,
the release of arachidonic acid when cells are incubated with EAMS (endotoxin-
activated mouse serum, an attractant for mouse macrophages), methylation of the
lysine and arginine residues of protein, and protein carboxymethylation were all
inhibited by both 3-deazaadenosine and 3-deazaaristeromycin. From these studies
we conclude that none of these reactions are required for chemotaxis by RAW264
cells.
In contrast, the synthesis of a small number of proteins, identified after
separation by two-dimensional polyacrylamide gel electrophoresis, does show
the necessary inhibitor specificity for involvement in RAW264 chemotaxis.
Quantitation of 100 of the more prominent proteins on the gels by computerized
densitometry showed that in cells treated with 3~deazaadenosine the synthesis
of approximately ^0% of the proteins was inhibited by more than 50?, whereas
in cells treated with 3-deazaaristeromycin the synthesis of these proteins was
not significantly inhibited. The correlation of the inhibition of a subset of
proteins with the inhibition of chemotaxis was strengthened by the finding that
other inhibitors of chemotaxis inhibited the synthesis of the same subset of
proteins. These inhibitors are 3-deoxyadenosine and the combination of erythro-
9-(2-hydroxy-3-nonyl)adenosine (EHNA), adenosine and homocysteine. A common
feature of the inhibitors of chemotaxis described above is that they all can
inhibit the synthesis of functional mRNA. In this regard, we have also found
that inhibitors of protein synthesis and translation, such as cycloheximide,
puromycin and actinomycin D, inhibit chemotaxis.
We have proposed as a working hypothesis that treatment of RAW264 cells with
3-deazaadenosine, 3'""deoxyadenosine, and the combination of EHNA, adenosine
andhomocysteine inhibits the synthesis of functional mRNA coding for one or
morechemotactic proteins. In support of this hypothesis, we have found that
3-deazaadenosine is a more potent inhibitor of polyadenylated mRNA than
3-deazaari3teromycin and that AdoHcy and 3-deazaAdoHcy do not inhibit ui vitro
translation.
Time-lapse video cinematography shows that motility and EAMS-induced
morphological changes are similar in 3~deazaadenosine-treated and control
cells. These observations suggest that in cells treated with 3~deazaadenosine,
signal processing after attractant binding to the chemoreceptor is inhibited.
886
Z01 MH 00942-06 LGCB
Additional studies to examine directly the effects of 3-deazaadenosine on
attractant binding or to investigate the steps in signal transduction have
been hindered by the lack of chemically defined attractants. The attractants
described for RAW254 cells, EAMS and LDCF (lymphocyte-derived chemotactic
factor), are both complex molecular mixtures with multiple biological
activities. On the other hand, human monocytes and neutrophils are known to
exhibit chemotaxis to FMLP (N-formylmet-leu-phe) , a commercially available
synthetic attractant. For these reasons hybrid cells were isolated from fusions
between human leukocytes and thioguanine-resistant RAW264 cells, and some of the
hybrids exhibited chemotaxis to FMLP and structurally related N-formylpeptides.
The WBC26M-9 cell line has been cultured for more than 6 months without loss of
chemotaxis to FMLP demonstrating that a stable cell line has been obtained.
Chemotaxis of WBC26M-9 and human leukocytes to FMLP are similar in several
respects. The concentrations of N-formylpeptides that elicit the optimal
chemotactic response in WBC264-9 cells are similar to the optimal chemotactic
concentrations reported for human leukocytes. WBC254-9 migrates more quickly
to FMLP than to EAMS, and the time course of WBC264-9 migration to FMLP is
similar to that of human leukocytes. It also appears that WBC254-9 chemotaxis
to FMLP and to EAMS may be regulated independently.
However, a study of the binding of radiolabeled FMLP to WBC264-9 cells indicated
that WBC264-9 cells contained fewer receptors than human leukocytes do, although
it should be noted that the receptor number is sufficient for chemotaxis. To
reduce the technical problem of nonspecific binding, the number of receptors on
membrane preparations was determined. From this data it was calculated that
there are approximately 1200 receptors per WBC264-9 cell, compared to reported
values from 2000 to 120,000 per cell for human leukocytes. The apparent
dissociation constant for FMLP binding. to WBC2614-9 membranes was 2 nM, in
agreement with values reported for the high affinity site of human leukocytes.
It has been proposed that the high affinity receptors for FMLP mediate
chemotaxis.
Studies on the human FMLP receptor have been carried out in collaboration
with Dr. L. Harvath. Our laboratory's principal contribution has been the
preparation and analytical determination of chemical derivatives of FMLP.
These studies have shown that human monocytes exhibit chemotaxis for both FMLP
sulfoxide and sulfone, whereas human neutrophils do not exhibit chemotaxis to
either of the oxidized peptides. In contrast, both human neutrophils and
monocytes migrate to FMLP, and both cell types generate superoxide anion,
secrete enzymes and polarize when stimulated with FMLP, FMLP sulfoxide or FMLP
sulfone. These data suggest that the FMLP receptor complex or chemotaxis
transduction mechanism is different in human neutrophils and monocytes.
In collaboration with Dr. Harvath, we have also developed flow cytometric
procedures that allow us to determine the subpopulation of leukocytes in whole
blood that bind a fluorescent FMLP derivative and to determine the rate of
binding of fluorescent FMLP to human neutrophils.
In addition to chemotactic receptors, one or more guanine nucleotide proteins
(N-proteins) are required for chemotaxis by RAW264 and WBC264-9 cells. This
887
Z01 MH 00942-06 LGCB
conclusion is based on the observation that chemotaxis of either RAW264 or
WBC264-9 cells is inhibited upon incubation of the cells with either cholera
toxin or pertussis toxin. In all cases entry of the toxin is required and
there is a correlation between toxin-catalyzed ADP-ribosylation of an N-protein
and the inhibition of chemotaxis.
Although both cholera toxin and pertussis toxin can affect cAMP levels, our
evidence indicates that cAMP is not involved in chemotaxis. This was shown
by elevating cAMP with either isoproterenol or forkolin to levels comparable
to those achieved with cholera toxin. Chemotaxis of cells treated with
isoproterenol or forkolin was not inhibited, showing that increased levels
of cAMP per se do not inhibit chemotaxis.
In agreement with observations in several other laboratories, we found that
the major membrane protein ADP-ribosylated by cholera toxin is distinct from
that ADP-ribosylated by pertussis toxin. This was shown by the different
electrophoretic mobilities of the proteins and the difference in the nucleotide
specificity of the ADP-ribosylation reactions. However, under certain condi-
tions, cholera toxin also appeared to ADP-ribosylate a membrane protein with a
molecular weight similar or identical to the substrate for pertussis toxin.
Further immunochemical studies that employed a battery of antisera specific for
several pertussis toxin substrates indicated that RAW254 cells have only one
major pertussis toxin substrate identified as Ni~2. A similar protein was also
identified in bovine brain. It is likely that Ni-2 is the guanine nucleotide
binding protein that couples chemotactic receptors to an effector protein such
a phospholipase C or ion channels.
Significance of Biological Research to the Program of the Institute:
Several reports have shown that stress-induced neuropeptides modulate
immunological activities and that leukocytes have receptors for beta- endorphin
and other neuropeptides. Chemotaxis is an important component of the
immunological response, and it has been shown that human monocytes exhibit
chemotaxis to met-enkephalin and beta-endorphin. Injection of beta-endorphin
into the rat cerebral ventricle results in the immigration of macrophage-like
cells, indicating that chemotaxis to beta-endorphin can occur in vivo.
Identification of the steps involved in chemotaxis would provide a basis for
the development of strategies to counteract stress-induced immunological
dysfunction.
Guanine nucleotide binding proteins are important components of signal
transduction by both hormones and chemoattractants. Brain is also one of
the richest sources of guanine nucleotide binding proteins, suggesting that
these proteins may be important regulators of brain function. Studies of
interactions between receptors, guanine nucleotide binding proteins, and various
effector systems, such as adenylate cylase, should improve our understanding of
signal transduction mechanisms in cells.
Mammalian cell chemotaxis is also important in the development of the nervous
system, inflammation and wound healing, and chemotaxis is a behavioral response
888
Z01 MH 00942-06 LGCB
at the cellular level. Studies of bacterial chemotaxis from the laboratories
of Koshland and Adler have shown that bacteria have "memory" and adapt to
their environment, and progress has been made in explaining these concepts in
molecular terms. The mammalian cell line model for chemotaxis that we have
developed provides a mammalian system to test concepts developed from bacterial
chemotaxis and to study the biochemical reactions involved in signal transduc-
tion.
Proposed Course of Research:
Future work will be directed toward the identification of biochemical components
of chemotaxis. These problems will be approached by a combination of bio-
chemical and molecular biology techniques.
Publication:
Aksamit, R.R.: A human-mouse hybrid cell line that stably expresses chemotaxis
to N-formylmethioniyl-leucyl-phenylalanine. Biochem. Blophys. Res. Commun. 138:
1001-1008, 1986.
Patent :
Aksamit, R.R,: A human-mouse hybrid cell line expressing monocyte-macrophage
properties, ref E-404-85; patent pending.
889
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 to September 30, 1987
PROJECT NUMBER
ZOl MH 00943-06 LGCB
TITLE OF PROJECT (BO chvuefn or tou. TW* muat htonontUM tutw—n m* bor<l»ft.)
Pathways of Methionine and Threonine Metabolism and Their Control In Higher Plants
PRINCIPAL INVESTIGATOR (iMt othtr pfoltaiontl (mnonrmi tuttow th^ Princpitl InvttOgator.) (Nam*. tiOt, laboratory, and inslrtuia attiliauon)
P.I. J. Giovanelli Research Chemist
S.H. Mudd
LGCB NIMH
A.H. Datko
Chief, Section on Alkaloid
Biosynthesis LGCB NIMH
Biologist LGCB NIMH
COOPERATING UNITS (» any)
None
LAa/BRANCH
Laboratory of General and Comparative Biochemistry
SECTION
Section on Alkaloid Biosynthesis
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.2
PROFESSIONAL
1.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
a (a1) Minora
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (U»t u«ndvcf unmiuiMa lypt. Do not txamd tfw tfiac* pnnkhd.)
At least 80% of thie total activity of aspartokinase in crude extracts of
Lemna paucicostata was inhibited by lysine^ with the remaining activity inhibited
by threonine. Inhibition by lysine was synergistically increased by
S-adenosylmethionine, V(*iich by itself had no effect. Inhibition by lysine
and threonine was additive, not cooperative. Aspartokinase activity extracted
from Lemna was an order of magnitude greater than that reported by other
workers for other plant tissues, and in large excess (approximately 20-fold)
of the in vivo requirements for synthesis of the aspartate family of amino
acids. Either lysine-sensitive or threonine-sensitive aspartokinase activities
alone can support the combined in vivo flux into the aspartate family of
amino acids. Severe. inhibition of both forms of enzyme activity was required
to reduce this flux below the normal requirement. No evidence was obtained
for repression/derepression of aspartokinase in plants grown with amino
acids of the aspartate family. A major conclusion from these combined data
is that, contrary to suggestions of other workers, the step catalyzed by
aspartokinase does not appear to be the overall rate-limiting one for entry
of 4-carbon units into the aspartate pathway. Further, the findings confirm
the absence of major "channeling" of lysine-sensitive or threonine-sensitive
aspartokinases into separate biosynthetic branches of the aspartate family.
The findings also help explain why little, if any, feedback regulation of
threonine synthesis occurs in plants supplemented with threonine alone,
v\*iile complete feedlaack regulation of threonine synthesis occurs in plants
supplemented with both threonine and lysine.
891
HiS ijiri ;aev. ';i'04)
OI>C> •! J-il«
Project Nd. ZOl MH 00943-06 LGCB
Project Description:
Aspartokinase catalyzes the first cantnitting step in the synthesis of the
aspartate family of amino acids (lysine, threonine, isoleucine and methionine),
and has been suggested to act as the overall rate-limiting step in biosynthesis
of these amino acids. The enzyme was studied in extracts of Lemna grown
under control conditions or supplemented with one or more of the aspartate
family of amino acids. The high-lights of these studies are:
(1) Using a sensitive and specific assay developed for these studies,
aspartokinase was found to be cortprised of two activities. At least 80%
of the total activity of crude extracts was strongly inhibited by
lysine , but not by threonine. Inhibition by lysine was synergistically
increased by MoMet, which by itself had no effect. The remaining (lysine-
insensitive) activity was inhibited by threonine. Inhibition by lysine
and threonine was additive, with no indication of concerted inhibition
by these amino acids as reported in a number of bacteria eind another
species of Lemna (L. minor) .
(2) Activity of aspartokinase extracted from Lemna was an order of magnitude
greater than that reported by other workers for other plant tissues, and in
large excess (approximately 20-fold) of the in vivo requirements for synthesis
of the aspartate family of amino acids. Either lysine-sensitive or
threonine-sensitive aspartokinase activities alone can support the combined
in vivo flux into the aspartate family of amino acids. Severe inhibition
of both forms of enzyme activity was required to reduce this flux below
the normal requirement. Activities of lysine- and threonine-sensitive
aspartokinase determined in gel-filtered extracts were similar for control
plants and plants grown with a variety of supplements containing lysine,
threonine, and methionine, indicating that aspartokinase is not subject
to represssion/derepression by amino acid products of the aspartate
family. A major conclusion fron these coribined data is that, contrary
to suggestions of other workers, the step catalyzed by aspartokinase
does not appear to be the overall rate-limiting one for entry of 4-carbon
units into the aspartate pathway. The adequacy of either lysine- or
threonine-sensitive aspartokinase activities for nornal flux requirements
corroborates previous studies of labeling patterns obtained with [ l^Cjhanoserine
that argue against "channeling" of lysine-sensitive or threonine-sensitive
aspartokinase into separate biosynthetic branches of the aspartate family.
Further, the finding that both lysine- and threonine-irihibited aspartokinase
activities must be severely inhibited to cause significant reduction of
flux through the aspartokinase step helps explain why little, if any,
feedback regulation of threonine synthesis occurs in plants supplemented
with threonine alone, while complete feedback regulation occurs in plants
supplemented with both threonine and lysine.
892
Project No. ZOl MH 00943-06 ICCB
Significance to Bianedical Research and the Program of the Institute:
Our combined in vivo and hi vitro studies v/ith Lemna reveal many unusual
features in the regulation of synthesis of the aspartate family of amino acids.
An inportant practical application of these studies is that we are now
able to propose novel and logical strategies for iiiproving the nutritional
content of these amino acids in plants by genetic engineering.
Proposed Course of Future Research:
Regretably, we will not be able to pursue a number of intriguing avenues of
research, since this project will be abolished with the Section in October, 1987.
Publications :
Giovanelli, J. : Cystathionine B -Lyase fron Spinach. In W.B. Jacoby and
O.W. Griffith (eds). Sulfur and Sulfur Pmino Acids, Vol. 143, pp. 443-449,
1987, Academic Press, New York.
Giovanelli, J. : Sulfur Amino Acids of Plants: An Overview. In W.B. Jacoby and
O.W. Griffith (eds). Sulfur and Sulfur Amino Acids, Vol. 143, pp. 419-426, 1987,
Academic Press, New York.
893
DLFARTMENT OF HEALTH AND HUWAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, igS-e to September 30, 1987
PROJECT NUMBER I
i
ZOl MH 02321-02 LGCB '
i TITLE OF PROJECT (BO characters or less Title must til on one line between the borders.)
DNA Methylation and Gene E
PRINCIPAL INVESTIGATOR (List other prolessiona: personnel belov, the Principal Investigator. ) (Name, title, laboratory, and institute aftiliation)
P.I. G. L. Cantoni Chief, Laboratory of General LGCB
and Comparative Biochemistry
Others:
R. Razin
o. A^ostini
T. Gomi
Visiting Scientist, The Hebrew University
Jerusalem, Israel
Guest Researcher LGCB
Visiting Fellow LGCB
COOPERATING UNITS (if any)
Department of Cellular Biochemistry, The Hebrew University, Hadassah Medical
School, Jerusalem, Israel; Department of Human Biopathology , University of Rome,
La Sapienza, Rome, Italy
LAB/BRANCH
Laboratory of General and Comparative Biochemistry
SECTION
Section on Proteins
INSTITUTE AND LOCATION
NIMH, ADAMHA, Bethesda, Maryland 20892
TOTAL MAN-YEARS;
3.5
PROFESSIONAL:
OTHER;
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues E2 (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
When Friend Erythroleukemia cells (FELC) are exposed to a variety of chemical
agents capable of inducing terminal differentiation their DNA undergoes a
genome-wide demethylation in the absence of DNA replication. Considerable
evidence has accumulated to indicate that demethylation of specific genes, or
of portion of specific regions of the DNA, is correlated with gene expression.
The transient genome-wide demethylation observed during FELC differentiation
must be an expression of the fact that the overall pattern of DNA methylation
changes during differentiation with some genes becoming active in transcription
and others becoming silent. The mechanism of DNA demethylation is completely
unknown: theoretically, inhibition during at least two cycles of DNA replication
of maintenance methylase, an enzyme capable of methylating hemimethylated DNA,
could result in DNA demethylation and changes in the DNA methylation pattern.
However the inhibition of maintenance methylase can not be involved in the
genome wide, transient demethylation that is observed in the early phases of
FELC differentiation, since this occurs in the absence of DNA duplication.
895
PHS 6040 (Rev 1'84)
GPO SI 4-BIB
ZOl MH 02321-02 LGCB
Project Description:
When Frifciid Erythroleukemia cells (FELC) are exposed to a variety of chemical
agents capable of inducing terminal differentiation their DNA undergoes a
genome-wide demethylation in the absence of DNA replication. Considerable
evidence has accumulated to Indicate that demethylation of specific genes, or
of portion of specific regions of the DNA, is correlated with gene expression.
The transient genome-wide demethylation observed during FELC differentiation
must be an expression of the fact that the overall pattern of DNA methylation
changes during differentiation with some genes becoming active in transcription
and others becoming silent. The mechanism of DNA demethylation is completely
unknown: theoretically inhibition of maintenance methylase, an enzyme capable of
methylating hemimethylated DNA, during at least two cycles of DNA replication
could result in DNA demethylation and changes in the DNA methylation pattern.
However the inhibition of maintenance methylase can not be involved in the
genome wide, transient demethylation that is observed in the early phases of
FELC differentiation, since this occurs in the absence of DNA duplication.
In an attempt to elucidate the mechanism by which active demethylation takes
place, we have considered three possible mechanisms: i) removal of the
methyl group from 5-methylcytosine by direct demethylation; ii) removal of
5-methylcytosine and its replacement with cytosine through an enzymatic
mechanism not previously described; iii) removal of a stretch of DNA that
would include the 5-methylcytosine moiety by the conventional excision-repair
mechanism. The first mechanism may be excluded a priori since it would require
a reductive cleavage of a C — C bond by a biochemically unprecedented and
improbable reaction. Experiments were designed to distinguish between these
two mechanism. The results that we obtained indicate conclusively that
transient DNA demethylation is due to a unique and novel mechanism whereby
5-methylcytosine is specifically replaced by cytosine. The ratio of
5-methylcytosine: cytosine in DNA extracted from cells 12 hours after induction
is strikingly lower than that from untreated cells or from cells induced for 24
hrs. The timing of this transient demethylation indicates that the phenomenon
occurs in the absence of DNA replication. The removal of 5-methylcytosine and
its replacement by cytosine is specific both with regard to the base and its
position in the sequence of deoxynucleosides in DNA. This conclusion is bases
on the demonstration that the cytosine residues that become incorporated into
DNA in replacement of 5-methylcytosines were incorporated specifically into
methylatable (or CpG) sites.
3-Deazaadenosine has been extensively studied in this lab for its ability to
function as a substrate of AdoHcyase and give rise in vivo to a congener of
Adenosylhomocysteine endowed with specific biochemical characteristics. In
order to examine the relationship between biological methylation and the novel
5-methylcytosine replacement reaction the effect of the administration of 3-DZA
on FELC cell differentiation was examined.
It was found that treatment of FELC with 3-DZA and homocysteine during the
first 20 hours after induction with HMBA or DMSO will completely inhibit the
expression of of the differentiated state (measured at 72-96 hours). By
contrast when treatment with 3DZA was delayed until 24 hours after induction
896
ZOl MH 02321-02 LGCB
differentiation was not affected. The effect of 3DZA was specific (adenosine
nor deazaaristeromycin were active had no effect) and required the presence of
homocysteine, a result that indicates conclusively that the effect is mediated
by adenosylhomocysteinase. The striking correspondence in the timing of the
inhibition of differentiation produced by 3DZA and the replacement of
5-methylcytosine by cytosine adds weight to the hypothesis that this limited
and specific modification of DNA structure is correlated with gene expression.
Further work with intact cells is under way in order to delineate with greater
precision the sequence of events that take place within 24 hours after induction
and that commit the cells to differentiation. Hopefully these experiments
will lead to studies in cell free systems and characterization of the enzyme
mechanisms involved.
Significance of the Program to the Institute:
Biological methylation underlies many different physiological events. A role
for biological methylation has been proposed for such diverse phenomena as
memory and chemotaxis in bacteria, repair of cellular damage due to aging,
fruit ripening, membrane and receptor function, synthesis and maturation of
messenger RNA and gene expression.
In addition there is a growing body of experimental evidence that suggests that
alteration in specific methylation reactions may be important in disease. The
results from well controlled clinical studies have documented the efficacy of
S-Adenosylmethionine in depression, in chronic arthritis and in liver diseases.
The biochemical basis for these effects is entirely unknown and the search for
suitable explanations for these pharmacological effects presents a formidable
challenge .
All methylation reactions utilize S-Adenosylmethionine as the methyl donor and
are inhibited by S-Adenosylhomocysteine. The great number and variety of
reactions involving these intermediates has provided both an opportunity and an
obstacle to the elucidation of the controlling role of methylation reactions in
the different phenomena listed above. Continued efforts directed to unraveling
the physiological significance and control of methylation reactions is
fundamental to progress in this important area of research.
Publication:
None.
897
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOIMH 01037-19 LMB
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (BO characters or less. Title must tit on one line between the borders.)
The Role of the Cell Membrane in Cellular Organization: A Molecular Study
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: D. M. Neville, Jr. Chief, Sec. on Biophy. Chem. LMB, NIMH
Others: T.H.Hudson Staff Fellow LMB, NIMH
J. W. Marsh Staff Fellow LMB, NIMH
K. Srinivasachar Visiting Associate LMB, NIMH
K.-H. Jung Visiting Fellow LMB, NIMH
COOPERATING UNITS (if any)
B/BRANCH
Laboratory of Molecular Biology
ECTION. -„. , . , -,,
Section on Biophysical Chemistry
LOCATION
ethesda, Maryland 20892
TOTAL MAN-YEARS;
5.3
PROFESSIONAL:
3.3
2.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The general aim of this project is to determine the chemical interactions and energetics such as
membrane potential and pH and ifin gradients which are involved in the insertion of proteins into
cellular membranes and/or the translocation of proteins across cellular membranes. The events are
studied from the initial receptor binding to the final physiologic response or pathological response
in the case of toxins such as ricin. colicins. diphtheria and tetanus toxins. Utilizing basic data from
such studies immunotoxins (toxins linked to monoclonal antibodies') are constructed to serve as a
new class of pharmacologic reagents to eliminate unwanted cell types such as cancer cells or T-4
lymphocytes in AIDS infections, or to manipulate specific cells such as Tcell subsets to correct
imbalences which exist in autoimmune diseases which can affect the CNS such as multiple
sclerosis and lupus and cause psychosis. In addition immunotoxins continue to prove useful in
deminishing the incidence of graft-versus-host-disease following bone marrow transplantation and
thus will also have utility in enzyme replacement therapy and organ transplantation.
899
PHS 6040 (Rev, 1/84)
GPo gi 4-sie
ZOl Mil 01037-19 LMB
Project Descriptions:
The general aim of this project is to determine the chemical interactions and energetics such as
membrane potential and pH and ion gradients which are involved in the insertion of proteins into
cellular membranes and/or the translocation of proteins across cellular membranes. The events are
studied from the initial receptor binding to the final physiologic response or pathological response in
the case of toxins such as ricin, colicins, diphtheria and tetanus toxins. Utilizing basic data from
such studies immunotoxins (toxins linked to monoclonal antibodies) are constructed to serve as a
new class of pharmacologic reagents to eliminate unwanted cell types such as cancer cells or T-4
lymphocytes in AIDS infections, or to manipulate specific cells such as T cell subsets to correct
imbalences which exist in autoimmune diseases which can affect the CNS such as multiple sclerosis
and lupus and cause psychosis. In addition immunotoxins continue to prove useful in deminishing
the incidence of graft-versus-host-disease following bone marrow transplantation and thus will also
have utility in enzyme replacement therapy and organ transplantation.
Major Findings:
Diphtheria toxin translocation across the cell membrane is driven by a plasma membrane voltage
gradient or a pH gradient. In contrast, ricin translocation is not coupled to these gradients but is
tightly coupled to the availabiUty of ATP.
A tissue culture model system for studying the effects of tetanus toxin on the inhibition of
neurotransmitter release has been developed utilizing NG-108 cells. The productive receptor
appears to be a ganglioside present in very low amounts (500 molecules per cell).
Diffusion barriers to immunotoxin exist between the peritoneal cavity and the intravascular
compartment. This enhances the effects of LP. administered immunotoxins on tumor loads within
the peritoneal cavity where 3-4 log kills are achievable. Irradication of specific immune system
subsets lying within the vascular compartment is more readily accomplished by using the
intravascular route.
Significance to Biomedical Research and the Program of the Institute:
The identification of voltage gradients, pH gradients and ATP stores as the driving forces for
protein toxin insertion and translocation prompts one to look for physiological insertions and
translocation driven by the same forces. The toxins are thus model systems in which protein
insertion into and translocation across membranes can be quantitatively studied. Protein insertion
into membranes results in altered membrane functions. For example, differential protein insertion
into neuronal membranes is believed to play an important role in establishing and maintaining
neuronal synaptic connections.
The development of an animal model system for the evaluation of in vivo immunotoxin
manipulation of T cell subsets is a first step in evaluating these agents for their therapeutic potential
in the treatment of autoimmune diseases and AIDS.
Of particular interest to NIMH is the recently demonstrated association of serum and CSF
autoantibodies directed at the carboxy terminus of ribosomal phosphoproteins and episodes of
psychosis in patients suffering from systemic lupus erythematosus (Bonfa et al. 1987).
Of great promise is the possibility that immunotoxins directed at T4a"'" T cells and macrophages
could, early in the course of AIDS, eliminate the HTV infected cells before the virus spreads to cells
whose eradication would prove harmful, i.e., neurons. It is also important to determine if the
900
ZOl MH 01037-19 LMB
maintenance CNS HIV infection (and resulting progressive dementia) requires a feeder population
of easily infected cells, i.e., systemic T-4a+ macrophages and T cells.
Proposed Course:
Identification of the energy sources for toxin translocation permit toxin translocation to be studied in
simple in vitro systems utilizing mixtures of cell fractions, cell "sap" and energy sources and
inhibitors of these sources. Isolation of toxin enriched vesicles should provide the cellular
components which document cellular sorting and packaging of proteins and provide an enrichment
of the protein translocation machinery.
By using voltage sensitive dyes which report membrane potential changes via fluorescent changes,
we hope to be able to map the insertion of arrays of membrane proteins both spatially and
temporally in living cells following a variety of pertubations. State-of-the-art image processing
hardware and software operating on digitized images is being assembled to perform this task. This
is a new methodology which should be appUcable to studying membrane insertions of pathological
materials (toxins) and physiological insertions (ion conducting channels).
The development of immunotoxins for human in vivo use is being pursued by devising various
strategies to reversibly block the toxin binding site of the immunotoxin. The site must be blocked
outside the cell to achieve specificity but must be unblocked inside the cell where it is utilized for
efficient translocation thus providing high efficacy to the reagent.
Immunotoxins constructed with intact diphtheria toxin, which have specificity and efficacy in mice,
will be directed to eliminate helper and suppressor murine T cell subsets. The effects of these on
the functioning of the immune system and the retention of immunologic memory will be assessed.
As soon as immunotoxins are capable of achieving efficacy and specificity in humans are
developed, the murine studies will be repeated in monkeys which share many of the same T cell
subsets with humans. This wiU provide the necessary animal studies prior to clinical trials.
Publications:
Marsh, J.W. and Neville, D.M., Jr.: Kinetic comparison of ricin immunotoxins: Biricin conjugate
has potentiated cytotoxicity. Biochemistry 25: 4461-4467, 1986.
Hudson, T.H. and Neville, D.M., Jr.: Temporal separation of protein toxin translocation from
processing events. J. Biol. Chem. (in press).
Hudson, T.H. and Neville, D.M., Jr.: Enhancement of immunotoxin action: Manipulation of the
cellular routing of proteins. In: ^rankel. A., Ed.) Immunotoxins. Martinos Nijhoff Publ., Boston
(in press).
Marsh, J.W. and Neville, D.M., Jr.: Development of an immunotoxin with in-vivo efficacy for
murine systems. NY Acad. Sci. (in press).
Neville, D.M., Jr.: Immunotoxins for mv/vo therapy: Where are we? NY Acad. Sci. rin press).
Neville, D.M., Jr. and Marsh, J.W.: Methods for quantifying immunotoxin efficacy. In:
(Frankel, A., Ed.) Immunotoxins. Martinos Nijhoff Publ., Boston (in press).
Marsh, J.W., Srinivasachar, K., and Neville, D.M., Jr.: Antibody-toxin conjugation. In:
(Frankel, A., Ed.) Immunotoxins. Martinos Nijhoff Publ., Boston (in press).
901
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOIMH 01035-19 LMB
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. We must tit on one line between the txxders.)
TVip Prnrp.«;«s nf T A/sngp.ny
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: H. A. Nash Chief, Sec. on Molecular Genetics LMB, NIMH
Others: P. Kitts Visiting Associate LMB, NIMH
M. Bruist Research Associate LMB, NIMH
C.-C. Yang Visiting FeUow LMB, NIMH
COOPERATING UNITS (if any)
Laboratory of Molecular Genetics, NICHD; Department of Microbiology, University of
Illinois, Urbana, IL; and Cell Genetics Department, South San Francisco, CA
LAB/BRANCH
Laboratory of Molecular Biology
SECTION
Section on Molecular Genetics
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.75
PROFESSIONAL:
3.75
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have investigated the mechanism by which the DNA of bacteriophage lambda integrates into
the chromosome of its Rscherichia coli host. We studied the role of DNA homology between
the recombining sequences and ruled out two plausible alternatives for this requirement. We
also determined that the two recombination partners interact with recombination proteins very
differently. The bacterial partner obtains its recombinase only by collision with a nupleoprotein
assembly formed on the viral partner. The genes for a second recombination protein have been
placed on an overexpression vector and thus enabled the synthesis of large quantities of active
protein.
903
PHS 6040 (Rev. 1/84)
GPO 91 4-910
ZOIMH 01035-19 LMB
Objectives:
Rearrangement of genetic information by breakage and reunion of DNA sequences is an important
feature of all living organisms. DNA rearrangements are used to alter the genetic content of a cell in a
precise, controlled and potentially reversible way. The life cycle of retroviruses, the generation of
components of the immune system and the expression of alternate genes all depend upon
recombination between specific sites in DNA. This project has as its goal the elucidation of the
molecular mechanism of a prototypical site-specific recombination: the integration of the DNA of
bacteriophage lambda into the chromosome of its E. coli host. We want to know how specific
recombination proteins can locate special sequences in DNA, bring them together and carry out a
cycle of events that includes breaking the DNA, switching the broken strands to new partners and
reseating of the new joints. Our understanding of this reaction not only serves as a model for
understanding other members of its family but also a broad spectrum of complex biological
processes that involve interactions between more than one protein species with multiple targets in
DNA.
Major Findings:
Integration of the chromosome of bacteriophage lambda involves the interaction between two pieces
of DNA - the viral attachment site, attV, and bacterial attachment site atiB. These sites have in
common a 15 base pair core sequence within which the crossover takes place. We have shown that
the viral Int protein can cut DNA within the core to initiate strand exchange and can subsequentiy
reseal the broken DNA. Each strand of DNA within the core is cut by Int at a unique place but the
two cuts are separated by 7 base pairs. Others have shown that efficient recombination requires that
this 7 base pair "overlap" sequence be identical in two attachment site. It has long been speculated
that this requirement reflects a mechanism of recombination in which double strand breaks are
introduced into each attachment site, thereby generating breaks with protruding single stranded ends.
The model asserts that recombination involves annealing of one end from each parent, a process that
is expected to require that both parents have the identical overlap sequence. We have now proven
that this model is wrong. Our test was based on in vitro Int-promoted crosses in which one
attachment site is a heteroduplex. Specifically, we constructed sites in which the overlap region
contains one or more non-complementary pairs. The double-strand break and annealing mechanism
predicts that crosses with such heteroduplex sites should yield one completed recombinant and one
broken site. Instead, we find that non-reciprocal recombination is uncommon and that the typical
outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products
are resealed. Moreover, the occasional appearance of non-reciprocal products is explained by our
finding that Int can cleave heteroduplex attachment sites after recombination is completed. Taken
together, our data strongly indicate that lambda integrative recombination does not proceed by the
homology -dependent annealing of cohesive ends. Alternatives that demand homology to enable
attachment sites to come together are consistent with our results, as are models that invoke the
initiation of recombination by the exchange of one strand for each parent (rather than a double strand
break) followed by a homology-dependent migration of the resulting Holhday structure.
Heteroduplexes have also been used to tell us about the state of atiE when it participates in a
recombination reaction. Specifically, we want to know whether arrB encounters attV when it is
decorated with Int protein that it has obtained from free solution or whether it begins recombination
as a naked piece of DNA, deriving its Int from the many copies that are tightiy bound to att?. We
had previously shown that heteroduplexes of an attachment site that, like att?, has multiple Int
binding sites are efficientiy cleaved by Int. This is because when Int cuts a single strand of the
heteroduplex the broken ends can not be resealed due to lack of complementarity. Ultimately double
strand breaks accumulate in the heteroduplex and become a permanent record for the binding of Int
904
ZOIMH 01035-19 LMB
protein. In contrast to sites like attP with multiple Int targets, attB heteroduplexes are scarcely cut
by Int. This suggests that Int fails to bind efficiently to attB, an hypothesis supported by chemical
protection studies. These footprinting experiments show that, although attB does have binding sites
for Int, they are very weak and can not compete for Int protein with the strong sites on attP or even
with those found in non-specific DNA. Although, cleavage of attB heteroduplexes by Int is
inefficient, the reaction is dramatically stimulated by addition of attP. This cleavage reaction
therefore provides a novel assay for the interaction between attB and attP, a device we have used to
show that synapsis does not depend upon homology in the overlap region between attP and attB.
This results rules out one of the two possibilities that we discussed above and strongly supports the
remaining hypothesis, i.e., that the role for homology is in the branch migration of HoUiday
structures.
In addition to Int, the virus-encoded protein that carries out breakage and reunion, integrative
recombination requires a host protein. This protein, which we named IHF (for integrative host
factor) is of general interest. First, mutants of IHF show alterations in many processes that are
important for E. coli, its plasmid and phages. Moreover, EHF protein has been directly implicated
as an essential component of in vitro reactions as diverse as the initiation of transcription, the
packaging of viral DNA, and the transposition of movable genetic elements. Second, IHF may
represent a novel form of DNA sequence-specific binding protein that recognizes an assymetric
sequence through contact with base pairs in the minor grove of DNA. To assist future structural and
chemical studies of IHF we have constructed strains that overproduce the protein. EHF is composed
of two dissimilar subunits, each containing about 100 amino acids. The genes for these two subunits
lie far apart on the E. coli chromosome and are expressed under separate control mechanisms. We
find that when each gene is overexpressed separately, the resulting polypeptides are either unstable
or insoluble. By contrast, overexpression of both genes conjointly leads to the accumulation of large
amounts of active IHF. Extracts of such cells provide the starting material for a rapid purification
procedure that results in milligram quantities of apparently homogenous IHF.
Significance to Biomedical Research and the Program of the Institute:
Two features of lambda integrative recombination make it an important reaction to try to understand.
First, during lambda integration DNA gets broken and rejoined into a novel arrangement. Related
rearrangements of DNA are well known in molecular biology and more examples are being
discovered at a rapid rate. It is widely believed, and some early successes support the notion, that
lessons learned in one system will be applicable to many. Thus, our progress in delineating the role
of homology during lambda integrative recombination and our analysis of the different way that two
recombination partners interact with the recombinase will be widely appreciated by others studying
both site-specific and homologous recombination in prokaryotic and eukaryotic systems. The
second feature of integrative recombination that makes it a significant model system is the
complexity of its protein-DNA interactions. It has been widely accepted that important steps in
higher organisms such as turning gene expression on or off during development and activating
origins of replication during cell division are multicomponent processes that involve the interaction of
many proteins binding to many DNA sites. The integrative recombination reaction is one such
multicomponent system in which the basic elements are well defined and the components have all
been well characterized at the biochemical level. Thus, as we uncover the logic that underlies this
reaction, we provide insight to a broad spectrum of molecular biologists.
905
ZOIMH 01035-19 LMB
Proposed Course:
The experiments refuting the cohesive end model for homology dependence in integrative
recombination are completed and a paper describing the results has just been published. The use of
heteroduplex substrates to analyze the state of attB in recombination is being prepared for
publication. The major conclusion of this work is that attB enters into recombination as a naked
piece of DNA and acquires its recombination protein not from solution but from its recombination
partner. We would like to test this unusual proposal by preparing attP nucleoprotein complexes,
freeing them from unbound protein and determining the kinetics with which such complexes
recombine with attB. We also plan to modify attP and test which features of this multiprotein
assembly are required for capture of attB. To study the structural features of IHF in more detail,
we are exploiting our overproducer strain to generate large quantities of the pure protein. There has
already been some initial success at obtaining crystals suitable for X-ray diffraction. We also plan to
undertake a wide variety of footprinting studies to clarify the way in which IHF contacts DNA.
These studies will be complemented by our planned measurement of the stoichiometry with which
IHF binds to its specific site.
Publications:
Nash, H.A.: Virus-host interactions in site-specific recombination of bacteriophage lambda.
Genetic Chemistry: The Molecular Basis of Heredity. Robert A. Welch Foundation Conference on
Chemical Research. Vol. 29, Houston, Texas, 1985, pp. 285-296.
Gardner, J.F. and Nash, H.A.: Role of Escherichia coli IHF protein in lambda site-specific
recombination: A mutational analysis of binding sites. J. Mol. Biol. 191: 181-189. 1986.
Richet, E., Abcarian, P., and Nash, H.A.: The interaction of recombination proteins with
supercoiled DNA: Defining the role of supercoiling in lambda integrative recombination. Cell 46:
1011-1021, 1986.
Gellert, M. and Nash, H.: Communication between segments of DNA during site-specific
recombination. Nature 325: 401-404, 1987.
Nash, H.A., Bauer, C.E., and Gardner, J.F.: The role of homology in site-specific recombination
of bacteriophage lambda: Evidence against anneahng of cohesive ends. Proc. Natl. Acad. Sci. USA
84: 4049-4053, 1987.
Nash, H.A., Robertson, C.A., Flamm, E., Weisberg, R.A., and Miller, H.I.: Production of E.
Coli integration host factor, a protein with non-identical subunits. J. Bact. (in press).
Kitts, P.A. and Nash, H.A.: Homology dependent interactions in phage X site-specific
recombination. Nature (in press)
906
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 02228-03 LMB
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Genetic Neurobiology of Drosophila
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: H. A. Nash Chief, Sec. on Molecular Genetics LMB, NIMH
Others: K.Weber Graduate Student Harvard Univ.
COOPERATING UNITS (if any)
jEaboratory of Molecular Biology
SECTION
Section on Molecular Genetics
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.25
PROFESSIONAL:
0.25
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have isolated mutants of the fruit fly Drosophila melanogaster that show an altered response to
general anesthetics. We have mutangenized flies with ethylmethane sulfonate and found amongst
their offspring mutants that are hypersensitive or resistant to halothane. These mutants form the
starting point for a genetic and a molecular biological study of the mechanism by which anesthetics
interfere with pain and consciousness.
907
PHS 6040 (Rev. 1/84)
GPO B14-Sie
ZOl MH 02228-03 LMB
Objectives:
Genetic analysis has been a successful strategy for unraveling complex processes in a variety of
organisms. In recent years there has significant success in using genetic techniques to illuminate
developmental, physiological, and biochemical aspects of the nervous system of the fruit fly,
Drosophila melanogaster. The goal of this project is to extend such studies into to previously
unexplored territory. We are especially interested in using genetics to analyze the mechanism of
general anesthesia. To this end, we have undertaken the isolation of mutants with altered sensitivity
to anesthetics. Amongst such mutants should be some that change the putative target of the agent.
Analysis of these mutants by physiological and molecular biological techniques should help in
understanding both the anatomic and biochemical basis of consciousness and the perception of pain.
Major Findings:
Success at isolating mutants depends on two components: efficient mutagenesis and a sensitive
screening procedure. In the past year we have learned how to successfully apply a classical
chemical mutagenesis protocol. From the frequency of sex-linked lethal mutations and easily
scored eye color variants in our mutagenized population, we estimate that each locus in the genome
of these offspring have a one in a thousand chance of being mutated. Since Drosophila is estimated
to have five thousand to ten thousand genetic loci, one expects that, if a particular gene is not
essential for survival, then screening twenty thousand offspring should yield a handful of mutations
in that gene. Because of the ease of screening for recessive mutations, we have focused our efforts
on sex-linked genes. Mutagenized males are crossed with attached-X virgin females and the
subsequent generation is screened en masse for the response to anesthesia.
To screen the offspring, we have adapted a device introduced by K. Weber (Harvard University) to
quantitate the response of flies to intoxication by alcohol. This "inebriometer" consists of a vertical
glass column fitted with nylon mesh baffles that impede the fall of partially anesthetized flies. We
have learned how to provide a reliable, constant dose of volatile anesthetic to the inebriometer and
have screened our mutagenized population for an altered response to anesthetic.
By focusing on the first and last flies to exit the column, we identify potential hypersensitive and
resistant mutants. Those flies that show a consistent response upon retesting are individually mated
to generate a clonal line. We have now identified several such lines that are reproducibly
hypersensitive or resistant to a fixed concentration of the clinical agent, Halothane. In most cases,
only male flies are affected, as expected for a recessive sex-linked mutation. The severity of the
alteration to anesthesia varies amongst the different tines from mild to moderate. We conclude that
the anesthetic response is amenable to a genetic analysis.
Significance to Biomedical Research and the Program of the Institute:
Analysis of the conscious state is a subject of interest to neurobiologists. One wants to know what
anatomic structures are essential and what neurophysiological mechanisms contribute to awareness.
Anesthetics have long been recognized as potentially useful reagents in investigating the conscious
state. However, their wide distribution in the brain has hindered the formulation of decisive tests.
Identifying the genes that control or encode anesthetic targets should help in this endeavour. The
distribution of gene products in the brain could provide information on the anatomical basis of the
conscious state and the nature of the gene products could tell us about important cellular
mechanisms. Our success in isolating mutants with altered sensitivity to anesthetics represents an
important first step in this analysis.
908
ZOl MH 02228-03 LMB
Proposed Course:
We will continue to collect more anesthetic mutants; this exercise is potentially useful up to the point
where one begins to recover the same mutants repeatedly, i.e., until one saturates the genome.
However, we already have enough mutants on hand to begin a more detailed pharmacological and
genetic characterization. We want to quantify the degree of sensitivity to Halothane, test the
response to other anesthetics, and devise tests of the response to anesthesia that will be applicable to
single flies. The latter is important for the genetic characterization of the mutants. We want to map
them precisely, determine their genetic complimentation both with wild-type and other mutant
alleles, and begin the search for genetic suppressors.
909
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT l\;UMBER
ZOl MH 00934-15 LMB
EBJ0DCPVERED,„„^ „
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less. Title must fit on one line befween the boniers.)
The Biochemical Basis of Peptide Receptor Activity
PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: W. A. Klee Chief, Sec. on Regulatory Proteins
Others: D.L.Newton Staff Fellow
J.-Y. Ye Visiting Associate
R. C. Rice Research Chemist
A. E. Jacobson Research Chemist
M. Nirenberg Chief, Lab. Biochem. Genetics
P. Hargrave Professor of Opthamology
LMB, NIMH
LMB, NIMH
LMB, NIMH
LC, NIADDK
LC, NIADDK
LBG, NIHLB
University of Florida
COOPERATING UNITS (if any)
Laboratory of Neurophysiology, NINCDS; Laboratory of Chemistry, NIADDK; and Laboratory
of Biochemical Genetics, NIHLB
LAB/BRANCH
Laboratory of Molecular Biology
SECTION
Section on Regulatory Proteins
INSTITUTE AND LOCATION
NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.0
PROFESSIONAL:
3IL
AJL
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues 0 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space pmvided.)
In the past year we have continued our studies on reconstituted opiate receptors in purified
systems. Purified G-proteins and adenylate cyclase were reconstituted into liposomes from
detergent solutions by dialysis in the presence of phospholipids. In the reconstituted vesicles
adenylate cyclase activity is stimulated by Gs and the stimulated activity is inhibited by Gi or by the
beta-gamma subunit complex of bovine transducin. Further progress has been made in our efforts
to obtain useful amounts of purified opiate receptors, and in characterizing the physical and
biochemical properties of G-proteins.
We have identified a monoclonal antibody, directed against a defined region of the amino acid
sequence of bovine transducin, which also recognizes opiate receptors from NG108-15
neuroblastoma x glimoa hybrid cells. A peptide corresponding to the epitope of the antibody
inhibits precipitation of opiate receptors by the antibody. A peptide from the homologous region of
the porcine brain muscarinic receptor not only blocks the antibody, but also activates a number of
G-proteins both in membranes and in solutions of the purified proteins. We have thus identified the
signal transmitting domain of G-protein coupled receptors.
911
PHS 6040 (Rev. 1/84)
GPO BI4>SIB
ZOIMH 00934-15 LMB
Project Description and Major Findings:
Receptors on the cell surface, such as those for the opiates, are coupled to enzymes, such as
adenylate cyclase, on the inside of the membrane via the mediation of GTP-binding regulatory
proteins (G-proteins). In this manner, information is transmitted to a cell from neighboring cells
and from the fluid environment. Our goal is to dissect this system into its component parts, and
study each of the proteins and other components of the system both in isolation and as a
reconstituted functional entity. In the past few months, the Section has achieved a major break-
through that has resulted in identification of the receptor domain that activates G-proteins and the
demonstration that peptides corresponding to this domain stimulate G-protein activity in the absence
of any other substances.
We have chosen to concentrate upon opiate receptors in the cultured neuronal cell line, NG108-15.
These cells are richly endowed with opiate receptors of a single type, namely d. The receptors were
shown to be coupled, as inhibitors, to adenylate cyclase both in these cells and in brain tissue.
Activation of the receptors with opiates or opioid peptides reduces cellular cyclic AMP levels and
thereby lowers the extent of phosphorylation of many cellular enzymes. In analogy to the addictive
process, the cells become tolerant to and dependent upon opiates after prolonged exposure. This
adaptive process is due to a gradual increase in adenylate cyclase activity which serves to maintain
normal cyclic AMP levels in the continued presence of opiates. With opiates such as morphine,
adaptation occurs in the absence of changes in receptor number. Other opioids, such as the
enkephalins, produce receptor down-regulation as well as increased adenylate cyclase activity upon
chronic exposure.
We have over the past few years developed procedures for the solubilization of receptors from
membranes by extraction with the zwitterionic detergent CHAPS, and have isolated affinity labeled
opiate receptors in a homogeneous state. Such receptors, because of the presence of covalently
linked opiates are more useful for structural than for functional studies. They have proven to be
particularly good tools for antibody screening experiments as well.
All receptors share two essential properties: they bind ligands, and transmit the information of
whether or not an activating ligand (agonist) is bound. Many receptors send information to one of
several GTP-binding regulatory proteins (G-proteins) which have very similar amino acid
sequences. This family of G-proteins includes at least 3 types of Gi, Go, and transducin. Receptors
coupled to these proteins include, among others, opiate, muscarinic and bradykinin receptors and
the photon receptor, rhodopsin. Activation of G-proteins by agonist occupancy of these receptors
ultimately results in activation, or inhibition, of one of several enzymes including phospholipase C,
cyclic GMP phosphodiesterase and adenylate cyclase. We reasoned that receptors of this class,
which must all interact with very similar regulatory proteins, might share structural features
responsible for these interactions.
The availability of a battery of monoclonal antibodies directed against defined regions of rhodopsin
(developed by Dr. Paul Hargrave and his collaborators at the University of Florida), allowed an
experimental test of the hypothesis. For this test we used opiate receptors from NG108-15
neuroblastoma X glioma hybrid cells specifically substituted with the synthetic opiate [3H]-3-
methylfentanylisothiocyanate (superFIT). We found that one of the 46 anti-rhodopsin monoclonal
antibodies tested also recognizes opiate receptors. The epitope against which this antibody is
directed corresponds to a cytoplasmic segment of the rhodopsin molecule immediately following the
seventh (putative) transmembrane helix. A peptide corresponding to this region, rhodopsin 310-
321, blocks interaction of the antibody with both rhodopsin and opiate receptors. An amidated
peptide corresponding to the homologous region of the procine brain muscarinic receptor, residues
422-431 (NIOL) also blocks the antibody. Interestingly, this peptide activates Gi function in S49
912
ZOIMH 00934-15 LMB
cell membranes, where it inhibits adenylate cyclase and stimulates low Km GTPase. The peptide
also activates G-proteinss in membranes of NG108-15 cells. In solution and at concentrations
below about 200 |jM, the peptide stimulates the GTPase activity of both purified transducin and
purified Go. These experiments suggest that NIOL corresponds to the receptor domain directly
responsible for information transmission and show that it can work in a completely defined system.
Thus, many experiments can now be performed using only purified proteins and peptides that
should greatly clarify the mechanism of receptor action. As more receptor sequences become
available much will be learned using this approach about receptor specificity and selectivity.
Receptor preparations which have not been irreversibly modified are needed for functional
reconstitution studies. To this end, we have prepared several affinity columns consisting of opiates
covalently linked to cross-linked agarose beads to which opiate receptors bind. In combination with
lectin affinity chromatography this type of procedure has allowed the partial purification of opiate
receptors from NG108-15 cell membranes. The yields obtained in these procedures have, so far,
been low when receptors are assayed by opiate binding activity. One reason for the apparently low
yields has recently been discovered by Yk. Newton, who has found that the solubilized receptors
are composed of a mixture of high and low affinity molecules. The low affinity receptors had been
missed up until now because of technical problems in their assay.
Several GTP-binding regulatory proteins have been characterized, but, with the exception of that of
Gs, the functions of these proteins have not been clearly established. The G-proteinss are
members of a closely related family and are each composed of three subunits: a, |3, and y. The
major differences in structure among the several G-proteinss are found in the a subunits. The P
subunits are all identical or nearly so and the P-y subunit complexes of the most diverse of the G-
proteinss have been found to substitute for one another. We have purified two such proteins, Gi
and Go from bovine brain and prepared antibodies which recognize the a subunits of one or the
other protein. It has recently become clear that there at least three forms of Gi, two of which are
present in brain. Liver, on the other hand, contains primarily a single form of Gi and hardly any
Go. We have therefore purified the liver protein and are in the process of characterizing it better.
This material will significantly aid our studies of receptor signaling domains.
The G-proteins are present in fairly low amounts in most tissues. Even in brain, which is fairly rich
in G-proteins, the presence of at least 4 such proteins complicates isolation of really pure samples.
In some of our studies we therefore use transducin, the G-protein of retina, which is easily available
in mg amounts as a homogenous protein. Dr. Ye has developed methods for the study of the
isolated subunits of transducin. Separation of the a from the Py subunits of the protein is readily
accomplished in good yield and with retention of native properties. Dr. Ye has studied the physical
properties of the isolated subunits by measuring fluorescence and circular dichroism both under
native and denaturing conditions. In addition, he has demonstrated that the subunits contain both
disulfide and sulfhydryl functions. This unusual circumstance may be important to the function of
the protein and certainly complicates its study. For example, separation of P from the y subunit
requires prior denaturation in urea or guanidine hydrochloride. Reconstitution of the two to a
functionally active entity requires a reversal of the denaturation, presumably under controlled redox
conditions. In order to minimize problems associated with disulfide interchange and other slowly
reversible processes, we have developed an HPLC separation of the two subunits which can be
accomplished at low temperatures and within 12 minutes. These studies will not only increase our
understanding of transducin as a prototypic G-protein, but also serve as paradigms for the study of
the other, less readily obtained, members of the family.
913
ZOIMH 00934-15 LMB
Significance to Biomedical Research and the Program of the Institute:
A major problem in biology is understanding the mechanism of signal-response coupling across cell
membranes. Cells communicate with one another and with their environment largely through
chemical messengers which are sensed by cell surface receptors and thereby elicit other chemical
changes within the cell. The opiates, and related substances, are important transmitters of
information in the nervous system. An understanding of how brain cells transmit and use such
information is essential to the design of rational therapy for mental illness.
Proposed Course:
We plan to continue our efforts to understand the molecular basis of signal transduction, with
particular emphasis on opiate receptor and related mechanisms. In the next year we hope to be able
to use our newly discovered insight into the nature of the signaling domain of receptors to learn
more of the nature of receptor G-protein activation mechanisms. The approach may also prove
useful to elucidate those aspects of receptor structure which determine affinity for particular G-
proteins. It does not seem unreasonable to hope that a completely synthetic receptor may be
prepared that reproduces not only the signaling properties of hormone action but also the high
affinity interaction with specific G-proteins as well.
Publications:
Burke, T.R., Jr., Jacobson, A.E., Rice, K.C., Silverton, J.V., Simonds, W.F., Streaty, R.A.,
and Klee, W A.: cis-(+)-3-methylfentanyl isothiocyanate, a potent site-directed acylating agent for
5 opioid receptors. Synthesis, absolute configuration, and receptor enantioselectivity. J. Med.
Chem.29: 1087-1093, 1986.
Lessor, R.A., Bajwa, B.S., Rice, K.C., Jacobson, A.E., Streaty, R.A., and Klee, W.A.:
Potential irreversible narcotic antagonist-based ligands derived from 6,14-endo
ethenotetrahydrooripavine with 7-(Methylfumaroyl) amino, (Bromacetyl)amino, or isothiocyanate
electrophiles: Chemistry, Biochemistry and Pharmacology. J. Med. Chem. 29: 2136-2141, 1986.
Schonenberger, B., Jacobson, A.E., Brossi, A., Streaty, R., Klee, W.A., Flippen-Anderson,
J.L., and Gilardi, R.: Comparison of (-)-Eseroline with (+)-Eseroline and dihydroseco analogues in
antinociceptive assays: Confirmation of rubreserine structure by X-ray analysis. J. Med. Chem.
29: 2268-2273, 1986.
Milligan, G., Streaty, R.A., Gierschik, P., Spiegel, A.M., Klee, W.A.: Development of opiate
receptors and GTP-binding regulatory proteins in neonatal rat brain. J. Biol. Chem. 262: 8626-
8630. 1987.
914
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, i;
PROJECT NUMBER
ZOl MH 01031-19 LNC
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
The Conversion of Phenylalanine to Tyrosine
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Seymour Kaufman
Michael Davis
Jennifer Tipper
Yohsuke Minatogawa
Hans-Ulrich Siegmund
Chief LNC NIMH
Senior Staff Fellow LNC NIMH
Senior Staff Fellow LNC NIMH
Visiting Scientist LNC NIMH
Visiting Fellow LNC NIMH
COOPERATING UNITS (H any)
LAB/BRANCH
Laboratory of Neurochemistry
INSTITUTE AND LOCATION
ADAMHA, NIMH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
5.2
PROFESSIONAL
4.2
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
n (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Phenylalanine hydroxylase activity, measured at neutral pH, can be
markedly increased by a brief exposure of the enzyme to alkaline pH.
Phenylalanine given to rats leads to an enhanced phosphorylation and
activation of hepatic phenylalanine hydroxylase.
915
PHS 6040 (Rev. 1/84)
GPO BI4-«lt
ZOl MH 01031-19-LNC
Major Findings:
During the course of our studies of the regulation of phenylalanine
hydroxylase, we have found that a brief exposure of the enzyme to alkaline pH
(pH 8.5-9.5) results in a marked (~10-fold) activation of the enzyme when it
is assayed at neutral pH in the presence of its natural cofactor, tetrahydro-
biopterin. These results indicate that on exposure to alkaline pH, the
conformation of the enzyme changes to one that has higher catalytic activity
and that this activated state persists for a time even when the enzyme is
returned to neutral pH. We showed that this activated species of the enzyme
does indeed have an altered conformation by demonstrating that the
fluorescence of the enzyme changes when it is activated in this manner. We
previously reported there is a similar correlation between activation of the
enzyme and changes in its fluorescence spectrum during the course of
activation by its substrate, phenylalanine.
A study of the mechanism of the alkaline pH activation of phenylalanine
hydroxylase showed that it is a complex process. We have found that
preincubation of the enzyme at alkaline pH primes the enzyme for substrate
activation so that activation by phenylalanine of this primed species occurs
at a much greater rate than that of the resting enzyme. But significantly, we
have also shown that the primed species also has a two-fold greater intrinsic
hydroxylase activity than the native enzyme. This latter finding represents
the first example of an increase in the enzyme's intrinsic activity. It is
significant because it disproves the currently widely-accepted generalization
that asserts that all modes of activation of the hydroxylase simply reflect
enhanced activation of the enzyme by its substrate.
We have also obtained evidence which indicates that not only are all
modes of activation of the enzyme not simply reflections of substrate
activation, but that even some examples of substrate activation are indirect
and are the consequence of another kind of activation. It has been reported,
e.g. that administration of phenylalanine to rats activates the enzyme.
Although this activation has been attributed entirely to substrate activation,
our results indicate that a significant part of this activation is due to
phenylalanlne-mediated phosphorylation of the hydroxylase.
Tetrahydrobiopterin, discovered in this laboratory approximately
twenty-five years ago, is the physiological cofactor for the aromatic amino
acid hydroxylases. Twenty years ago we showed that the auto-oxidation of this
cofactor at neutral pH in phosphate buffer resulted in the formation of
quinonoid dlhydrobiopterin which rapidly rearranges to 7 , 8-dihydrobiopterin.
(Only the quinonoid compound is a substrate for dihydropteridine reductase,
the enzyme which regenerates tetrahydrobiopterin m vivo) . Later it was shown
that whereas quinonoid dlhydrobiopterin also rearranges in acid to
7, 8-dihydrobiopterin, under basic conditions 7 , 8-dihydropterin is formed i.e.,
the dihydroxypropyl side chain of biopterin is lost during the chemical
rearrangement. More recently, an Australian group has reported, in apparent
contradiction of our earlier finding, that at neutral pH quinonoid
tetrahydrobiopterin is converted almost exclusively to 7 , 8-dihydropterin. Our
reinvestigation of this chemical rearrangement has clarified the apparent
916
ZOl MH 01031-19-LNC
contradiction between our earlier results and those workers. Using three
different methods for identifying the two putative products, (HPLC,
multicomponent analysis of ultraviolet spectra, and chemical oxidation and
analysis of the products), we have found that both pathways proceed but to
differing extents depending on the reaction conditions i.e., the type of
buffer, temperature, as well as the pH, all play a role in whether the side
chain is lost during the chemical rearrangements. Under the conditions of our
original experiments (neutral pH in phosphate buffer), our original results
were replicated, i.e., the major ultimate product of BH, oxidation is indeed
the corresponding 7,8-dihydrobiopterin. At higher pH values, the side-chain
of blopterin does come off. Since the Australian workers studied different
conditions than we did initially, they incorrectly concluded that the two sets
of results were mutually exclusive. Our findings should help to delineate the
conditions under which human tissues can be stored prior to analysis of
tetrahydrobiopterin.
Significance to Biomedical Research and Proposed Course of Project:
Our new results on the regulation of phenylalanine hydroxylase have
provided important insight into the relationship between activation of the
enzyme by its substrate, phenylalanine, and other types of activation. The
widely-accepted dogma asserts that all types of activation of the hydroxylase
are merely different manifestations of substrate activation. According to
this view, for example, activation by phosphorylation or activation by
exposure to alkaline pH would simply prime the enzyme to be more rapidly
activated by its substrate. Our results show, however, that with the latter
type of activation, at least, part of the activation is due to an increase in
the intrinsic activity of the hydroxylase that is quite independent of
substrate activation, and part of it is substrate-mediated. These results,
therefore, show for me first time that the accepted dogma is incorrect.
We have also shown that even some of the other accepted ideas about
substrate activation must be modified. Our results indicate, for example,
that an earlier published paper describing the direct in vivo activation of
pheriylalanine hydroxylase by phenylalanine is not due entirely to direct
substrate activation. Rather, as we had previously postulated, part of the
phenylalanine effect is due to a phenylalanine-mediated increase in
phosphorylation 6f the enzyme.
These studies add support to the idea that substrate activation of
phenylalanine hydroxylase is synergystically and reciprocally related to
actlvatibn by phosphorylation, as well as to other forms of activation.
We plan to continue to explore the ways in which rat liver phenylalanine
hydroxylase is regulated. We also plan to expand these studies to include the
differential regulation of kidney phenylalanine hydroxylase using both
bidchemical and recombinant DNA techniques.
917
ZOl MH 01031-19 LNC
Publications:
1. Iwaki, M. , Phillips, R. S. and Kaufman, S. Proteolytic modification of
the amino-terminal and carboxyl-terminal regions of rat hepatic
phenylalanine hydroxylase. J. Biol. Chem. . 261: 2051-2056, 1986.
2. Rao, D. N. and Kaufman, S. Purification and state of activation of rat
kidney phenylalanine hydroyxlase. J. Biol. Chem.. 261: 8866-8876, 1986.
3. Kaufman, S. Regulation of the activity of hepatic phenylalanine
hydroxylase. In: Advances in Enzyme Regulation Weber, G. , ed. Pergamon
Press, Oxford, New York, Vol. 25: pp 37-64, 1986.
4. Davis, M. , Kaufman, S. and Milstien, S. A modified ferrozine method for
the measurement of enzyme-bound iron. J. Biochem & Biophys Meth. , 13:
39-45, 1986.
5. Kaufman, S. Enzyme control by phosphorylation: Aromatic amino acid
hydroxylases. Hi: The Enyzmes . Boyer, P.D. and Krebs, E. G. , eds.
Academic Press, Orlando, Fl., Vol. 18: 218-281, 1986.
6. Kaufman, S. NADH dihydropteridine reductase from sheep liver. In:
Methods in Enzymology, Colowick, S. P. and Kaplan, N. 0., eds. Academic
Press, Orlando, Fl, Vol. 142: pp 97-102, 1987.
7. Kaufman, S. The metabolic role of tetrahydrobiopterin. 2b- Chemistry
and Biology of Pteridines, Cooper, B. A. and Whitehead, V. M. , eds.
Walter de Gruyter, Berlin, pp. 185-200, 1986.
8. Davis, M. D. and Kaufman, S. The effect of dietary iron on the activity
of rat liver phenylalanine hydroxylase. Xn: Chemistry and Biology of
Pteridines, Cooper, B. A. and Whitehead, V. M. , eds. Walter de Gruyter,
Berlin, pp. 363-367, 1986.
9. Parniak, M. A., and Kaufman, S. Alterations in cof actor -dependent
activity of phenylalanine hydroxylase as a function of pH. In:
Chemistry and Biology of Pteridines. Cooper, B. A. and Whitehead, V. M. ,
eds. Walter de Gruyter, Berlin, pp. 355-358, 1986.
10. Kaufman, S. The enzymology of the aromatic amino acid hydroxylases. In:
Amino Acids in Health and Disease: New Perspectives, Kaufman, S., ed.
UCLA Symposia on Molecular and Cellular Biology held at Keystone, CO. ,
May 30-June 4, 1986. Alan R. Liss, Inc., New York, pp. 205-23332, 1987.
918
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 01032-19 LNC
TITLE OF PROJECT (BO cheracteiz or less. We must tit on one line between the borders.)
Biosynthesis of Catecholamines
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliation)
PI Seymour Kaufman
Thomas Nelson
Dominique Pigeon
Chief
Staff Fellow
Visiting Fellow
LNC NIMH
LNC NIMH
LNC NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neurochemistry
INSTITUTE AND LOCATION
ADAMHA, NIMH, Bethesda, Maryland 20892
TOTAL MAN- YEARS:
2.2
PROFESSIONAL:
1.2
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
n (a2) Interviews
□ (b) Human tissues S (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A new rapid isolation procedure has been developed for the purification of
tyrosine hydroxylase from brain and adrenal tissue.
A phosphatase that catalyzes the dephosphorylation-deactivation of tyrosine
hydroxylase has been partially purified from rat brain. Its activity is affected
by the pterin coenzyme for tyrosine hydroxylase.
919
PHS 6040 (Rev. 1/84)
GPO 914>aiS
ZOl MH 01032-19LNC
Project Description:
We have continued our studies of brain and adrenal tyrosine
hydroxylase using our new rapid, high-yield purification procedure. We
have also devised several new methods for determining protein-bound
phosphate (Pi) that are far more sensitive than previously published
procedures. Using the new hydroxylase purification procedure in
conjunction with these very sensitive Pi assays, we have determined, for
the first time, the amount of protein-bound Pi in both pure brain and
adrenal tyrosine hydroxylases. Since the enzyme was isolated in the
presence of phosphatase inhibitors, the values of protein-bound Pi, about
0.07 moles/mol of hydroxylase subunit, probably reflect the Pi content of
the enzyme in the resting state. With this value for the endogenous Pi
content, together with a determination of the change in hydroxylase
activity as a function of Pi incorporated (mediated by cAMP-dependent
protein kinase), we were able to obtain, for the first time, a detailed
picture of the way hydroxylase activity varies with the total content of
protein-bound Pi. With the brain enzyme, hydroxylase activity is not a
linear function of Pi content, a result that suggests a small allosteric
effect.
We have also partially purified from rat brain a phosphatase that
catalyzes the dephosphorylation (and deactivation) of phosphorylated
tyrosine hydroxylase. This phosphatase appears to be regulated in
opposite ways by GTP, the precursor of BH, , and by BH, itself; the former
compound inhibits, whereas the latter compound stimulates the phosphatase.
Significance to Biomedical Research and Proposed Course of Project:
The characterization of a phosphatase that catalyzes the
dephosphorylation-mediated deactivation of tyrosine hydroxylase should
enable us to obtain a much more complete picture of how the state of
phosphorylation of tyrosine hydroxylase affects its activity. The
demonstration that the phosphatase is inhibited by GTP and activated by
BH, suggests a heretofore unsuspected way in which the coenzyme for the
hydroxylase, BH, , can affect the activity of the hydroxylase. This
observation has already suggested new interpretations for some puzzling
old data in the literature.
We plan to continue to characterize this phosphatase and to study its
interaction with tyrosine hydroxylase.
Publications :
1. Nelson, T. and Kaufman, S. Two enzymatic methods for determination
of the phosphate content of phosphoproteins . Anal. Biochem. 161:
352-357, 1987.
2. Nelson, T. and Kaufman, S. Interaction of tyrosine hydroxylase with
ribonucleic acid and purification with DNA-cellulose or poly
(A)-sepharose. Archives of Biochem and Biophys 257: 1987.
920
TITLE OF PROJECT (80 characters or less. Title must fit on orte line between the tx>rders.)
Phenylketonuria and Other Diseases Caused by Defects in Biopterin-Dependent Enzymes
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 01038-19 LNC
PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator) (Name, title, laboratory, and institute attillation)
PI
Seymour Kaufman
Sheldon Milstien
Stanley Rapoport
Chief
Research Chemist
Chief
LNC NIMH
LNC NIMH
LN NIA
COOPERATING UNITS (If any)
Lab. of Neurosciences, National Institute on Aging
LAB/BRANCH
Laboratory of Neurochemistry
INSTITUTE AND LOCATION
TOTAL MAN- YEARS:
0.7
PROFESSIONAL:
0.7
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues 1] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A determination of neopterin and biopterin levels in amniotic fluid has
been used in the prenatal diagnosis of a possible case of a defect in
tetrahydrobiopterin synthesis.
Alzheimer patients have low CSF levels of tetrahydrobiopterin.
921
PHS 6040 (Rev. 1/84)
CPO 9I4-»I«
ZOl MH 01038-19LNC
Project Description:
The goal of this research project is the detailed description, at the
molecular level, of diseases caused by defects in components of the
aromatic amino acid hydroxylating systems.
Major Findings:
We have successfully carried out one of the first prenatal diagnoses
of a defect in the de novo synthesis of tetrahydrobiopterin. The method
used was based on our previous demonstration (Nixon et al. J. Neurochem.
35: 898-904, 1980) that this disease can be detected by a determination in
urine of neopterin (N) and biopterin (B), patients with the disease having
an elevated N/B ratio. We reasoned that the same metabolic abnormality
would be found in amniotic fluid. In this case, the N/B ratio indicated
that the fetus (17 to 18 weeks of gestation) was not homozygous for the
trait and advised that the pregnancy be carried to term. The baby was
normal at birth and continues to show no signs of the disease. There has
been only one other example of prenatal diagnosis of this condition
carried out by a Swiss group, which independently utilized the same
concept and similar methodology.
We, as well as others, have found that patients with Alzheimer's
disease have a significant decrease in the concentration of BH, in their
cerebrospinal fluid. Direct measurements of the activities of the BH,
biosynthetlc enzymes in the cortex of a single case of senile dementia of
the Alzheimer type have so far not detected any decreases compared to one
control.
Significance to Biomedical Research and Proposed Course of Project:
Our findings that patients with Alzheimer's disease have decreased
concentrations of BH, in their CSF but not in their blood has required a
major revision of the idea that these patients suffer from a generalized
deficiency of BH, . Further studies are being carried out to determine
whether it will be possible to identify the cause of the decreased BH,
levels in this disease. We also plan to explore the question of whether a
deficiency of BH, is a consequence or a cause of the disease.
We plan to try to isolate cDNA clones to the four enzymes involved in
BH, synthesis and to use these clones for prenatal diagnosis of variant
forms of phenylketonuria caused by defects in these enzymes.
Publications :
1. Kaufman, S. Tetrahydrobiopterin and hydroxylation systems in health
and disease. Neurochem & Neuropharmacol, 142: 1-28, 1986.
2. Kaufman, S. Unsolved problems in the diagnosis and therapy of
hyperphenylalaninemla caused by defects in tetrahydrobiopterin
metabolism. J. Pediatr 109: 572-578, 1986.
922
ZOl MH 01038-19 LNC
3. Irons, M. , Levy, H. L. , O'Flynn, M. E. , Stack, C. V., Langlais, P.
J., Butler, I. J., Milstien, S. and Kaufman, S. Folinic acid therapy
in the treatment of dihydropteridine reductase deficiency. J.
Pediatr, UO: 61-67, 1987.
4. Kaufman, S. Classical phenylketonuria and its variants caused by
defects in biopterin metabolism. In: Amino Acids in Health and
Disease: New Perspectives. Kaufman, S., ed. UCLA Symposia on
Molecular and Cellular Biology held at Keystone, CO. , May 30-June 4,
1986. Alan R. Liss, Inc., New York, p. 517-538, 1987.
5. Kay, A. D. , Milstien, S., Kaufman, S., Creasy, H. , Hoxby, J. V.,
Cutler, N. R. and Rapoport, S. Cerebrospinal fluid biopterin is
decreased in Alzheimer's disease. Archives of Neurol. 43: 996-999,
1986.
923
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30. 1987
PROJECT NUMBER
ZOl MH 01039-19 LNC
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the txirders.)
Pteridine Biosynthesis
PRINCIPAL INVESTIGATOR (List other professional personnel 6e/o* the Principal Investigator.) (Name, title, laboratory, and institute atfitlation)
Sheldon Milstien
Seymour Kaufman
Research Chemist
Chief
LNC
LNC
NIMH
NIMH
COOPERATING UNITS (U any)
UVB/BRANCH
Laboratory of Neurochemistry
INSTITUTE AND LOCATION
ADAMHA, NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.7
PROFESSIONAL:
0.7
OTHER:
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A scheme has been developed to isolate and characterize all of the enzymes
of tetrahydrobiopterin (BH^) biosynthesis from rat brain. The wide distribution
of tetrahydrobiopterin and these enzymes in the brain as well as the lack of
effect of specific neurotoxins on some of the enzymes in the pathway suggests
that tetrahydrobiopterin and/or these biosynthetic enzymes may have other roles
than previously thought. Work is now under way to prepare antibodies to each
of these enzymes to use for molecular cloning to obtain DNA probes to investigate
the physiological roles.
925
PHS 6040 (Rev. 1/84)
GPO ■14-SI*
ZOl MH 01039- 19 -LNC
Project Description:
It is now clear that the availability of tetrahydrobiopterin, the
cofactor required for the hydroxylation of tyrosine and tryptophan, can be
a determining factor In the synthesis of those neurotransmitters which are
derived from the hydroxy lated amino acids. Thus, any physiological
condition which alters the metabolism of tetrahydrobiopterin could have
profound CNS effects. In order to better understand the mechanisms by
which tetrahydrobiopterin levels are regulated as well as investigating
its role in the pathophysiology of such disorders as Alzheimer's disease
and variant forms of PKU, we are isolating the enzymes which catalyze the
de novo biosynthesis of tetrahydrobiopterin. Antibodies are being
prepared to all of the proteins which can then be used for
characterization of the proteins in human tissues as well as to screen
genomic libraries to prepare cDNAs which will be very valuable in genetic
screening.
Major Findings:
The four enzymes which catalyze the de novo biosynthesis of BH, from
GTP, GTP-cyclohydrolase, 6-PPH, synthase, 6-PPH, reductase, and
sepiapterin reductase have all been isolated and characterized from rat
brain. The enzymes, as well as BH, itself, are fairly evenly distributed
in the rat brain. There is a good correlation between the concentration
of BH, and the activity of GTP-cyclohydrolase and the activities of
tryptophan and tyrosine hydroxylases. However, treatment of rats with
specific nlgro-strlatal toxins "causes a concommltant decrease in BH, ,
tyrosine hydroxylase and GTP-cyclohydrolase without having any significant
effect on 6-PPH, synthase, 6-PPH, reductase, or sepiapterin reductase.
These results suggest either that the terminal enzymes in the BH
biosynthetic pathway are located in other neurons or that only a small
fraction of these enzymes is co-localized with tyrosine hydroxylase and
that the major portion of these enzymes located in other neurons must have
other functions.
Significance to Biomedical Research and Proposed Course of Project:
The characterization of the four enzymes involved In the de novo
synthesis of BH, will help to precisely localize the enzyme defects in
this pathway that can lead to abnormal brain development and function. We
plan to continue to try to prepare antibodies to all of the enzymes. Such
antibodies will be used in attempts to isolate cDNA clones for each of the
enzymes .
Publications :
I. Mllstlen, S. and Kaufman, S. The biosynthesis of tetrahydrobiopterin
in rat brain. In: Chemistry and Biology of Pteridlnes, Cooper, B.
A. and Whitehead, V. M. , Walter de Gruyter, Berlin, pp. 169-181,
1986.
926
ZOl MH 01039-19-LNC
Mllstlen, S. and Kaufman, S. The oxidation of apomorphine and other
catechol compounds by horseradish peroxidase. Relevance to the
measurement of dihydropteridine reductase activity. Biochim.
Biophys. Acta, 923:333-338, 1986.
927
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1986 through September 30, 1987
PROJECT NUMBER
ZOl MH 01040-19 LNC
TITLE OF PROJECT (BO characters or less. Title must tit on one line between ttie torders.)
Molecular Biology of the Pterin-Dependent Hydroxylases and Ancillary Enzymes
PRINCIPAL INVESTIGATOR (Ust otimr prolessional personnel below the Phncipal Investigator.) (Name, title, laboratory, and institute atfiliatlon)
PI Seymour Kaufman
Sheldon Milstien
Bruce Citron
Y. C. Liu
John Donlon
Chief LNC NIMH
Research Chemist LNC NIMH
Senior Staff Fellow LNC NIMH
Visiting Fellow LNC NIMH
Visiting Scientist LNC NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neurochemistry
INSTITUTE AND LOCATION
ADAMHA, NIMH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.2
PROFESSIONAL:
2.2
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
n (a1) Minors
n (a2) Interviews
n (b) Human tissues S (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A sufficiently extensive cDNA clone of rat liver phenylalanine hydroxylase
has been isolated and sequenced.
A composite DNA molecule containing this cDNA and the E. coli lac promoter,
operator, and translation initiation site has been constructed and placed in a
host that is inducible for this operon.
Elevated phenylalanine hydroxylase mRNA levels have been observed in
diabetic rats.
929
PHS 6040 (Rev. 1/84)
SPO 91 4-sia
ZOl MH 01040-19LNC
Project Description:
Through a combined, interdisciplinary approach, we are studying the
structure-function relationships of the aromatic amino acid hydroxylases and
related enzymes to explain the precise biochemical mechanisms involved in the
reactions which these enzymes catalyze. Importantly, with the aid of
molecular biology, we can more readily modify these enzymes in very specific
ways to clearly define the molecular interactions required for proper
catalysis. We also plan to use cDNA clones of these enzymes as probes to
study aspects of the molecular genetics of certain neurological and
psychiatric disorders.
Major Findings:
Phenylalanine Hydroxylase
We have isolated many cDNA clones to rat phenylalanine hydroxylase and
several of these have been sequenced to identify a few partial N-terminal and
C-terminal cDNA inserts and one nearly full length cDNA isolate. The full
length cDNA contains a 1916 base-pair insert which includes the coding
sequence from asparagine 8 to the C-terminal serine 452 and continues to the
poly(A) sequence 568 nucleotides further downstream.
Studies of hormonal regulation of phenylalanine hydroxylase activity in
diabetic rats have indicated that a three fold increase of enzyme activity in
the liver seems to be correlated with a five-fold increase of phenylalanine
hydroxylase mRNA.
Tyrosine Hydroxylase and Tryptophan Hydroxylase
Several putative cDNA clones have been isolated from the human brain stem
and also the basal ganglion. These have been partially characterized by
restriction mapping and southern hybridization.
Dihydropteridine Reductase
An intact DNA fragment has been isolated from the full length cDNA clone,
which contains only the dihydropteridine reductase gene.
6-pyruvoyl-tetrahydropterin synthase
Antibody specific for this protein has been produced and shown to
identify the enzyme band on a Western blot.
Significance to Biomedical Research and Proposed Course:
Phenylalanine Hydroxylase
Phenylketonuria results form an untreated deficiency of phenylalanine
hydroxylase. A major symptom of this disease is severe, irreversible mental
retardation. This indicates that individuals carrying a mutant allele that
930
ZOl MH 01040-19LNC
yields a partially deficient enzyme should have some propensity for mental
disorders. We will be using cDNA probes to characterize alterations in
messenger RNA levels that have biological effects.
Tyrosine Hydroxylase
This enzyme is the primary control point for the neurotransmitter pathway
implicated in several diseases such as Parkinsonism. Recent findings have
been consistent with at least one type of mutation in this gene being
responsible for at least one type of manic depressive disorder. Using cDNA
probes, we will study the transcriptional response of a wild-type copy of this
gene in neuroblastoma cells to a wide variety of stimuli. These probes will
also be used to characterize the modifications in tyrosine hydroxylase that
lead to some of the diseases explainable by altered catecholamine metabolism.
Tryptophan Hydroxylase
The neurotransmitter, serotonin, is an end product of the metabolism of
tryptophan by tryptophan hydroxylase. Decreased activities of all three
aromatic amino acid hydroxylases produce pronounced central nervous system
disorders broader than the symptoms due to any characterized or postulated
abnormality of any one hydroxylase. Conversely, there is a large array of
mental diseases for which no biological explanation exists. Aberrant
tryptophan hydroxylase expression might be responsible for some of these
disease states and we will attempt to identify these cases using hybridization
probes .
Dihydropteridine Reductase (DHPR) and 6-pyruvoyl-tetrahydropterin
synthase (PTS).
All three aromatic amino acid hydroxylases have an absolute requirement
for the cof actor, tetrahydrobiopterin. DHPR and PTS are involved in the
production of this cofactor and individuals deficient in one of these
activities have atypical phenylketonuria. This is a severe disease involving
mental retardation, CNS problems, movement disorders, etc. Several patients
have been identified having marked deficiencies in each of these enzymes. We
will first characterize the genetic basis in the known cases and also use
these probes to identify changes in less severe and less obvious classes of
such disorders.
Publications:
1. Citron, B. A., Chaudary, P. V., Rao, D. N. and Kaufman, S. Evidence for
transcription and potential translation of the human 1.9 kb Hindi II
repetitive element. Nucleic Acids Res, 14: 3137-3142, 1986.
2. Lockyer, J., Cook, R. G. , Milstien, S., Kaufman, S., Woo, S. L. C. and
Ledley, F. D. Structure and expression of human dihydropteridine
reductase. Proceed. Natl Acad Sci. 84= 3329-3333, 1987.
931
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT MUMBER
PERIOD COVERED
October 1, 1986 to September 30. 1987
Z01 m 00^81-21 LMP
TITLE OF PROJECT ^80 characters or less. Title must tit art one Ime tiefween the borders.)
Mechanical, Thermal and Ootical Signs of Excitation in the Nervous System
PRINCIPAL INVESTIGATOR (List other protessionel personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfiliation)
PI: Ichiji Tasaki- Chief, Unit of Neurobiology LNP, NIMH
Others: Nobuko Tasaki Guest Worker LNP, NIMH
COOPERATING UNITS (it ar\y)
LAB/BRANCH
Unit on Neurobiology, Laboratory of Neuronhysiology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects D (b) Human tissues 0 (c) Neither
D (a1) Minors
D (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Due to the transfer of orincipal investigator to the Laboratory of Tell
Biology, this project has been assigned number Z01 MH 02396-01 LCB.
933
PHS 6040 (Rev. 1/84) =''0 ■''•en
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01092-09 LNP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders.)
The Frontal Lobe and the Cerebral Control of Behavior
PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Principal Investigator ) (Name, title, laboratory, and Institute atliliatlon)
PI: Steven P. Wise
Others: Kiyoshi Kurata
Eilon Vaadia
Shraga Hocherman
Research Biologist
Visiting Fellow
Visiting Associate
Visiting Associate
LNP, NIMH
LNP , NIMH
LNP , NIMH
LNP , NIMH
COOPERATING UNITS (H any)
LAB/BRANCH
Laboratory of Neurophysiology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH Animal Center, Poolesville, Maryland 20837
TOTAL MAN- YEARS:
2.4
PROFESSIONAL:
2.4
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The primate frontal lobe consists of three main parts: the primary motor cortex
(MI), the prefrontal cortex, and the nonprimary motor cortex. Previous work on
this project has shown that the nonprimary motor cortex can be divided into at
least two fields: the supplementary motor cortex (SM) and the premotor cortex (PM).
Definition of these cortical fields depended on an analysis of neuronal responses
to peripheral inputs, thresholds for evoking movements with intracortical electri-
cal stimulation, the properties of single neurons during the performance of several
visuomotor tasks, and cytoarchitectonics. After defining the areas, we directed
our studies, using the techniques of behavioral neurophysiology developed in this
laboratory, toward the nonprimary motor cortex. Our results support the hypothesis
that PM and SM contribute to sensorially-ref erenced behaviors especially those
guided by abstract or arbitrary sensory cues. These studies have provided new
insight into the processes by which animals, including humans, prepare for action
in relation to partially predictable environmental events. The preparation for
receipt of informa (attention) and for future action (motor set) represent higher
brain functions amenable to both quantitative and qualitative neurophysiological
analysis and appear to be a property conferred by the frontal cortex.
935
PHS 6040 (Rev. 1/M)
OPO ti4-«ia
ZOl MH 01092-09 LNP
Objectives:
The goal of this project is to gain a better understanding of functional organi-
zation of the primate frontal lobe and to contribute to the development of a
unified hypothesis of its role in directing behavior. In order to understand
the biological basis of mental illness, it is necessary to have a better under-
standing of the processing underlying the behavioral flexibility of higher mam-
mals i.e. , the ability to respond with virtually any motor action of which
the animal is capable to virtually any sensory input that the animal can discrim-
inate, to plan for future action, and to formulate and achieve reasonable goals.
We believe that the mechanisms of the frontal cortex underlie these important
higher brain functions. Our approach to an analysis of frontal lobe mechanisms
consists of a multidisciplinary experimental strategy involving the formulation
and testing of hypotheses with the techniques of behavioral neurophysiology de-
veloped in this laboratory.
Methods:
Fourteen rhesus monkeys have been trained to perform visually guided motor tasks
over the nine-year course of this project. Each one of the eight tasks described
below is a separate subproject:
(1) One monkey was operantly conditioned to depress one of four keys lo-
cated in a perimeter at arms length. While the monkey pressed one key, another
of the four keys, selected randomly, was illuminated after a randomly varied
delay period. This key thereby became the next target. An auditory cue near the
target could be substituted for illumination of the target. A barely discernable
visual cue near the target key, appearing after another variable delay, signaled
the monkey to move and depress the target. The monkey was required to make the
movement within a short period of time, near the limit of reaction time. The
purpose of this subproject was to make an initial survey of neuronal activity
patterns in PM to help define it. Electrical stimulation and cytoarchitectonic
techniques were used in this subproject. Another important aspect of this sub-
project was a comparison of neuronal activity when visual vs. auditory signals
instruct the monkey to make the same movement.
(2) Two monkeys were conditioned to align two spots of light on a screen.
One of these spots was controlled by a computer (the target spot), the other by
the arm movements of the animal (the position spot). The monkey was required to
align the spots within a small accuracy "window." In five-sixths of the trials,
after a short period of time, the target spot jumped to one of six locations.
The monkey had to maintain his arm position unchanged until the target spot dim-
med, at which point it was required that the monkey flex or extend his forearm
rapidly and accurately in order to realign the position spot with the target
spot. In one-sixth of the trials, the computer selected a situation in which
physically identical stimuli signaled the animal to make ji£ movement. This
experiment was designed for two purposes: to contrast neuronal activity in MI
and PM, and to distinguish neuronal activity when identical stimuli signal
the execution vs. the withholding of movement.
936
ZOl MH 01092-09 LNP
(3) Two monkeys were operantly conditioned to depress the central of three
keys located on a panel at arm's reach. After a period of time, either the left
or right key became illuminated. Three experimental conditions ensued: (a) the
left or right key remained illuminated and served as the target for the subse-
quently triggered movement, (b) the light was turned off before the monkey was
allowed to execute the movement, forcing the monkey to remember the proper
target, or (c) the target light was switched before the monkey was allowed to
execute the movement. This experiment was designed to further test the rela-
tionship of neurons in PM to the motor set of the animal. In addition, we
could compare cell activity when the monkey performed the task described above
and a self-paced movement between the left and right keys. Subproject 3 was
designed to test the competing hypotheses that set-related activity in PM
reflects a continued visual stimulus and that it reflects the preparation for
movement.
(4) One monkey was conditioned to execute a single limb movement as well
as a short sequence of two limb movements in the same direction. The monkey
was seated in front of a panel of three keys as in subproject 3. Each trial
started with the monkey pressing the leftmost of the three keys. Two experi-
mental conditions ensued: (a) the center key was illuminated, thus indicating
that a single movement was to be made to depress the center key, or (b) both
the center and right lights were simultaneously illuminated to indicate that
a short motor sequence was to be initiated to depress both keys, in order.
This subproject was designed to test the hypothesis that PM is especially im-
portant in guiding simple sequences of movement.
(5) Two monkeys were conditioned to respond to two different sorts of
visuospatial instruction signals. One type of instruction signal was compar-
able to that described in the subprojects described above, i.e. , the visual cue
itself was directional; indeed it was part of the target. This situation could
be contrasted with one in which the instruction cues contained no directional
information. A blue lamp meant to move the limb to the right and a yellow lamp
to move to the left. Thus the relationship of the stimulus to the movement in
the latter situation was abstract or arbitrary. The hypothesis was that if PM
activity reflects the preparation for movements there should be little or no
difference in activity in the two situations.
(6) Two monkeys were conditioned to make the same movements under two dif-
ferent experimental conditions: (a) when visual instructions of the type de-
scribed for subprojects 1, 3 and 5 guided the movement, and (b) when the identi-
cal cues were irrelevant or nonexistent and the monkey guided its behavior via
internal (i.e., nonsensory) processes. This study was designed to test the
hypothesis that PM is especially important when sensory signals instruct a
movement and to contrast activity in PM with that in SM, which has been hypo-
thesized to play a special role when internal processes instruct a movement
(P.E. Roland et al, J. Neurophysiol. , 1980, 43: 118; J.C. Eccles, Arch. Psychiat.
Nervenkr. . 1982, 231: 423; G. Goldberg, Behav. Brain Sci. , 1985, 8: 567).
(7) Two monkeys were trained to respond to one visual stimulus with a hand
movement and to another visual stimulus with a foot movement. The cell activity
in PM before hand movements was compared to that before foot movements. Before
937
ZOl MH 01092-09 LNP
either movement, the monkey was required to withhold movement for a variable
delay period. This study was designed to examine the internal organization of
the premotor cortex, in particular whether it was topographic organization.
(8) The activity of cells in PM of one monkey was compared in two experi-
mental conditions: (a) when a visual cue instructed the monkey about where the
target of the next limb movement should be, as well as when to execute the move-
ment, and (b) when a visual cue indicates to the monkey only when to execute the
movement. In condition b, the monkey had no environmental cue upon which to
base its choice of two potential targets. Instead, the monkey simply had to
guess and was prevented from adopting the strategy of moving always to one tar-
get (thereby receiving rewards on half the trials) by an algorithm that randomly
selected what target would be rewarded. That algorithm decreased the probability
that a movement to a given target would be rewarded by 0.1 every time the monkey
made as many as three consecutive movements to that target. This study was de-
signed to examine the neuronal activity in PM that precedes the instructional
cues, in particular to test the hypothesis that such activity reflects the prep-
aration for movement.
In each of the subprojects, single-unit activity and behavioral data were col-
lected on-line with PDF 11/03 and 11/23 computers and analyzed off-line with PDP
11/23 and 11/73 computers. Presently, we are analyzing the data with a dedicated
PDP 11/73 in the laboratory, using a program written by Karl Arrington, formerly
of the LNP support staff.
Major Findings;
The activity of about 3600 neurons have been examined in this project to date,
and 1583 of these have been studied in detail. Three major sets of findings
developed from our work on this project and three others have spun off to other
projects: ZOl MH 01097-01 LNP headed by Melvyn Heyes; ZOl MH 01096-03 LNP headed
by Andrew Mitz; and ZOl MH 02376-01 LCB headed by Charles G erf en.
1. Comparison of Primary and Nonprimary Motor Cortex. SM neurons were found
to be much less responsive to peripheral somatosensory inputs than MI neurons
in the same monkeys. The lack of profound somatic sensory responsiveness in
these parts of the somatic sensorimotor cortex supports the hypothesis that SM
plays its most significant roles in the guidance of movement by internal pro-
cesses (such as memory) rather than by feedback from mechanoreceptors of the
limbs. We have begun an explicit test of this idea (subproject 6). MI seems to
be specialized for control of movement, in part, by cutaneous and noncutaneous
mechanoreceptors, and adjustment for internal or external events that cause
deviations from the animal's goals.
2. Cortical Field Definition and Internal Organization of Nonprimary Motor
Cortex. Our findings have enabled us to improve the current understanding of
cerebral localization and functional specialization in the agranular frontal
cortex (SM, PM, plus MI) and certain adjacent parts of the cortex. Of special
importance has been the effort to determine anatomical correlates of physiolo-
gically defined cortical regions. Microelectrode methods revealed that the
938
ZOl MH 01092-09 LNP
boundary between MI and SM corresponds to the boundary between two anatomically
defined parts of the agranular frontal cortex (termed areas 4 and 6). This
differed from the accepted published maps at the time this study was undertaken.
This line of inquiry has spun off to Mitz's project, as mentioned above. Similar
work has clarified the location of the boundary between PM and MI, and this work,
too, is being elaborated as part of Mitz's project. In this fiscal year, we
found in this project that most PM neurons contribute to the preparation for and
execution of specific limb movements rather than movement per se and thus has a
coherent internal organization. Further, the differential distribution of neu-
rons with activity related to hindlimb vs. forelimb movement (subproject 7) sup-
port previous indication that PM is topographically organized (K. F. Muakkassa,
and P. L. Strick, Brain Res. , 1979, 177: 176; K. Kurata, K. Okano and J. Tanji,
Exp. Brain Res. . 1985, 60: 188).
3. Premotor Cortex Physiology. The findings described above led to the elab-
oration of a related, third set of findings, those concerning the functional
specializations of PM and SM. PM can be distinguished from the MI representation
by its markedly increased threshold for evoking movements with intracortical
microstimulation. Further, and of most interest to us, a substantial population
of neurons change their activity in relation to motor set. The pattern of ac-
tivity termed "set-related" appears to be specifically correlated with the motor
preparation of the animal (subproject 1). This hypothesis has been supported
in six ways: (a) set-related units show changes in activity when visual signals
cue a movement, thus establishing a specific motor set, but not when the same
signals instruct the monkey to withhold movement (subproject 2); (b) if the
visual instruction changes (to establish a different motor set), the unit activ-
ity rapidly changes to reflect the new set (subproject 3); (c) when the instruc-
tion is removed (but the set remains the same), the unit activity continues to
reflect the set rather than the sensory signals (subproject 3); (d) the set-re-
lated activity before the first of a series of two movement is the same as that
before the same movement when it is executed by itself (subproject 4); (e) set-
related activity is usually the same when directional (left or right) a instruc-
tion stimulus and an arbitrary (yellow or blue) instruction stimulus instruct
the same movement (subproject 5); and (f) set-related activity is usually the
same when the monkey plans a movement on the basis of visual (external) stimuli
as when the monkey plans the same movement on the basis of mnemonic (internal)
information (subprojects 6 and 8). In addition, it has been found that these
and other premotor cortex units change their activity in advance of predictable
environmental events, and that such activity which we call pre-cue activity,
reflects or contributes to attention toward the instructional information trans-
mitted by the cue, as well as its timing (subprojects 2, 3, 6 and 8). Taken
together our findings improve the understanding of the set-related processes of
PM and accord with the hypothesis that PM plays an important role in behaviors
in which a movement must be retrieved from memory on the basis of highly flex-
ible, arbitrary cues.
The past year has seen the completion of data collection on subprojects 6, 7,
and 8. We had accepted for publication a full-length paper on subproject 5 in
Experimental Brain Research. In addition, this fiscal year saw the publication
of results from subprojects 2 and 3 (Wise et al. 1986) and a complete review of
the literature on the motor cortical fields of rodents, a basis for comparison
939
ZOl MH 01092-09 LNP
with the primate research performed in the present project (Wise and Donoghue,
1986). Toward the end of the fiscal year, we submitted certain of the results
of subproject 6 for publication and we are currently preparing full-length
manuscripts based on the results of subprojects 7 and 8. As of this writing,
we hope that these papers will be submitted for publication before the end of
the fiscal year. In addition, at least one and possibly more papers are expected
to result from these data in the next fiscal year.
Significance to Biomedical Research and to the Program of the Institute:
Studies of functional localization in higher-order motor cortical fields, such
as the premotor cortex and supplementary motor cortex, are important to under-
standing the cortical control of motor acts of the least automatic kind, in both
health and disease, and especially for understanding the way in which sensory
signals are converted, by the brain, into organized motor acts. A much improved
knowledge of the nonprimary areas of the cerebral cortex and their relation to
higher-order control of motor behavior may yield insight into higher brain func-
tions of all types. More generally, much of this project is devoted to basic
study of the frontal cortex, a presumed site of higher brain functions such as
reason, attention, forethought, and perception, as well as fine motor capabili-
ties. In this context, the recent findings of Weinberger and his group at St.
Elizabeth's Hospital are of great interest. They have hypothezied that disease
of the frontal lobe is one cause of schizophrenia (D.R. Weinberger, K.F. Berman,
and R.F. Zee. Archiv. Gen. Psychiatry, 1986, 43: 114-12A). We hope to link our
growing understanding of the frontal lobe mechanisms underlying "motor set", the
ability of animals to plan and select an action in advance of the time that it
needs to be performed, with the activity of frontal cortex neurons. Such ad-
vances would be of fundamental importance in understanding the roles of the
frontal lobe in mental health and disease.
Proposed Course of the Project:
The project is being elaborated in two directions: (1) an explicit examination
of the functional oragnization of the prefrontal cortex (PF) in collaboration
with Eilon Vaadia and Shraga Hocherman) and (2) a continuation of our study of
the premotor cortex (PM) and its role in the selection and control of behavior.
1. A monkey will be operantly conditioned to perform a visually guided behavior
involving three features: (a) if two signals, presented sequentially, are the
same (i.e. , red plus red or green plus green) then, after a self-timed delay
period, the monkey is required to displace the target key displaying that color
cue (red or green, respectively); (b) if the two signals are different (i.e. ,
one is red and the other green), then the monkey must either withhold any limb
movement. This task is directed toward further testing the hypothesis that PM
neuronal activity reflects the preparation for action (e.g. , S.P. Wise et al. ,
Brain Res. , 1983, 260: 301) whereas PF functions in comparing incoming informa-
tion against memories and in the application of behavioral rules.
This new subproject will extend our work on PM into the PF cortex and to explore
directly (and in the same animal) differences in activity in these two cortical
regions. In the past fiscal year, we have trained a monkey to perform this task
940
ZOl MH 01092-09 LNP
and have begun the data collection. Vaadia and Wise continued data collection
until Vaadia 's departure in August, at which time Hocherman began his work on
this subproject. We expect the first phase of this work to be completed next
fiscal year.
2. The behavioral neurophysiological studies of PM have many more planned sub-
projects than time and manpower to perform them. Accordingly, one important
line of investigation was spun off to Mitz's project (ZOl MH 01096-03): an exam-
ination of frontal lobe function in relation to memory. The only past studies
of memory-related neuronal activity in frontal cortex have either been indirect,
equivocal or based on complex and poorly localized summed potentials rather than
the activity of individual neurons. Accordingly, and in view of the recent
proposal by U. Halsband and R.E. Passingham (Behav. Brain Res. . 1985, 18: 269)
that PM functions in retrieving a movement from a long-term memory store, we
propose to test this hypothesis by examining PM activity both before and after
learning an arbitrary stimulus-movement association. We predict that there
will be little or no task related activity following the stimulus until the
association is learned.
We are planning two initiatives in the present project. In one, we plan to
examine frontal cortex neuronal activity to determine the frontal cortical loca-
tion of neurons subserving sensory memories and the locations of analagous repre-
sentations of motor memories. The experimental design to achieve our objective
can be summarized: the monkey must learn to associate a color (there must be
two) in a certain location in space (there must be four) with a specific motor
act (there must be two). In one condition, the monkey is compelled to remember
the sensory stimulus to be able, after a delay period, to execute the correct
behavior. In the other condition, the monkey must remember the motor act as-
sociated with the visuospatial stimulus. It will be time consuming to condition
monkeys to perform this difficult task, but we believe that it will be a very
rewarding study if successfully completed. In terms of cerebral localization,
the distinction being tested, that between the sensory and the motor, between
input and output, between perception and action, between memories and the behav-
ioral uses of those memories, is one of the most important problems in neurobiol-
ogy, one which is best approachable with the tools of behavioral neurophysiology.
Publications:
Kurata, K and Wise, S.P.: Premotor cortex of rhesus monkeys: Set-related
activity during two conditional motor tasks. Experimental Brain Res, in press,
1987.
Wise, S.P.: Neuroanatomical substrates of premotor centers. Progress in Brain
Research 64: 111-114, 1986.
Wise, S.P.: The motor cortex. In Adelman, G. (Ed): The Encyclopedia of
Neuroscience, Springer-Verlag, in press, 1987.
941
ZOl MH 01092-09 LNP
Wise, S.P. and Donoghue, J .P . : The premotor cortex of rodents. In Peters, A.
and Jones, E.G. (Eds.): Cerebral Cortex, Vol 5, Plenum, New York, 1986, pp.
243-270.
Wise, S.P., Weinrich, M. , and Mauritz, K.-H. : Movement-related activity in the
premotor cortex of rhesus macaques. Progress in Brain Research, 64: 117-131,
1986.
942
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1. 1986 to September 30. 1987
PROJECT NUMBER
ZOl MH 01096-03 LNP
TITLE OF PROJECT (BO charactars or less. Title must lit on one line between the tmrders.)
Spatial Organization of the Primate Motor Cortex
PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and institute atlillatJon)
PI:
Others:
Andrew R. Mitz
Steven P . Wise
Moshe Godschalk
Senior Staff Fellow
Research Biologist
Visiting Associate
LNP , NIMH
LNP , NIMH
LNP , NIMH
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neurophysiology
INSTITUTE AND LOCATION
NIMH. ADAMHA. NIH Animal Center. Poolesville. Maryland 20837
TOTAL MAN- YEARS:
1.9
PROFESSIONAL:
1.9
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
□ (a1) Minors
D (a2) Interviews
n (b) Human tissues S (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this project is to examine the internal organizations and the inter-
relationship among motor areas of the frontal lobe, including the primary motor
cortex (MI), the supplementary motor cortex (SM), the frontal eye fields (FEF) and
the premotor cortex (PM). The model species chosen for study is the rhesus monkey,
because these motor areas have been best characterized in this species. In the
first part of this project intracortical microstimulation is being employed to
examine the efferent topography of the motor areas.
In the second part of the project single-unit recording will be employed to examine
the possible involvement of the premotor cortex in learning new stimulus-response
relationships. It is the goal of this part of the project to determine the roles
played by each cortical motor area in recall and execution of learned motor behav-
iors.
943
PHS 6040 (Rev. 1/84)
SPO >i4>aia
ZOl MH 01096-03 LNP
Objectives:
Movement-related single-unit activity in the primary motor area (MI), supple-
mentary motor area (SM), premotor area (PM), and frontal eye field (FEF) suggest
that these cortical regions are involved in the programming and execution of
movement. Anatomical evidence further suggests that these areas are extensively
interconnected, and we hypothesize that these cortical fields act together to
generate appropriate motor commands. Although certain features of the anatomical
and physiological organization of these motor areas are known, many questions
remain about the detailed topography in each of these areas. The objective of
this project is to explore the topographic organization of these motor areas and
correlate their functional organization with aspects of their cytoarchitectonics.
A further goal is to characterize and distinguish each area's contribution to de-
scending motor commands .
Methods Elmployed:
1. Microstimulation mapping of cortical motor areas. Intracortical microstimu-
lation in awake or lightly sedated animals has become a standard method for ex-
amining motor topography in MI, FEF, and other motor areas. However, this tech-
nique had not been successful for exploring the somatotopy of SM or PM.
The problem of SM mapping has been overcome in this project by developing a
modified intracortical stimulation technique. This technique utilizes platinum-
iridium electrodes with about 1000 ^tm^ of metal exposed at the tip, compared to
standard microelectrodes with exposed tips of 100-250 nm2 . in addition to the
electrode geometry, careful selection of stimulus parameters and the use of
biphasic current pulses has contributed to successful SM mapping.
Rhesus monkeys were implanted with stainless steel chambers to exposed the dura
mater over SM, PM, and FEF. Electrodes were inserted through the dura and
electrical stimuli were delivered periodically as the electrode descended through
the cortex. The stimulus parameters were 0.2 ms duration for each phase of the
biphasic pulses, 330 pulses/second, 31-pulse trains, and 65 \ia search currents.
Movements evoked at each stimulation site were identified by two observers and
recorded.
2. Oculometry. In some instances eye movements were observed and recorded
manually. Later, eye movements were recorded as electro-oculograms via implanted
silver/silver-chloride electrodes or non-invasively with infrared oculometry.
When the latter techniques were used, two channel directional information (left-
right and up-down) was either recorded on separate magnetic tape channels or com-
bined as an X-Y vector on a storage oscilloscope and photographed.
3. Quantitative cytoarchitectonics. The boundary between cytoarchitectonic areas
4 and 6 was first estimated qualitatively. Then, a computerized cell plotting
system connected to a high-power microscope was used to locate, identify, and
measure individual cells on a series of histological sections. Cell body areas
were measured in 21 sections separated by about 500 ^im, and covering a 10 mm
rostrocaudal extent. Cell measurements were made from an area 1 mm in dorso-
ventral extent and centered midway between the dorsal surface of the hemisphere
and the cingulate sulcus. Each cell body greater than 20 [nn in any dimension was
944
ZOl MH 01096-03 LNP
circumscribed under 400 X magnification. The coordinates of the cell boundary
were recorded by the computer, which then computed the area of the cell body.
Any stained portion of the proximal dendrites was included in the cell area
measurement .
4. Chronic single-unit recordings. Movement-related units in the motor areas
were isolated using chronic recording techniques in an operantly conditioned
monkey. The behavioral paradigm chosen dissociated the direction of hand dis-
placement from the direction of wrist rotation by training the animal to make
wrist flexion/extension movements from either a pronated or supinated posture.
When the forearm was pronated, wrist flexion was a downward movement of the hand;
when the forearm was supinated, wrist flexion was an upward movement. Isolated
units from MI were tested in both forearm positions. Paradigm events, wrist
position, and unit firing times were recorded in real-time using a Plessey Micro
II (LSI 11/23) minicomputer. After each recording session, perievent spike his-
tograms were generated from the data to compare the event-related activities of
individual task-related neurons.
A new behavioral paradigm is being used to evaluate PM unit activity before and
after stimulus-response associations are learned. New hypotheses concerning the
functions of the PM suggest that one of its roles is to recall an appropriate
motor program based upon recognition of a stiiriulus. To study the validity of
this approach, monkeys will be trained to move a lever to one of 3 positions
based upon 3 color figures. The set of color figures will be changed peri-
odically to restart the learning process. Single-units will be studied as the
animal learns which figure matches which target position. Unit activity will
also be recorded during performance of a well-learned stimulus-response associa-
tion. The hypothesis will be supported if the response of a PM unit changes as a
figure-target relationship is established to resemble the activity in response to
the well-known stimulus.
Major Findings:
1. Controversy concerning the existence and nature of SM topography has stemmed
from methodological problems associated with electrical stimulation of SM.
Results from previous microstimulation studies of SM have led to the suggestion
that this area lacks a motor topography. The new methodological approach, out-
lined above, has yielded a topographic map of motor organization. In this map,
orofacial movements, including movement of the pinnae, lips, tongue, and jaw, and
conjugate eye movements to the contralateral visual hemifield, were observed most
rostrally. Adjacent to and overlapping with this orofacial and eye movement
region was a region from which forelimb movements were evoked. Evoked forelimb
movements usually included action at 2 or more joints. Caudal to and overlapping
the sites evoking forelimb movements was a third region of the SM from which
hindlimb and tail movements were evoked. The hindlimb and tail region of SM
merged with the hindlimb region of MI without a distinguishable boundary. The
overall rostrocaudal extent of the SM covered 12-14 mm.
The region of frontal cortex over which saccadic eye movements can be evoked with
low stimulus currents had been thought to be limited to the FEF and, based on our
recent results and those of others (Schlag and Schlag-Rey, J. Neurophysiology,
1987, 57:179), rostral SM. Using the new stimulation technique, however, we have
9A5
ZOl MH 01096-03 LNP
evoked eye movements from sites along a thin strip of cortex extending between
these two fields. Movements evoked nearer the FEF were most like those evoked
from the FEF, saccades of a fixed displacement and direction, regardless of ini-
tial ocular position. Closer to SM, evoked movements were most like one class
described by Schlag and Schlag-Rey for the SM, eye movements depended upon the
initial ocular position and always terminated near the same final position. Thus
the FEF and rostral SM eye movement areas may be part of a larger cortical area
concerned with eye movement control.
Preliminary simulation in and around the arcuate sulcus indicate that forelimb
movements can be reliably evoked from PM. A detailed topographic analysis of the
evoked movements must await the results from the histological examination in
progress.
2. Quantitation of cell sizes along the medial wall of the cortex has allowed an
objective evaluation of the cytoarchitectonic transition from area 4 to area 6.
The rostrocaudal distribution of cell bodies greater than 600 piti^ and of cell
bodies greater than 1200 nin2 show that these populations decrease from a peak to
low densities over a 4 to 5 mm range. Neurons with areas of 300-500 [om^ do not
have the same rostrocaudal change in density. The qualitatively-determined
boundary between area 4 and area 6 corresponds to the rostrocaudal level at which
the largest pyramidal cells appear in substantial densities. The location of
this boundary determined relative to the tail representation (of MI, SM, or both)
agrees well with that described previously.
3. Findings from the first single-unit study was inconclusive in one monkey. The
task chosen, while appropriate for dissociation of direction of hand displacement
from the direction of wrist rotation, could not test the experimental question.
Electromyographic analysis of the flexion/extension task showed that the extensor
carpi radialis muscle is active for upward movements in both postures. Therefore
it is not possible to conclude that cells discharging before upward hand move-
ments, independent of the direction of movement around the wrist, code for spa-
tial trajectory of hand movement, as hypothesized for PM. This problem of inter-
pretation points out the difficulty of behavioral neurophysiological work in the
distal forelimb. On the basis of this experience, the principal investigator
decided to abandon this approach, and the monkey was studied instead in an
anatomical paradigm developed by Charles Gerfen, LCB/NIMH.
Significance to Biomedical Research and to the Program of the Institute:
The project represents an effort to explore the organization of the frontal cor-
tex. SM is the primary cortical target of the basal ganglia. Examining the in-
ternal organization of PM, FEF, and SM, and their interactions with the other
frontal areas is of importance in gaining an overall understanding of the frontal
cortex and its role in health and disease.
Proposed Course of the Project:
The current project involves a systematic study of three motor areas, MI, SM, and
PM and the frontal cortex areas involved in eye movements . Before the start of
this project, the somatotopy of MI had been well established. Stimulation map-
ping has demonstrated SM topography and a heretofore unstudied eye movement area.
946
ZOl MH 01096-03 LNP
The first priority is to complete PM stimulation mapping. Further work on PM
topography will depend upon the outcome of this mapping study. If the results
from stimulation show that the PM forelimb representation resides between the FEF
and the forelimb representation of MI, then the mapping work will be complete.
If, however, the maps contradict current ideas concerning PM organization, the
simulation mapping can be combined with an anatomical tracer study. In such a
study either the forelimb or the hindlimb representation of PM and MI are
elucidated with electrical stimulation mapping and then tracer dyes are injected
into the representations. This combination anatomical and physiological methods
to study PM topography in macaque monkeys is possible with the new stimulation
method described above.
Another possible line of study would be to explore the eye movement area found
between the rostral SM and and the FEF. As discussed above, some ocular move-
ments evoked by stimulation in this area depend upon the initial eye position.
It would be valuable to test the visual receptive fields of isolated single units
with the eyes in different positions to see if some units have different recep-
tive fields with different eye positions. However, eye movement studies are most
easily studied with the aid of scleral search-coil oculometry, a technology not
immediately available in this laboratory.
A new project is underway to record single-unit activity in the PM during move-
ments in response to learned stimuli. As discussed above, PM may be involved in
recall of motor programs. If this can be shown, a central role for PM in the
generation of learned movements will have been elucidated. Further experiments
would be aimed at examining the transition from the unlearned to the learned con-
dition. If units in the PM are insensitive to the difference between novel
stimuli and those associated with a specific motor response, then current inter-
pretation of PM lesion experiments (by others) must be re-examined.
Publications :
Mitz, A. R. and Wise, S. P. (1987) The somatotopic organization of the supplemen-
tary motor area: Intracortical microstimulation mapping. J. Neuroscience,
7:1010-1021.
947
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01097-01 LNP
PERIOD COVERED
October 1. 1986 to September 30. 1987
TITLE OF PROJECT (80 characters or leas Title must lit or) one line tMtween tt>e borders.)
Activity of Corticostriatal Neurons In Motor Cortex of Primates During Wn'sr. Movement
PRINCIPAL INVESTIGATOR (Uat other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute atlilialion)
PI:
Others:
Melvyn P . Heyes
Steven P . Wise
Visiting Associate
Research Biologist
LNP , NIMH
LNP , NIMH
COOPERATING UNITS (H hny)
UAB/BRANCH
Laboratory of Neurophysiology
INSTITUTE AND LOCATION
NIMH. ADAMHA. NIH Animal Center. Poolesville. Maryland 20837
TOTAL MAN-YEARS:
0.4
PROFESSIONAL:
0.4
CHECK APPROPRIATE BOX(ES)
n (a) Human subjects
D (al) Minors
D (a2) interviews
D (b) Human tissues B (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the apace provided.)
Although it is well known that the major input to the striatum originates in the
cerebral cortex, virtually nothing is known about the type of information conveyed
by the corticostriatal pathway. The part of the striatum that receives input from
the primary motor cortex is the putamen. Putamen neurons discharge at or after
the onset of EM3 activity and reflect the direction of movement per se, whereas
many neurons in the primary motor cortex discharge prior to the onset of EM3 acti-
vity and reflect individual muscle activity independent of the direction of move-
ment. To investigate corticostriatal (CS) activity during movement, we developed
a strategy to identify CS neurons and quantitate their activity, their timing with
respect to movement, and their relationship to the pattern of muscle activation in
rhesus monkeys. Our observations indicate that motor information is conveyed via
the CS pathway and that CS neurons more closely resemble primary motor cortex
neurons than putamen neurons with respect to their timing and relationship to the
patterns of muscle activity. It is possible, therefore, that motor disorders could
result from excess in CS activity that causes abnormal contraction of muscles and
limb position in space.
949
PHS 6040 (Rev. 1/84)
apo ti4-*i*
ZOl MH 01097-01 LNP
Objectives;
Anatomical studies have established that the major afferent input to the striatum
originates in the cerebral cortex. However, virtually nothing is known about the
type of information that may be conveyed by the corticostriatal (CS) pathway.
The purpose of this project, then, was to develop a strategy of identify CS neu-
rons in the cortex and evaluate the type of information they conveyed to their
targets. Because movement is a very well defined and easily quantifiable output
of the brain, the activity of CS neurons in the motor cortex was monitored during
the performance of a simple motor task.
Electrophysiological studies have shown that movement-related modulations
of neuronal activity in the putamen begin only briefly before movement begins,
occurring at the same time or after the earliest muscle EbG activity causing
the movement. In contrast, many neurons in the motor cortex, particularly those
projecting through the pyramidal tract, begin their activity modulations sub-
stantially before the earliest changes in muscle EMJ activity associated with
movement. In addition, neuronal activity in the putamen is relatively unaffected
by torque loads assisting or opposing movement, whereas the activity of neurons
in the motor cortex are significantly modified by loads. In this respect the
output of the motor cortex reflects the activity of individuals muscles during
the movement, whereas putamen neurons tend to reflect the pattern of movement.
For example, when a flexion movement is performed against a load, agonist muscle
activity is potentiated compared to when the same movement is performed against
no load. In contrast, if the load applied assists the movement, the agonist
muscle no longer may be active and the actual movement may be executed by regu-
lation of antagonist muscle activity only. In this situation, motor cortex
neuronal activity would be potentiated by opposing loads and attenuated by as-
sisting loads, whereas neuronal activity in the putamen would be relatively
insensitive to the loads. The activity of CS neurons under these circumstances
was the subject of the present study.
Methods:
Two adult male rhesus monkeys were trained to make repeated flexion and extension
movements of their right wrist between three target windows (A, B and C) each 8°
wide in a repeated A,B,C,B,A sequence. The monkey viewed a line of 32 red light
emitting diodes (LED) 16 above and 16 below a large green LED. When the wrist
was positioned within the correct window, the green light was turned on. Move-
ment to the next window was initiated after 750 ms, 1000 ms or 1250 ms (randomly
selected) period following a reward (see below) by turning off the green LED and
turning on a red LED whose direction and distance from the green LED informed
the monkey of the direction and magnitude of the required movement. A correct
movement was defiend as a movement to the designated window within 750 ms of the
trigger signal and maintenance of that position for 1500 ms; a movement in the
wrong direction or an overshoot of the target window by more than 3 degrees
triggered a return to the original window and a repeat of the trial. Correct
movements were rewarded with fruit juice (0.1 ml). Once each monkey had mastered
the paradigm, (>67% success) torque loads were applied.
950
ZOl MH 01097-01 LNP
The timing of movement with respect to neuronal activity was monitored. In
addition the effects of torque loads that either assisted or opposed movement
in a particular direction were tested. Studies of E>G activity during load
application showed that assisting and opposing loads attenuated and accentuated,
respectively, the E>G activity of agonist muscles.
The recording electrode was slowly advanced through the cortex while the putamen
was being stimulated once every second. This strategy allowed detection of field
potentials and unit responses from the CS neurons. When a putative CS neurons
was detected, it was isolated and the discriminated signal used as trigger for
the stimulation of the putamen. A neuron was accepted as a CS neuron when their
response was absent following a collision with a spontaneously occuring cell dis-
charge within its collision window and when the response to putamen stimulation
at 333 Hz followed each of five stimuli with a constant antidromic latency.
Once a CS neuron had been unequivocably identified, its activity was studied
during movement. In monkey #1, the highest priority was to determine the timing
of movement-related discharges of CS neurons. In monkey #2, the priority was
to determine the effects of assisting and opposing loads on unit activity of
movement-related CS neurons.
Major Findings:
Three principal findings have emerged from the analysis of corticostriatal cell
activity: they discharge in relation to movement, the changes in their discharge
rates preceed muscle EVC activity, and their activity during steady state holding
and movement is significantly affected by loads both with and against the direc-
tion of movement.
1. Relationship to movement.
A total of 48 CS neurons were identified in the first monkey and 46 were identi-
fied in the second monkey. The antidromic latencies in the two monkeys were
comparable 1.12 + 0.40 (SD) ms and 1.36 + 0.51 ms. Assuming a length of 30 mm
for the CS axons, the conduction velocity of our sample averaged 16 m/s.
Forty-nine CS neurons were isolated long enough to record their activity during
the wrist flexion/extension task. Neuronal activity changes were related to
wrist movement in 25 of these neurons, 11 in the first monkey and 14 in the
second monkey.
2. Timing of activity changes.
em; activity and the timing of CS cell activity modulations were studied most
extensively in the second monkey. ElyG records were obtained during the recording
sessions and again after the completion of the single-unit data collection. The ,
increase in ElyC activity of the earliest prime-mover muscles preceded movement
onset by 93 ms. The time from the premovement change in firing rate (unit activ-
ity onset) to the onset of movement was 120 + 32 ms in 11 CS cells from the first
monkey (range 52 to 175 ms) and 110 + 31 ms in 14 CS neurons from the second
951
ZOl MH 01097-01 LNP
monkey (range 50 to 150 ms). Of the 13 CS neurons in the second monkey that
we were able to study in detail, 8 were clearly active before the earliest
task-related changes in EM3 activity.
3. Effects of assisting and opposing torque loads.
Analysis of the effects of torque loads on the activity of CS neurons during
movement is in the early stages and will be completed near the end of this
fiscal year. However, some results have emerged from our preliminary analysis.
During steady-state holding and movement, tonic and phasic discharge rates
respectively were increased by loads against the preferred direction of wrist
movement in 15 of 25 CS neurons.
Significance to Biomedical Research and to the Program of the Institute;
Studies of the functional relationship between the cerebral cortex and the stria-
tum are important to understanding how abnormalities in the balance of striatal
neurotransmitters could cause clinical symptoms. Diseases such as torsion dys-
tonia, Huntington's disease and glutaric aciduria type I may be viewed both as
an inappropriate contraction of muscle groups producing an abnormal positioning
of the limbs and trunk in space. The physiological properties of the CS and
putamen neurons suggest that these symptoms may reflect an imbalance in CS and/or
putamen neuronal activity. In Huntington's disease, it is of interest that psy-
chiatric symptoms develop first and motor disturbances appear once neural degen-
eration is well under way. Thus, the physiological interaction of frontal cortex
with the striatum is of fundamental importance in understanding a variety of
brain abnormalities that adversely affect mental health.
Proposed Course of the Project:
Data collection has been completed on this project, and analysis will continue
into the next year. Once neurophysiological and histological analyses are com-
plete, we intend to prepare the results for publication. No further data col-
lection is intended for this project.
Publications:
None.
952
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01098-01 LNP
PERIOD COVERED
February 1. 1987 to September 30, 1987
TITLE OF PROJECT (BO characlBrs or less Title must tit on one line t>etween tfw borders.)
Anatomical Analysis of Neuronal Circuits
PRINCIPAL INVESTIGATOR (Ust other prolesslonal personnel below ttte Principal Investigator ) (Name, title, laboratory, and Institute altlllation)
PI;
Chisato Asanuma Stanfield Staff Fellow
LNP , NIMH
COOPERATING UNITS (H tinyj
LAB/BRANCH
Laboratory of Neurophysiology
INSTITUTE AND LOCATION
NIMH. ADAMHA. NIH Animal Center, Poolesville. Maryland 20837
TOTAL MAN-YEARS:
0.67
PROFESSIONAL:
0.67
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues S (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Previous studies of the dendritic structure of neurons have predominantly relied
on the century-old Golgi methods and the technique of injecting biological markers
into single neurons in vivo. For technical reasons, however, it has been difficult
to effectively combine these methods with other neuroanatomical labeling techniques
The recently introduced method of intracellularly injecting a fluorescent dye,
Lucifer yellow, in fixed tissue slices is capable of providing a complete picture
of neuronal geometry and has the potential for use in conjunction with other neuro-
anatomical techniques. This project involves intracellular injection of Lucifer
yellow in combination with retrograde labeling techniques and immunohistochemistry ,
and its objective is to extend the analysis of neuronal connectivity and transmit-
ter specificities to a finer level than previously possible. Initially two ques-
tions will be addressed: (1) is there a point-to-point reciprosity between thalamo-
cortical axon collaterals that innervate the thalamic reticular nucleus and the
neurons in that nucleus which project back to the thalamus; and (2) where on the
dendrites of reticular nucleus neurons do thalamocortical axon collaterals termi-
nate?
953
PHS 6040 (R«v. 1/84)
OPO tw-cit
ZOl MH 01098-01 LNP
Objectives;
This project is designed to elucidate, at the single-cell level, the struc-
tural details of the dendritic arborizations of identified neurons and the exact
locations of identified afferent terminations on those neurons. The specific
objectives are: (a) to develop the necessary techniques; (b) to determine the
degree of reciprosity between thalamocortical axon collaterals that innervate
the thalamic reticular nucleus with the neurons in the reticular nucleus that
project back to the thalamus; and (c) to describe the exact distribution of
thalamic afferents on the dendrites of reticular nucleus neurons.
Methods:
This investigation involves the development of the methods for combining intra-
cellular Lucifer yellow injections with immunohistochemistry and with the retro-
grade transport of fluorescent dyes, and applications of the method to a series
of studies on thalamocortical circuitry in rats.
Initially, selected foci in the ventral lateral nucleus or the mediodorsal
nucleus of the thalamus are injected simultaneously with the retrogradely trans-
ported fluorescent dye, fluoro-gold, and the anterogradely transported plant
lectin, Phaseolus vulgaris leucoagglutinin (PHA-L). These thalamic nuclei have
been selected since, together, they represent the most prominent thalamic inputs
to the frontal cortex. Following an appropriate survival period, the rats are
perfused by a buffered aldehyde fixative solution and the brains sectioned on a
vibratome. Selected sections are secured on glass slides and examined in a
fluorescent microscope. Neurons in the thalamic reticular nucleus retrogradely
labeled with fluoro-gold are impaled under visual guidance with glass micropi-
pettes containing a 2% Lucifer yellow solution. As the dye is iontophoresed
into the cell, it quickly diffuses throughout the neuron's dendritic arbor.
Following the injection procedure, the sections are removed from the slides and
run through standard immunohistochemical procedures for visualization of the
anterogradely transported P HA-L, using rhodamine isothiocyanate as the label on
the secondary antibody.
Major Findings:
The efforts of this project thus far have been devoted to establishing the
necessary facilities and testing, successfully, the feasibility of the study.
A series of experiments have been conducted in rats in order ascertain that the
use of the retrograde tracer fluoro-gold, immunohistochemical procedures for
visualizing the plant lectin PHA-L, and the intracellular Lucifer yellow filling
procedure are compatible with each other in the same tissue. Further, several
technical parameters have been assessed, such as the ratio of paraformaldehyde
to glutaraldehyde in the fixative mixture required to optimize the intracellular
filling of neurons with Lucifer yellow while not compromising the subsequent
immunohistochemical results, the perfusion parameters, the temperature at which
the tissue should be kept at for obtaining optimal filling of neurons with
Lucifer yellow, and the types of stablization precautions which are needed on
the microscope in order to be able to maintain the pipette within the neuron
long enough to inject sufficient amounts of Lucifer yellow.
954
ZOl MH 01098-01 LNP
The preliminary experiments conducted thus far indicate that this study
is feasible. Retrograde labelling of neurons with fluoro-gold, intracellular
injections of neurons with Lucifer yellow, and anterograde labeling of axon
terminals with PHA-L immunohistochemistry are all compatible with each other in
the same tissue and the use of each of these techniques do not compromise the
effectiveness of either of the other techniques.
One slight problem which we have encountered has been that the fluoro-gold
and PHA-L have a tendency to precipitate each other out of solution if mixed
together. As a result of this, we have had to modify our procedures to make the
initial injection of the fluoro-gold and of the PHA-L separately through double
barrelled pipettes instead of together through a single barrel pipette.
Although we have most of the necessary equipment set up and ready for these
experiments, we are still awaiting the arrival of a fluorescent microscope. We
expect to receive it before the end of this fiscal year.
Significance to Biomedical Research and to the Program of the Institute:
A knowledge of the structural details of each of the circuits interconnec-
ting the thalamus and cortex is an important step in furthering our understand-
ing of how these circuits process neural information and mediate higher brain
functions. This knowledge includes the dendritic geometry of identified neurons,
their axonal targets, the location, on the dendrites, of identified inputs, and
the transmitters specific to the neurons studied.
The traditional Golgi methods and the technique of injecting horseradish
peroxidase (HRP ) into single neurons have, in the past, been the predominant
means used for examining the dendritic architecture of neurons. However, it
has been difficult to effectively use these methods in conjunction with other
tracing or immunohistochemical techniques. The recently introduced fluorescent
dye, Lucifer yellow, designed and first synthesized by Mr. Walter Stewart of
the Laboratory of Analytical Chemistry, NIADK, diffuses quickly throughout the
cell into which it is injected and allows the visualization of the cell's en-
tire dendritic arbor. This method of visualizing the dendritic structure of
neurons has the potential for use in conjunction with a variety of other neuro-
anatomical techniques, and should bring our understanding of the structural
details of neuronal circuits to a finer level than possible in the past.
While a comprehensive understanding of all structural details inherent in
any given neuronal circuit, along with an understanding of their significance
in the context of specific functions, remains a major challenge, especially for
those neuronal circuits mediating higher functions of the type which are relevant
to mental diseases, a systematic and detailed structural study of the thalamic
circuits underlying the transmission of external signals from subcortical centers
to the frontal cortex should provide some clues concerning some basic input mod-
ulating mechanisms such as the control of attention and arousal. Both of these
neural functions are, of course, quite relevant to many mental illnesses, and
several recent hypotheses put forth suggest that neural circuits in the thalamus
may play a critical role in these functions.
955
ZOl MH 01098-01 LNP
Proposed Course of the Project:
The initial focus of this project will be on the interrelation of the
thalamic reticular nucleus with other parts of the thalamus. Although long held
to be the final link in the ascending 'non-specific' activating system mediating
the desynchronization of the cortical EBG (i.e. , arousal), the results of recent
studies indicate that the thalamic reticular nucleus may instead be part of a
feedback circuit that modulates the transmission of signals through specific
thalamic relay nuclei. This feedback circuit has been postulated to be involved
in the modulation of the relative amplitude of externally generated stimuli
(Ahlsen et al. , Exp. Brain Res. , 1985, 58:134) and in the control of selective
attention (Skinner and Yingling, Prog. Clin. Neurophysiol. , 1977, 1:30; Crick,
Proc. Natl. Acad. Sci. , 1984, 81:4586). However, some investigators continue to
maintain that the thalamic reticular nucleus mediates general levels of arousal
(Steriade et al. , J. Neurosci. , 1986, 6:68). Since neurons in the thalamic
reticular nucleus receive inputs from collaterals of thalamocortical relay axons
and since neurons in the thalamic reticular nucleus project back to the thalamus,
a study combining the retrograde transport of fluorescent dyes and the antero-
grade transport of the PHA-L from the thalamus with injection of Lucifer yellow
into thalamic reticular nucleus neurons is ideally suited for evaluating whether
the specificity that would be predicted of a circuit involved in the modulation
of selective attention exists in this particular circuit.
Upon successful completion of the first set of experiments, the project will
be extended to include some investigations of cortical neurons. In particular,
we will study those cortical neurons that project back to the thalamus and other
neurons that project to the opposite cerebral hemisphere. In each case, the
reciprocal afferents which would be prelabeled with PHA-L (the thalamocortical
axon collaterals in layer 6 of the cortex, and the terminal arborizations of
commissural axons, respectively) are known to arborize selectively in quite close
proximity to the corresponding cortical efferent neurons, although the details
of these relationships are, at present, unclear. A fine grained anatomical study
of the type outlined above, directed at these cortical efferent circuits should
provide a more comprehensive picture of the details of these relationships, and
could further our understanding of the functional significance of the cortico-
thalamic feedback pathway and our understanding of interhemispheric information
transfer.
Publications:
None.
956
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MH 01099-01 LNP
PERIOD COVERED
October 1, 1986 to September 30, 1987
TITLE OF PROJECT (80 characters or leas Title must lit on one line lietween the txirders.)
Neurochemical Interactions Between Cortical and Striatal Dopaminergic Activity
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute atfillation)
PI:
Melvyn P . Heyes
Others: Ikuro Namura
Stephen Suomi
Visiting Associate
Visiting Fellow
Chief
LNP, NIMH
LCS, NIMH
LCE, NICHD
COOPERATING UNITS (H hny)
LAB/BRANCH
Laboratory of Neurophysiology
INSTITUTE AND LOCATION
NIMH, ADAMHA, NIH Animal Center, Poolesvllle, Maryland 20837
TOTAL MAN-YEARS:
0.5
PROFESSIONAL:
0.5
CHECK APPROPRIATE BOX(ES)
D (a) Human subjects
D (a1) Minors
D (a2) Interviews
D (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Disturbances in the balance of regional brain dopaminergic activity have been im-
plicated in the symptomatology of schizophrenia. Recent studies suggest that dys-
function of the dorsolateral prefrontal cortex (DLPFC) may cause the impaired mo-
tivation, shallow affect, and deficiencies in the performance of problem solving
tasks suffered by schizophrenics. Similar deficits are produced in primates by
dopamine depletion in the DLPFC. Together these observations suggest a role for
DLPFC dopaminergic neurons in cognitive functions subserved by this area. In one
series of experiments in rats, dopamine depletion in the medial frontal cortex
(MFC), a possible rodent homologue of the DLPFC of primates, resulted in increased
dopaminergic activity in striatum. Our observations in rats showed that dopamine
depletion in the MFC by local injection of MPP"*" or 6-OHDA was associated with in-
creased dopaminergic in nucleus accumbens, but not the caudatoputamen. This pro-
ject will now be extended to primates to further test the hypothesis that cortico-
striatal abnormalities underlie psychotic disorders in a species more closely
related and similar to humans.
957
PHS 6040 (Rev. 1/84)
ai*o ti4-«i*
ZOl MH 01099-01 LNP
Objectives;
Disturbances in the balance of regional brain dopaminergic activity have been
postulated to be responsible for the clinical manifestations of schizophrenia.
This hypothesis stems from the fact that drugs that are effective in treating
the hallucinations and delusions of schizophrenia are dopamine (DA) receptor
antagonists. The observation that the 'negative features' of schizophrenia are
exacerbated by DA receptor antagonists, is consistent with this notion.
Schizophrenics have impaired motivation, shallow affect and deficiencies in
the performance of problem solving tasks. While they perform such tasks,
schizophrenics display a paucity in blood flow in the dorsolateral prefrontal
cortex (DLPFC) (Weinberger et al: Arch Gen Psychiatr. 43: 114, 1986). Somewhat
comparable behavioral deficits result in humans following lesions in the DLPFC
and in primates following depletion of DA from the DLPFC (Bronzoski et al:
Science 205: 929, 1979), thus suggesting a primary role for the DA system in
cognitive functions subserved by DLPFC.
In rats, DA depletion in the medial frontal cortex (MFC) is associated with
increases in DA concentration in the caudatoputamen (CP ) and nucleus accumbens
(Pycock et al: Nature 286: 74, 1980), as well as that of its metabolites DOPAC
and HVA. If the dopaminergic system of the MFC of the rats is analogus to dopa-
minergic system in the DLPFC of primates, and if DA depletion in the DLPFC re-
sults in increased dopaminergic activity in subcortical dopaminergic systems in
humans as it does in rats, then it is possible that the positive features of
schizophrenia result from increased dopaminergic activity in the CP or nucleus
accumbens (Bannon and Roth: Pharmacol. Rev. 35: 53, 1983; Weinberger et al:
1986).
To date the results of Pycock and co-workers have not been reproduced in rats
or investigated in primates. Should a reciprocal relationship between frontal
cortex and subcortical dopaminergic activity be demonstrates in primates, these
animals may serve as a primate model for schizophrenia.
In this fiscal year, a preliminary study in rats was conducted in collaboration
with Dr. Ikuro Namura of the Laboratory of Clinical Science. DA was depleted
in the MFC by local injections of either 6-hydroxydopamine (6-OHDA) or N-methyl-
4-phenylpyridine (MPP"*").
Methods:
DA was depleted in the MFC of adult male Sprague-Dawley rats by bilateral injec-
tions of either MPP"*" or 6-OHDA into the MFC. Saline injection served as con-
trols. All of the 6-OHDA and half of the MPP'''-treated rats received an intra-
peritoneal injection of desmethylimipramine (DMI) thirty minutes prior to the
MFC injection in an attempt to protect noradrenergic neurons. The concentration
of DA, DOPAC, homovanillic acid (HVA), norepinephrine (NE), 5-hdyroxytryptamine
(5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was then measured by HPLC and
electrochemical detection in MFC, the dorsal region of the cingulate cortex,
striatum and nucleus accumbens two weeks later.
958
ZOl MH 01099-01 LNP
Ma.jor Findings:
Injections into the MFC were found to decrease DA levels both there and in the
adjacent cingulate cortex.
In the caudatoputamen, DA, DOPAC and HVA concentrations were unaffected in any
group. In MPP and DMI treated rats, DA concentration were increased by 22.8%.
However, the large standard deviation rendered this increase stastically insig-
nificant. In MPP'^+DMI treated rats the concentrations of DOPAC and HVA were
also increased in CP by 11.2% and 27% respectively, but this also was not signi-
ficant. No changes in the concentration of NE, 5-HT or 5-HIAA was observed in
the CP.
In contrast, in the nucleus accumbens, DA concentrations were increased in the
MPP'''group by 18.5% and in MPP^+DMI treated rats by 62.8%. DOPAC and HVA concen-
trations were also increased in these groups to similar extents. 5-HT concentra-
tions in nucleus accumbens were increased in MPP"*" treated rats but decreased in
6-OHDA treated rats. No changes in NE or 5-HIAA were observed in the nucleus
accumbens.
Significance to Biomedical Research and to the Program of the Institute:
This project tests one hypothesis about the cause of schizophrenia and include
an attempt to develop an animal model of the disease. In the nucleus accumbens
of both groups of MPP ''"-treated rats, significant increases in the concentrations
of DA, DOPAC, and HVA were found, indicating increases in dopaminergic activity.
This is consistent with the hypothesis and encourages us to move up to a promate
model.
Proposed Course of the Project:
Dopamine will be depleted in the DLPFC and medial prefrontal cortex (MPFC) of
monkeys by local injections of either 6-OHDA or MPP"*". These regions have been
chosen because of their high DA content, their inputs from the VTA and the like-
lihood that one of these regions is the homologue of the rodent MFC. Six rhesus
monkeys, that have been raised under closely monitored social conditions, will
be studied in the initial two groups.
Key behavioral observations are planned in collaboration with Dr. Stephen Suomi,
LCE, NICHD. These include a study of the effect of regional frontal dopamine
depletions on social hierarchies and their effect on affiliative behavior.
Publications:
None.
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959
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