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Full text of "Annual report : National Institute of Mental Health. Division of Intramural Research Programs"

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 

mi 



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 Ser vice 

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 
Z01MH00274 
Z01MH00276 
Z01MH00277 
Z01MH00279 
Z01MH00301 
Z01MH00326 
Z01MH00332 
Z01MH00336 
Z01MH00337 
Z01MH00339 
Z01MH00351 
Z01MH00382 
Z01MH00388 
Z01MH00396 
Z01MH00397 
Z01MH00400 
Z01MH0O422 
Z01MH00424 
Z01MH00427 
Z01MH00429 
Z01MH00433 
Z01MH00434 
Z01MH00447 
Z01MH00450 
Z01MH00452 
Z01MH00471 
Z01MH00478 
Z01MH00484 
Z01MH00486 
Z01MH00491 



Z01MH00503 
Z01MH00504 
Z01MH00507 
Z01MH00508 
Z01MH00509 
Z01MHn0672 
Z01MH00679 
Z01MH00680 
Z01MH00fi81 
Z01MH00682 
Z01MH00787 
Z01MH00796 
Z01MH00797 
Z01MH00798 
Z01MH00799 
Z01MH00881 
Z01MH00882 
Z01MH00887 
Z01MH00889 
Z01MH00903 
Z01MH00931 
Z01MH00934 
Z01MH00935 
Z01MH00936 
Z01MH00940 
Z01MH00941 
Z01MH00942 
Z01MH00943 
Z01MH00981 
Z01MH01031 
Z01MH01032 
Z01MH01035 
Z01MH01037 
Z01MH01038 
Z01MH01039 
Z01MH01040 
Z01MH01090 
Z01MH01092 
Z01MH01096 
Z01MH01097 
Z01MH01098 
Z01MH01099 
Z01MH01831 
Z01MH01833 



Z01MH01836 
Z01MH01850 
Z01MH01855 
Z01MH01860 
Z01MH02032 
Z01MHn2033 
Z01MH02035 
Z01MH02036 
Z01MH02037 
Z01MH02038 
Z01MH02039 
Z01MH02040 
Z01MH02152 
Z01MH02155 
Z01MH02156 
Z01MH02164 
Z01MH02169 
Z01MH02170 
Z01MH02171 
Z01MH02177 
Z01MH02178 
Z01MH02179 
Z01MH02180 
Z01MH02181 
Z01MH02182 
Z01MH02183 
Z01MH02184 
Z01MH02186 
Z01MH02187 
Z01MH02188 
Z01MH02189 
Z01MH02190 
Z01MH02191 
Z01MH02193 
Z01MH02197 
Z01MH02201 
Z01MH02205 
Z01MH02206 
Z01MHO2207 
Z01MH02216 
Z01MH02217 
Z01MH02219 
Z01MH02220 
Z01MH02222 



XVII 



RESEARCH PROJECT SERIAL NUMBER LISTING (Cont'd.) 



ZniMHn???3 
Z01MHn2225 

zniMHn??28 
zniMHn?22P 

ZniMHn2231 
ZniMH02232 
ZniMHn2233 
ZniMH02234 
Z01MHn2236 
ZniMHn2237 
ZniMHn2239 
Z01MH02240 
ZniMH02288 
ZniMHn2289 
ZniMH02290 
ZniMH02292 
ZniMHn2294 
ZniMH02295 
Z01MH02296 
Z01MH02297 
Z01MHn23n2 
Z01MH023n3 
ZniMH02307 
Z01MH02308 
ZniMH02321 
Z01MH02324 
ZniMH02325 
ZniMH02326 
ZniMH02327 
ZniMH02328 
ZniMH02340 
Z01MH02341 
ZniMHn234? 
ZniMH02343 
ZniMHn2344 
ZniMH02361 
ZniMHn2362 
Z01MHn2363 
Z01MHn2364 
ZniMHn2365 
ZniMHn2366 
Z01MH02367 
Z01MHn23fi8 
ZniMH02369 
ZniMH0237n 
ZniMHn2371 
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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 n nn7n-i4 rp 



PERIOD COVERED 



TITLE OF PROJECT (80 characterz or less, rule must lit on one line between the borders.) 

Psycholo gical 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) 

(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 

24 



ZOl MH 00070-14 BP 

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) . 



27 



ZOl MH 00070-14 BP 

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. 



31 



ZOl MH 00070-14 BP 

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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ZOl MH 00070-14 BP 
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 Psy chiatry, 1985 (Developments in Psychiatry, Vol. 7). 
Amsterdam, Elsevier, 1986, pp. 886-888. 

33 



ZOl MH 00070-14 BP 
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 

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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). 



45 



ZOl MH 00071-07 BP 

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 

46 



ZOl MH 00071-07 BP 

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. 



47 



ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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. 



49 



ZOl MH 00071-07 BP 

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 



50 



ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 
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 . 



57 



ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

.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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ZOl MH 00071-07 BP 

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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\ ZOl MH 00071-07 BP 

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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ZOl MH 00071-07 BP 

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. 



64 



ZOl MH 00071-07 BP 
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. 



65 



ZOl MH 00071-07 BP 

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. 



66 



ZOl MH 00071-07 BP 

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 
stres s 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. 



75 



ZOl MH 000452-12 BP 

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 Disorde r; 
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. 



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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 

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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. 

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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. 



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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, Behav i- 
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. 



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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 

96 



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. 

97 



ZOI MH 01090-1 I BP 
Significance to Biomedical Research and to t he 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 

98 



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, Mot oy Ar eas 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.) : 
Physiol ogical 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 

99 



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. 



101 



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. 



102 



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 



104 



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. 



105 



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. 



109 



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- 

120 



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- 

121 



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. 

122 



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 

Va n 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. 

Si gnificance 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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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. 



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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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ZOl MH 02206-03 CP 
Project Description and Methods: 

1. Study of the Psychobiological Effects of Lig ht 

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 N ormal 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 Psycho biological Ef fects 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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ZOl MH 02206-03 CP 

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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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 Biome dical 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. 



171 



ZOl MH 02206-03 CP 



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 Biomed ical 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: 







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.) 



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^HI flunitrazepam 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. 

Si gnificance 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 parent ill group, 
6/18 in the 1 parent ill group, and 4/7 in the 2 parent ill group. 

Si gnificance 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: 





PROFESSIONAL: 





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 Biomedi cal 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 Biomed ical 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 



Clinic a l Psychob io logy Br a nch 



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. 

Si gnificance 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 



Si gnificance 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. 

Si gnificance 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. 

Si gnificance 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. 

Si gnificance 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. 

Si gnificance 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. 

Si gnificance 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 

nct ober 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, ADAM HA, 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 , 198 6 through Septembe r 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„ 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 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 



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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 

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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 

O ctober 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. 



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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. 



265 



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. 

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ZOl MH 00337-08 LCS 

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 



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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 



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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 



PHS 6040 (Rev. 1/84) 



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. 



280 



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 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 . Bul l. 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 



(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. 



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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. 

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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. 



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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 . 



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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 . 



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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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315 



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 fronta l 
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 



320 



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 Researc h 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 Researc h 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 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 p r"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^ 1 Q«7 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

A Stlidy of Arlpnn<;inp Rprpntnr-;- TsolfltJ 



soi a tion a nd 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. 



M a ryland 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 "^ iii nr ^ I k'^*^-* * . ^ V I 

TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

r.nnce ptual 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 
attentio n deficit disorder, residual typ e (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 
Ty pPf 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 a ffective 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 a ffected- 
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 linkag e 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 
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 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. 



410 



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 
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 

416 



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