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RA 

790.6 

U5591 

1985 

v.l 


ANNUAL 
REPORT 


Division  of  Intramural  Research  Programs 
National  Institute  of  Mental  Health 

October  1,  1984  ■  September  30,  1985 


VOLUME  I 

SUMMARY  STATEMENTS 


U.S.  DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES 

Public  Health  Service 

Alcohol,  Drug  Abuse,  and  Mental  Health  Administration 

National  Institute  of  Mental  Health 

Division  of  Intramural  Research  Programs 


ANNUAL  REPORT 


DIVISION  OF  INTRAMURAL  RESEARCH  PROGRAMS 

NATIONAL  INSTITUTE  OF  MENTAL  HEALTH  L^''^-) 
If  0 

October  1,  1984  —  September  30,  1985 


VOLUME  I 
SUMMARY  STATEMENTS 


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ANNUAL  REPORT 

DIVISION  OF  INTRAMURAL  RESEARCH  PROGRAMS 

NATIONAL  INSTITUTE  OF  MENTAL  HEALTH 

October  1,  1984  -  September  30,  1985 

TABLE  OF  CONTENTS 
VOLUME  I  SUMMARY  STATEMENTS 

Page 

Director,  Division  of  Intramural  Research  Programs 1 

Biological  Psychiatry  Branch 9 

Clinical  Neurogenetics  Branch 21 

Clinical  Psychobiology  Branch 27 

Laboratory  of  Clinical  Science 39 

Child  Psychiatry  Branch 55 

Clinical  Neuroscience  Branch 57 

Laboratory  of  Neuropsychology 65 

Laboratory  of  Developmental  Psychology 85 

Laboratory  of  Psychology  &  Psychopathology 97 

Laboratory  of  Socio-Envi  ronmental  Studies 109 

Laboratory  of  Cell  Biology 121 

Laboratory  of  Cerebral  Metabolism 135 

Laboratory  of  General  &   Comparative  Biochemistry 141 

Laboratory  of  Molecular  Biology 147 

Laboratory  of  Neurochemistry 155 


Page 

Laboratory  of  Neurophysiology 157 

Research  Services  Branch 167 

Neuropsychiatry  Branch 1  79 

Laboratory  of  Preclinical  Pharmacology 185 


II 


ANNUAL  REPORT 
of  the 
DIRECTOR,  DIVISION  OF  INTRAMURAL  RESEARCH  PROGRAMS 
October  1,  1984  -  September  30,  1985 
Frederick  K.  Goodwin,  M.D. 

My  fourth  annual  report  as  scientific  director  caps  off  a  year  in  which 
the  program  enjoyed  strong,  stable,  supportive,  and  innovative  leadership  from 
Drs.  Frazier  and  Macdonald  at  the  Institute  and  ADAMHA.  This  year  also 
required  some  personal  soul  searching  as  I  considered  an  attractive  outside  job 
offer,  a  process  which  ultimately  reinforced  my  commitment  to  the  Intramural 
program  and  the  Institute.  Headquarters  underwent  a  major  reorganization  reflecting 
a  new  focus  on  the  Institute's  research  mission  and  while  we  were  not  directly 
affected,  I  participated  in  aspects  of  the  planning  process.  We  mourned  the  death 
of  one  of  our  most  distinguished  scientists  and  began  to  fill  gaps  left  by  the 
departure  of  two  others.  We  undertook  a  reevaluation  of  the  way  we  conduct  animal 
research  and  began  to  participate  in  an  ambitious  NIH-wide  effort  to  upgrade 
animal  facilities. 

In  early  July,  at  age  59,  Dr.  Ed  Evarts  suffered  a  fatal  heart  attack  in  his 
laboratory,  shortly  after  returning  from  an  extended  overseas  trip.  Ed's  meticulous 
work  at  NIMH,  beginning  in  1953,  set  the  standard  for  the  neurophysiological  mapping 
Of  the  functional  anatomy  of  the  brain.  Although  he  was  loath  to  stray  far 
from  the  lab,  Ed  served  as  Associate  Director  for  Basic  Science  during  the 
formative  days  of  my  directorship  and  lent  his  broad  scientific  vision  to  the 
reshaping  of  our  Program.  Hundreds  of  Ed's  friends  from  throughout  the  NIH 
community  and  around  the  world  attended  a  Memorial  Convocation  at  the  Masur 
Auditorium  in  late  July.  Among  17  colleagues  who  shared  their  recollections  of  Ed 
and  his  work  were  Drs.  W.  Thomas  Thach  of  Washington  University,  Emilio  Bizzi  of  MIT, 
Mahlon  Delong  of  Johns  Hopkins,  Jun  Tanji  of  Hokkaido  University,  Daniel  Tosteson 
of  Harvard,  Eric  Kandel  of  Columbia,  Gerald  Edelman  of  Rockefeller  University, 
Fred  Robbins  of  the  National  Academy  of  Sciences,  and  Julie  Axelrod,  Seymour 
Kety,  John  Eberhart,  and  Allan  Mirsky  of  our  own  "family."  Dr.  Steven  Wise 
has  taken  over  as  Acting  Chief  as  we  ponder  the  long-term  future  of  the 
Laboratory  of  Neurophysiology  and  he  has  performed  exceptionally  well. 

Similarly  under  review  at  this  writing  is  the  Laboratory  of  Socioenvi ronmental 
Studies,  whose  chief.  Dr.  Melvin  Kohn,  a  33-year  NIMH  veteran,  retired  this 
past  summer  to  become  Professor  of  Sociology  at  Johns  Hopkins  University.  In  a 
series  of  studies  begun  here  in  the  early  1960s,  Dr.  Kohn  pioneered  the  application 
of  statistically  sophisticated  methodologies  for  understanding  the  relationship 
between  job  conditions  and  psychological  functioning.  Dr.  Carmi  Schooler  is 
serving  as  Acting  Chief  as  we  reevaluate  the  future  course  and  leadership 
of  IRP  efforts  in  the  psychosocial  area. 

Also  retiring  from  Federal  service  this  past  year  was  Dr.  Erminio  Costa,  Chief 
of  the  Laboratory  of  Preclinical  Pharmacology  (LPP)  located  at  Saint  Elizabeths 
Hospital.  "Mimo"  and  10  of  his  colleagues  from  the  Lab  have  moved  to  Georgetown 
University,  where  a  new  neuroscience  institute  has  been  established  under  an 
agreement  with  Fidia  Research  Foundation,  an  affiliate  of  Fidia  S.P.A.,  a 
major  Italian  pharmaceutical  firm.  Since  its  inception  in  1968,  Dr.  Costa's 
lab  has  been  a  mecca  for  young  neuroscientists  from  around  the  globe.  Among  some 
175  who  trained  there  are  50  apiece  from  Italy  and  the  U.S.,  5  from  China,  7  from 


Scandinavia,  and  10  from  Eastern  Bloc  countries.  As  director  of  the  new  Fidia- 
Georgetown  Neuroscience  Institute,  Dr.  Costa  will  continue  collaborative  work 
underway  with  Dr.  Steven  Paul  and  others  at  NIMH.  Of  particular  interest  are 
studies  with  the  endogenous  anxiogenic  peptide  OBI,  which  was  discovered  and 
sequenced  by  Dr.  Alessandro  Guidotti  of  the  LPP  over  the  past  few  years.  I  was 
honored  to  have  had  the  opportunity  to  chair  the  distinguished  scientific  symposium 
marking  the  opening  of  the  new  Institute.  We  all  wish  Dr.  Costa  and  his  colleagues 
well  in  their  new  venture. 

As  I  mentioned  last  year.  Dr.  Costa's  departure  and  the  transfer  of  Saint 
Elizabeths  Hospital  to  the  District  of  Columbia  have  spurred  efforts  to  relocate 
the  remaining  elements  of  this  satellite  program  to  the  main  NIH  campus.  Following 
its  review  of  Dr.  Wyatt's  Neuropsychiatry  Branch  last  fall,  the  IRP  Board  of 
Scientific  Counselors,  in  recommending  the  relocation,  stressed  the  scientific 
advantage  of  conducting  the  Branch's  important  clinical  research  in  close 
proximity  to  the  Clinical  Center's  high  tech  positron  emission  tomography  (PET) 
and  magnetic  resonance  imaging  (MRI)  facilities.  Since  any  such  move  must  await 
new  construction  at  NIH,  during  the  interim  we  are  committed  to  maintaining  the 
continuity  and  critical  mass  of  personnel  and  resources  required  to  sustain  an 
efficient  research  center  at  the  William  A.  White  Building.  Some  Bethesda-based 
investigators  may  temporarily  move  to  WAW  to  take  advantage  of  the  comparatively 
spacious  quarters  and  vacated  laboratory  facilities  there. 

Meanwhile,  our  Clinical  Director,  Dr.  Rex  Cowdry,  is  developing  plans  for 
renovations  of  NIMH  inpatient  units  in  the  Clinical  Center  to  accommodate  an  adequate 
number  of  beds  for  the  eventual  consolidation  of  schizophrenia  research  programs 
on  campus.  Efficiencies  achieved  through  a  higher  ratio  of  bed  space  to  office 
space  on  the  units  will  permit  us  to  devote  about  a  third  of  our  total  beds  to 
schizophrenia  studies. 

Misleading  information  was  circulated  earlier  this  year  about  the  IRP's  invest- 
ment in  schizophrenia  research,  causing  unnecessary  concern  among  patients  and  their 
families.  I  did  my  best  to  dispel  these  unfortunate  misperceptions  in  several 
letters  and  talks,  principally  in  an  address  to  the  Annual  Meeting  of  the  National 
Alliance  for  the  Mentally  111.  In  addition  to  boosting  clinical  studies,  we 
continue  to  pursue  a  better  understanding  of  fundamental  brain  mechanisms  to 
advance  the  state-of-the-science  in  schizophrenia. 

At  this  writing,  the  best  hope  for  new  laboratory  space  on  the  NIH  campus  is 
riding  on  the  perceived  need  to  upgrade  our  animal  facilities  to  meet 
The  American  Association  for  Accreditation  of  Laboratory  Animal  Care  (AAALAC) 
standards.  While  we  can  give  no  quarter  to  extremists  whose  purpose  is  to  stop 
any  use  of  animals  in  medical  research,  it  is  important  that  we  optimize  our 
physical  facilities  for  animal  housing.  Dr.  Wyngaarden  has  committed  the 
NIH  to  meeting  AAALAC  standards  within  2  years.  All  intramural  programs  on  campus 
share  the  same  problem:  We  must  create  more  room  for  improved  animal  facilities 
in  a  cramped  research  setting  that  already  has  a  high  density  of  investigators 
per  unit  of  space.  Since  centralization  of  facilities  is  emerging  as  the  most 
efficient  way  to  do  this,  unprecedented  cooperation  between  the  Institutes  will 
be  required. 


Ably  representing  IRP  in  ongoing  deliberations  with  the  other  Institutes  is 
our  head  veterinarian  Dr.  Rob  Werner.  Although  Dr.  Mort  Mishkin's  Laboratory 
of  Neuropsychology  primate  facilities  in  Building  9  have  been  recently  renovated, 
that  World  War  II  vintage  temporary  structure  is  hardly  a  long-term  solution  to 
our  primate  research  and  space  requirements.  Accordingly,  we  are  hopeful  that  a 
proposed  new  non-human  primate  neuroscience  building  to  be  shared  by  NIMH,  NIDR, 
NEI,  and  NINCDS  will  make  available  enough  new  space  to  relocate  the  Saint 
Elizabeths  operation.  Discussions  are  also  underway  with  private  foundations 
concerning  a  possible  joint  venture  to  erect  a  new  building  on  campus.  In 
concert  with  Dr.  Werner  and  the  NIH  Institutes,  Drs.  Steve  Paul,  Steve  Wise, 
and  other  members  of  our  own  IRP  Animal  Care  Committee  have  been  weighing  alternative 
animal  care  arrangements.  One  plan  would  consolidate  all  small  animal  holding 
facilities  to  certain  areas  of  the  Clinical  Center.  The  challenge  will  be  to 
achieve  efficiencies  of  scale  that  will  not  unduly  hamper  research.  The  Committee 
has  also  drawn  up  contingency  plans  for  a  coherent  response  should  NIMH  become 
a  target  of  animal  rights  activists;  Steve  Wise  has  been  especially  effective  in 
articulating  rational  scientific  responses  to  this  threat.  The  Mobilization  for 
Animals  and  other  groups  of  that  ilk  claim  behavioral  studies  with  animals  can 
teach  us  nothing  about  human  mental  illness.  Yet  increasingly,  our  findings 
in  basic  animal  experiments  are  converging  with  hypotheses  emerging  from  clinical 
studies.  Consider  that  Dr.  Ed  Evarts'  discovery  in  monkeys  that  the  prefrontal 
cortex  plays  a  central  role  in  the  maintenance  of  set — the  preparation  for  voluntary 
motor  activity--di rectly  relates  to  Dr.  Daniel  Weinberger's  finding  of  altered 
cerebral  blood  flow  in  that  same  region  when  schizophrenics  are  given  a  task 
requiring  the  maintenance  of  set.  Similarly,  Dr.  Steven  Paul  and  colleagues 
have  recently  found  neurochemical  changes  in  human  volunteers  undergoing  experimental 
learned  helplessness  which  parallel  changes  seen  in  animals  subjected  to  the 
inescapable  shock  paradigm  and  mimic  alterations  seen  in  seriously  depressed 
patients.  Consider  also  Dr.  Robert  Post's  landmark  development  of  carbamazepine 
as  a  treatment  for  rapidly  cycling  manic  depressive  patients  based  on  animal  kindling 
and  sensitization  models.  Models  of  depression  and  anxiety  induced  by  separation 
in  hamsters  (by  Dr.  Jacqueline  Crawley)  and  in  monkeys  (by  Dr.  Steve  Suomi) 
produce  animals  with  neurobiological  profiles  and  symptoms  that  resemble  human 
illness.  Moreover,  the  animals  respond  remarkably  like  humans  to  antidepressant 
medications.  Through  years  of  meticulous  biobehavioral  experimentation  in 
monkeys.  Dr.  Mort  Mishkin  and  colleagues  have  been  able  to  trace  the  brain  circuits 
involved  in  memory,  setting  the  stage  for  understanding  the  neurobiological  basis 
of  cognitive  losses,  such  as  those  seen  in  Alzheimers  disease.  Another  example: 
the  MPTP  model  of  Parkinsonism,  which  led  to  a  breakthrough  in  Parkinsons  disease 
research,  vividly  illustrates  the  reasons  why  we  must  experiment  on  monkeys. 
This  story  will  be  the  subject  of  a  PBS  NOVA  progam  scheduled  to  air  in 
February  1986.  The  IRP's  Dr.  Sandy  Markey  and  former  staffers  Drs.  Stanley 
Burns  and  Glenn  Davis  will  be  featured  in  this  remarkable  story  of  scientific 
discovery.  In  June,  Dr.  Markey  convened  an  interagency  symposium  on  MPTP  which 
brought  together  many  of  the  hundreds  of  investigators  who  have  begun  experimenting 
with  the  neurotoxin  following  discovery  of  its  Parkinsonian  effects  in  primates 
by  IRP  investigators  a  few  years  ago. 


IRP  also  played  host  to  other  International  meetings:  in  June  Dr.  Cantoni 
organized  a  highly  successful  workshop  on  DNA  methylation  and  in  September  Drs. 
Bill  Potter  and  Steven  Paul  coordinated  the  Fourth  International  Meeting  on 
Clinical  Pharmacology  in  Psychiatry.  This  gathering  of  psychopharmacologi sts  from 
around  the  world  was  held  just  prior  to  the  World  Congress  of  Psychiatry  Meeting 
in  Philadelphia. 

As  in  past  years,  IRP  scientists  distinguished  themselves  in  1985  with  an 
impressive  array  of  awards  and  honors  from  outside  organizations.  They  include: 
Dr.  Julius  Axel  rod,  the  Leibniz  Gold  Medal  and  membership  in  the  East  German  Academy 
of  Sciences;  Dr.  William  Freed,  the  Arthur  S.  Flemming  Award;  Dr.  Mortimer 
Mishkin,  the  American  Psychological  Association's  Distinguished  Scientific 
Contribution  Award;  Dr.  Steven  Paul,  named  Outstanding  Young  Scientist  for  1985 
by  the  Maryland  Academy  of  Sciences;  Dr.  Robert  Post,  the  Gold  Medal  Award 
from  the  Society  for  Biological  Psychiatry  (the  youngest  person  ever  to  receive 
this  prestigious  award);  Dr.  Terry  Reisine,  the  FAES  Yoshio  Sato  International 
Award;  and  Dr.  David  Rubinow,  the  A.E.  Bennett  Award  from  the  Society  for  Biological 
Psychiatry.  I  was  honored  to  have  been  elected  to  the  Institute  of  Medicine  of 
the  National  Academy  of  Sciences. 

Among  IRP  recipients  of  Government  awards  were:  Dr.  Dennis  Murphy,  the 
SSS  Presidential  Meritorious  Rank  Award;  Dr.  Llewellyn  Bigelow,  the  PHS 
Meritorious  Service  Medal;  Dr.  John  Calhoun,  the  Presidential  Design  Award 
from  the  National  Endowment  for  the  Arts;  Dr  Rex  Cowdry,  ADAMHA  Administrator's 
Award  for  Meritorious  Achievement;  Erskine  Davis,  ADAMHA  Administrator's  Award 
for  EEO  Achievement;  Dr.  Philip  Gold,  PHS  Outstanding  Service  Medal;  Dr. 
Howard  Nash,  PHS  Superior  Service  Award;  and  Dr.  Steven  Paul,  PHS  Meritorious 
Service  Medal . 

Such  scientific  excellence  reflects,  in  part,  the  guidance  we  receive  regularly 
from  our  panel  of  outside  experts,  the  Board  of  Scientific  Counselors.  This 
year,  the  Board  reviewed  elements  of  the  Laboratories  of  Neurophysiology  and 
Cerebral  Metabolism  as  well  as  the  Neuropsychiatry  Branch.  The  timing  of  the 
latter  evaluation  was  particularly  opportune,  coinciding  with  the  planning 
of  future  initiatives  in  schizophrenia  research  as  well  as  the  previously  mentioned 
reassessment  of  our  Saint  Elizabeths  location. 

Special  thanks  to  our  outgoing  Board  members  this  year:  Drs.  Norman  Garmezy, 
Anita  Hendrickson  and  Robert  Moore.  All  served  with  distinction  and  each  will 
be  sorely  missed,  both  personally  and  professionally.  We  are  now  in  the  process 
of  filling  these  critical  vacancies. 

Unexpected  budget  cuts  impacted  both  extramural  and  intramural  research  this 
year,  a  problem  made  all  the  more  serious  by  the  Institute's  earlier  history: 
a  major  research  funding  gap  that  had  developed  during  the  decade  from  1967  to 
1977,  a  gap  which  hit  the  extramural  programs  especially  hard,  leaving  them 
with  a  nearly  50%  drop  in  real  dollars.  The  health  and  vigor  of  intramural 
and  extramural  communities  are  interdependent;  we  complement  each  others' 
strengths.  The  three  budget  cycles  since  I  have  been  Intramural  Director  have 
seen  extramural  funding  grow  at  approximately  four  times  the  rate  of  intramural 
growth,  a  record  which  speaks  for  itself.  In  addition,  the  IRP  takes  seriously  its 
obligation  to  use  its  strategic  location  in  the  Nation's  Capital  to  highlight 


the  need  for  increases  in  mental  health  research  funding,  most  of  which, 
appropriately,  should  continue  to  go  to  the  Extramural  program,  especially  for 
investigator-initiated  grants  and  centers.  In  that  connection,  I  recently  joined 
Ann  Landers  and  others  in  highlighting  the  needs  and  opportunities  of  mental  illness 
research  to  over  50  members  of  Congress  at  a  reception.  IRP  scientists  continue 
to  contribute  to  public  interest  in  the  brain  sciences  and  mental  illness  research 
through  numerous  media  interviews  granted  over  the  past  year.  I  believe  the  IRP 
must  bear  a  special  burden  of  visibility  in  this  regard.  As  the  principal  component 
of  the  Institute  directly  conducting  research,  we  represent  NIMH's  main 
presence  to  a  mass  media  which  is  structurally  biased  toward  the  concrete  and 
the  visual.  Media  reports  about  extramural  grantees  typically  mention  only 
their  university  affiliations,  rarely  NIMH  as  the  funding  source.  Thus, 
Intramural  research  accounts  for  most  press  reports  about  NIMH  supported  science. 
In  my  experience,  the  media,  even  the  Congress,  generally  do  not  distinguish 
intramural  and  extramural  research  and  thus  we  carry  the  major  responsibility 
for  the  scientific  image  of  the  Institute.  In  the  interest  of  responsible  reportage 
and  minimal  disruption  of  our  labs,  Jules  Asher,  our  public  affairs  specialist, 
Marilyn  Sargent,  Chief  of  the  Public  Affairs  Branch,  and  the  Laboratory/ 
Branch  Chiefs  exercise  discretion  in  screening  potential  media  contacts.  We 
are  also  building  up  a  video  tape  library  of  "stock"  footage  depicting  patients 
in  various  clinical  states  and  research  procedures  to  be  used  in  lieu  of  direct 
taping  by  television  crews  whenever  possible. 

Among  other  public  education  efforts  this  past  year  were  two  "Medicine 

for  the  Layman"  lectures  by  IRP  clinical  investigators:  Dr.  Wallace  Mendelson 

on  sleep  and  Dr.  Judith  Rapoport  on  childhood  psychiatric  disorders.  We 

also  hosted  special  briefings  for  representatives  of  the  National  Alliance  for 

the  Mentally  111,  Dr.  William  Roper,  Assistant  Director  for  Health  Policy  in  the 

President's  Office  of  Policy  Development,  and  Richard  Jacob  of  the  Office  of 

Management  and  Budget. 

The  IRP  Director's  Conference  Series  continued  to  facilitate  collaborations 
between  Intramural  investigators  with  cross-cutting  reviews  of  promising  IRP 
research  areas.  Among  themes  explored  this  past  year  were:  learning  and 
memory,  appetite  and  eating,  animal  models  of  behavior,  and  animal  and  clinical 
studies  of  risk  factors  in  the  young.  The  Director's  Conference  Planning 
Committee,  which  is  chaired  by  Dr.  Carl  Merril,  is  considering  inclusion  of  some 
outside  investigators  in  future  conferences. 

At  another  level.  Dr.  Macdonald's  Administrator's  Research  Forum  has 
contributed  substantially  to  bridge  building  between  the  Intramural  and  head- 
quarters staffs.  This  year,  in  addition  to  my  own  presentation,  the  Forum 
featured  the  following  IRP  scientists:  Drs.  Robert  Post,  Steven  Paul,  Judith 
Rapoport,  Jacqueline  Crawley,  Daniel  Weinberger,  Edward  Evarts,  Elliot  Gershon, 
Rex  Cowdry,  David  Rubinow,  Candace  Pert,  and  Mortimer  Mishkin.  Feedback  from 
these  presentations  has  been  uniformly  enthusiastic. 

This  past  year   was  the  first  in  which  I  presented  full  briefings  on  IRP 
research  to  the  National  Advisory  Mental  Health  Council  under  its  expanded  mandate. 
Reports  indicate  that  the  Council  found  the  exercise  to  be  as  rewarding  as  I  did. 
While  headquarters  has  undergone  a  comprehensive  reorganization,  this  year  we 
have  just  a  few  organizational  and  personnel  adjustments  to  report.  Dr.  Peter 
Roy-Byrne  is  now  serving  part-time  as  my  special  assistant  while  continuing 


his  studies  in  the  Biological    Psychiatry  Branch.     Dr.  Robert  Cohen's  Section 
on  Clinical    Brain    Imaging  has  been  transferred   from  the  Laboratory  of  Psychology 
and   Psychopathology  to  Dr.   Sokoloff's   Laboratory  of  Cerebral    Metabolism.     Clinical 
PET  scan  studies   are  thus  consolidated  with  the  basic  glucose  mapping  work  in 
animals  which  made  them  possible.     This  critical   mass  of   imaging  expertise  and 
technology  is  now  jelling  as  the  NIH/NIMH  multi-million  dollar  PET  scanning/ 
cyclotron  facility  becomes  operational. 

As  members  of  the   PET  Policy  Advisory  Committee,   Dr.   Lou   Sokoloff  and    I 

played   a   role  in  assuring  that  the  administrative  structure  of  the  Clinical    Center's 

Nuclear  Medicine  Department  is  well  positioned  to  take  advantage  of  this  new 

technology.  At  the  request  of  Dr.  Joseph  Rail,  NIH  Deputy  Director  for  Intramural 

Research,  I  convened  a  committee  of  the  world's  leading  PET  experts  last 

spring  to  review  the  Department's  plans  and  procedures.  The  committee's  report 

is  currently  under  review. 

The  job  of  the  Clinical  Director  has  grown  increasingly  complex.  Not  only 

is  Rex  Cowdry  responsible  for  overseeing  clinical  care  and  clinical  training  across 

our  many  separate  clinical  research  programs,  he  also  has  played  a  leading  role 

in  restructuring  the  formulas  for  assigning  costs  in  the  Clinical  Center.  As 

our  representative  in  all  matters  pertaining  to  the  Clinical  programs,  he 

has  performed  with  grace  and  effectiveness  under  difficult  circumstances. 

His  recent  election  as  Chairman  of  the  Medical  Board  reflects  his  high  standing 

among  the  other  Clinical  Directors. 

To  better  coordinate  intramural  research  within  the  Agency,  I  have  been 
chairing  a  regular  ADAMHA  Scientific  Directors  meeting  with  my  counterparts  in 
NIAAA  and  NIDA,  Drs.  Boris  Tabakoff  and  Jerome  Jaffe,  both  of  whom  joined 
the  Agency  this  past  year.  I  also  now  represent  the  NIAAA  Intramural  component 
on  campus  at  the  NIH  Scientific  Directors  meetings. 

With  personnel  ceilings  dropping,  alternative  mechanisms  for  supporting  young 
scientists  who  wish  to  enter  research  careers  and  train  in  our  laboratories 
are  becoming  increasingly  important.  This  year,  we  welcomed  our  first  crop 
of  fellows  under  the  National  Research  Service  Act  and  look  forward  to  an 
additional  group  to  begin  work  next  year  under  the  National  Research  Council 
Fellowship  Program.  Our  labs  have  also  begun  to  see  a  healthy  rotation  of 
research-minded  medical  students  in  the  Howard  Hughes  Medical  Foundation 
Fellowship  Program,  which  got  underway  here  this  year.  Dr.  Ed  Evarts  played  a  key 
role  in  setting  up  this  much  needed  effort,  which  has  identified  neuroscience  as 
one  of  four  major  program  areas.  In  Ed's  place.  Dr.  Mike  Brownstein  now 
represents  NIMH  on  the  Hughes  Fellowship  planning  board. 

This  increased  interest  in  the  brain  sciences  by  such  major  philanthropic 
organizations  as  the  Hughes  Foundation  is  especially  heartening  in  light  of  the 
lag  in  government  funding  of  mental  illness-related  research  noted  earlier.  Even 
a  modicum  of  private  funding  affords  a  Government  program  such  as  ours  the 
leverage  and  flexibility  to  quickly  take  advantage  of  new  research  opportunities 
and  fill  the  gaps  endemic  to  Federal  operations.  For  example,  our  still  small 
NIMH-IRP  (general  Purpose  Fund  through  the  Foundation  for  Advanced  Education 
in  the  Sciences  (FAES)  is  intended  to  provide  some  travel  support  for  younger 
investigators  to  scientific  meetings  which  our  limited  travel  budget  could  not 


afford.  This  year,  the  Fund  was  supplemented  with  proceeds  from  a  continuing 
medical  education  course  on  psychopharmacology  offered  through  the  FAES. 
Coordinated  by  Dr.  David  Jimerson,  the  course  featured  presentations  by  a 
number  of  IRP  clinical  investigators.  Also  much  appreciated  this  year  were 
a  few  generous  contributions  to  the  Fund  from  private  donors  interested  in 
forwarding  mental  illness  research. 


ANNUAL  REPORT  OF  THE  BIOLOGICAL  PSYCHIATRY  BRANCH 

National  Institute  of  Mental  Health 

October  1,  1984  through  September  30,  1985 

Robert  M.  Post,  M.D. ,  Chief 

The  Biological  Psychiatry  Branch  consists  of  inpatient  and  outpatient  clin- 
ical research  programs  and  a  series  of  basic  neuroscience  units  designed  to  ex- 
plore clinically  relevant  themes.   The  3-west  Clinical  Research  Unit  provides  the 
inpatient  resource  for  studying  patients  with  unipolar  and  bipolar  affective  dis- 
orders, schizoaffective  illness,  and  panic-anxiety  disorder.   Dr.  Thomas  W.  Uhde, 
as  Chief  of  the  Unit  on  Anxiety  and  Affective  Disorder,  heads  up  this  inpatient 
facility.   Dr.  Philip  W.  Gold,  Chief,  Section  on  Clinical  Neuroendocrinology, 
studies  endocrine  aspects  of  patients  with  affective  and  anxiety  disorders.   Dr. 
David  R.  Rubinow,  Chief  of  the  Unit  on  Peptides,  and  the  Consultation  Liaison 
Service  for  NIMH,  studies  neuroendocrine  and  neuropeptide  aspects  of  patients 
with  these  disorders. 

Dr.  Uhde  examines  similarities  and  differences  in  phenomenology,  clinical 
course,  biochemistry,  and  pharmacological  responsivity  in  patients  with  affective 
and  anxiety  disorders.   In  the  Section  on  Psychobiology,  he  also  conducts  studies 
of  a  series  of  anticonvulsant  compounds  which  are  proving  effective  alternative 
treatments  for  lithium  non-responsive  patients  with  affective  disorders.   The 
anticonvulsant  carbamazepine  (Tegretol)  has  now  been  generally  accepted  as  a 
clinical  treatment  option  for  manic-depressive  patients;  predictors  of  clinical 
response  and  possible  mechanisms  of  action  of  this  drug  are  being  intensively 
explored. 

Outpatient  studies  are  focused  on  several  different  clinical  populations. 
Dr.  Uhde  leads  a  major  outpatient  initiative  in  the  clinical  evaluation,  biologi- 
cal characterization,  and  treatment  of  patients  with  panic-anxiety  disorders. 
Dr.  Rubinow  is  engaged  in  the  longitudinal  assessment  and  treatment  of  patients 
with  menstrually-related  mood  disorders.   Patients  with  affective  disorders  are 
followed  both  by  the  Section  on  Clinical  Neuroendocrinology  and  by  the  Section  on 
Psychobiology  for  detailed  followup  evaluation  of  the  long-term  efficacy  of 
carbamazepine  once  patients  have  left  the  direct  clinical  care  of  the  NIMH.   Dr. 
Gerald  L.  Brown  studies  clinical  and  neurobiological  aspects  of  aggressivity  and 
suicidality  in  a  variety  of  outpatient  populations. 

Paul  Marangos,  Ph.D.  and  Jit  Patel,  Ph.D.  lead  efforts  to  study  biochemical 
variables  relevant  to  neuropsychiatric  disorders.   Dr.  Marangos  focuses  on  the 
study  of  neurotransmitter  and  receptor  systems,  while  Dr.  Patel  focuses  on  the 
sub-receptor  mechanisms  and  second  messenger  systems,  particularly  the  role  of 
phosphorylation  in  central  nervous  system  neural  excitability.   Agu  Pert,  Ph.D. 
leads  the  Unit  on  Behavioral  Pharmacology,  which  is  engaged  in  the  study  of 
neurotransmitter  and  peptide  interactions  in  behaviorally  relevant  animal  models. 
Susan  Weiss,  Ph.D.  coordinates  efforts  in  the  study  of  several  animal  models  in- 
cluding electrophysiological  kindling,  behavioral  sensitization  to  psychomotor 
stimulants,  and  the  long-term  behavioral  and  biochemical  consequences  of 
avoidable  and  unavoidable  stress. 


Unit  on  Anxiety  and  Affective  Disorders  (Dr.  Thomas  W.  Uhde,  Chief) 
Dr.  Uhde  has  developed  a  precise  methodology  for  describing  the  longitudinal 
course  of  patients  with  generalized  anxiety  and  panic-anxiety  disorders.   He  has 
found  that  the  majority  of  patients  experience  panic  attacks  initially  and  later 
develop  agoraphobia.   He  has  also  found  that  approximately  50%  of  panic-anxious 
patients  develop  endogenous  depression,  although  in  only  half  of  these  are  the 
symptoms  endogenomorphic  and  of  long  duration.   Using  the  relatively  specific 
pharmacological  probe,  clonidine,  an  alpha-2  adrenergic  agonist  that  inhibits 
firing  of  the  locus  coeruleus,  evidence  of  altered  noradrenergic  responsiveness 
has  been  documented  in  patients  with  both  panic-anxiety  disorders  and  depression 
compared  to  normal  volunteer  controls,  based  on  the  findings  of  a  blunted  growth 
hormone  response  in  both  these  patient  populations. 

Endocrine  testing  with  corticotropin  releasing  factor  (CRF)  has  also 
revealed  common  findings  in  patients  with  panic-anxiety  and  those  with  major 
depression  compared  with  normal  volunteer  controls.   Both  patient  populations 
show  a  blunted  ACTH  response  to  CRF,  although  the  panic-anxious  patients  do  not 
show  pathological  escape  from  dexamethasone  suppression  or  increased  urinary  free 
Cortisol  excretion,  as  do  those  with  major  depression.   These  data  are  examples 
of  the  theme  pursued  by  Dr.  Uhde ' s  Unit  to  explicate  clinical,  biological,  and 
pharmacological  similarities  as  well  as  differences  in  these  two  patient 
populations.   These  data  are  of  considerable  import  in  light  of  observations  of  a 
high  incidence  of  anxiety  in  depressed  patients  and  vice  versa,  as  well  as  the 
common  responsivity  to  tricyclic  and  monoamine  oxidase  inhibitor  antidepressant 
treatment  strategies. 

Panic-anxious  patients,  in  contrast  to  those  with  primary  affective  illness, 
do  not  show  the  same  degree  of  marked  shortening  of  REM  latency  and  show 
decreased  rather  than  increased  REM  latency.   Moreover,  they  show  an  increased 
amount  of  movement  time  during  EEG-monitored  sleep  recordings.   These  data  are 
illustrative  of  physiological  findings  that  differ  between  panic-anxious  and 
depressed  patients.   Panic-anxious  patients  also  do  not  show  the  same  high 
incidence  of  positive  clinical  response  to  one  night's  sleep  deprivation  as  do 
affectively  ill  patients.   Patients  with  panic-anxiety  disorder,  like  those  with 
primary  affective  illness,  were  noted  to  have  a  high  incidence  of  psychosensory 
symptoms  typically  reported  by  those  with  complex  partial  seizures.   However, 
these  anxious  patients  show  no  evidence  of  a  seizure  disorder  utilizing  EEC's 
with  nasopharyngeal  leads  following  sleep  deprivation,  photic  stimulation,  and 
hyperventilation . 

Another  major  investigative  strategy  involves  elucidation  of  the  clinical 
and  biological  mechanisms  involved  in  caffeine-induced  anxiety.   Panic-anxious 
patients  report  an  increased  sensitivity  to  caffeine;  Dr.  Uhde  and  his  collab- 
orators have  confirmed  these  observations  by  direct,  double-blind  pharmacological 
challenges.   Of  24  panic-anxious  patients,  9  (37.5%)  reported  panic  attacks 
following  ingestion  of  480  mg  of  oral  caffeine  (roughly  equivalent  to  5  or  6  cups 
of  coffee) ;  none  panicked  after  placebo.   In  contrast,  none  of  14  normal  controls 
demonstrated  panic  attacks  to  this  same  dose  of  caffeine.   Patients  showed 
greater  increases  in  plasma  lactate  levels  than  normal  controls,  and  those  who 
panicked  had  greater  increases  than  those  who  did  not  panic. 

AlprazolaiTi,  a  drug  with  potent  effects  at  the  "central-type"  benzodiazepine 
receptor,  has  been  reported  to  have  both  anti-panic  and  antidepressant 
properties.   Drs.  Uhde  and  Bierer  have  found  that  alprazolam  blocked 

10 


caffeine-induced  panic  attacks.   None  of  11  patients  administered  480  mg  of 
caffeine  panicked  while  on  blind  alprazolam  treatment,  in  contrast  to  the  37.5% 
of  patients  who  did  so  under  medication-free  conditions.   This  clinical  blockade 
of  caffeine-induced  panic  occurred  even  though  many  of  the  biochemical  and 
neuroendocrine  indices  of  caffeine-induced  effects  were  not  altered  by  alprazo- 
lam. 

Taken  together,  these  data  suggest  that  one  of  the  more  commonly  ingested 
and  potentially  abused  drugs  -  caffeine  -  in  moderate  to  large  doses  is  capable 
of  inducing  panic  attacks  in  panic-anxious  patients.   This  may  reflect  a  shift  in 
sensitivity  or  responsivity  to  caffeine  in  this  patient  population,  since  two 
normal  volunteers  who  were  challenged  with  higher  doses  (720  mg)  of  caffeine 
developed  panic  attacks.   Caffeine  may  be  one  cause  of  panic  attacks  in  the 
general  population  and  it  would  appear  that  caffeine  consumption  should  be 
restricted  in  patients  with  panic-anxiety  disorders.   Moreover,  understanding  the 
mechanisms  underlying  caffeine-induced  panic  may  help  to  elucidate 
pathophysiological  mechanisms  involved  in  the  panic-anxiety  disorders.   Drs.  Uhde 
and  Boulenger  have  completed  a  dose-response  study  of  caffeine  in  normal 
volunteers  and  found  dose-related  increases  in  anxiety,  mean  arterial  blood 
pressure,  plasma  lactate,  and  Cortisol.   Biochemical  mechanisms  potentially 
underlying  caffeine-induced  anxiogenic  effects  include  caffeine's  potent  blockade 
of  adenosine  receptors,  its  ability  to  act  with  benzodiazepine  receptors,  and, 
less  likely,  its  ability  to  potentiate  a  variety  of  neurochemical  systems  by  its 
action  as  a  phosphodiesterase  inhibitor.   It  is  of  interest  that  caffeine  appears 
to  be  producing  its  anxiogenic  effects  in  the  absence  of  elevating  plasma  MHPG. 
These  data  run  contrary  to  a  large  body  of  data  linking  alterations  in 
noradrenergic  function  to  parallel  alterations  in  degree  of  reported  anxiety. 
This  adrenergic  component  has  been  further  elucidated  by  Dr.  Uhde  and  his  group 
by  the  demonstration  that  relatively  low  doses  of  the  alpha-2  antagonist,  yohim- 
bine, which  increases  firing  of  the  locus  coeruleus,  induces  panic  attacks  in 
panic-anxious  patients  and  not  in  normal  controls.   Conversely,  the  alpha-2  agon- 
ist, clonidine,  which  decreases  firing  of  the  locus  coeruleus,  has  anti-anxiety 
effects. 

One  of  the  more  striking  findings  of  this  group  involves  the  study  of  the 
effects  of  caffeine  on  dexamethasone-induced  suppression  of  plasma  Cortisol.   In 
a  single-blind  comparison  of  the  effects  of  caffeine  (480  mg)  or  placebo  adminis- 
tered randomly  on  two  separate  days  at  2:00-2:30  PM  following  an  11  PM 
administration  of  dexamethasone  (1  mg)  the  night  before,  a  highly  significant 
effect  of  caffeine  on  post-dexamethasone  Cortisol  values  emerged.   Not  only  was 
there  a  highly  significant  increase  in  plasma  Cortisol  following  caffeine 
compared  to  placebo,  but  the  percentage  of  nonsuppressors  (by  the  conventional 
criteria  of  5  p.g/dl)  increased  from  14%  to  36%  following  caffeine  administration. 
These  data  suggest  the  possibility  that  a  dietary  artifact  such  as  caffeine  could 
account  for  some  of  the  differential  rates  of  escape  from  dexamethasone 
suppression  in  different  psychiatric  populations,  particularly  in  light  of 
observations  that  many  patients  with  panic  anxiety  disorder  report  giving  up 
drinking  coffee  because  of  its  CNS  stimulating  and  anxiogenic  properties.   In 
contrast,  depressed  patients  continue  to  drink  large  amounts  of  coffee,  possibly 
in  attempts  to  treat  their  depressive  symptoms.   Thus,  these  data  are  not  only  of 
potential  methodological  import,  but  again  focus  on  the  question  of  the 
mechanisms  involved  in  the  caffeine-induced  escape  from  dexamethasone 
suppression,  a  line  of  investigation  to  be  pursued  by  this  investigative  group. 


11 


Anecdotal  reports,  conventional  wisdom,  and  clinical  lore  all  raise  the 
possibility  that  hypoglycemia  may  play  a  role  in  some  patients  with  panic-anxiety 
disorders.   In  an  attempt  to  systematically  assess  this  supposition,  Drs.  Uhde, 
Vittone,  and  collaborators  have  investigated  the  clinical  and  biochemical 
response  to  a  glucose  challenge.   Utilizing  a  5-hour  glucose  tolerance  test 
paradigm,  they  observed  a  substantial  incidence  of  symptomatic  hypoglycemia  in 
the  panic-anxious  patients  but  this  was  generally  not  associated  with  the 
induction  of  panic  attacks.   These  data  are  of  interest  not  only  in  their  own 
right,  but  also  suggest  that  despite  the  many  induced  signs  and  symptoms  of 
hypoglycemia,  including  diaphoresis,  tremor,  and  lightheadedness,  panic-anxious 
patients  do  not  report  a  high  rate  of  panic  attacks.   These  data  highlight  the 
specificity  of  other  challenge  strategies,  including  lactate,  yohimbine,  and 
caffeine,  which  do  appear  to  induce  a  substantial  incidence  of  panic  attacks  in 
this  patient  population. 

Section  on  Clinical  Neuroendocrinology  (Dr.  Philip  W.  Gold,  Chief) 
Clinical  studies  with  the  newly-sequenced  corticotropin  releasing  factor 
(CRF)  are  illustrative  of  the  pioneering  work  of  Dr.  Gold  and  his  associates. 
These  investigators  have  substantially  advanced  the  state  of  knowledge  concerning 
the  normal  physiology  and  the  pathophysiology  of  hypothalainic-pituitary-adrenal 
function.   For  instance,  they  have  produced  the  most  compelling  evidence  to  date 
that  CRF  is  of  physiological  relevance  to  the  regulation  of  the  pituitary-adrenal 
axis  in  man.   Thus,  eight  1  ug/kg  pulses  of  human  CRF  given  over  24  hours  (in  a 
temporal  sequence  designed  to  mimic  naturally  occurring  ACTH  pulses)  restored  the 
normal  circadian  pattern  and  amplitude  of  Cortisol  secretion  in  patients  with 
hypothalamic  CRF  deficiency.   This  study  also  showed  that  the  ACTH  response  to 
CRF  was  enhanced  during  the  early  morning  Cortisol  surge,  suggesting  that  the 
pituitary  corticotroph  cell  shows  a  circadian  rhythm  in  its  response  to  CRF. 
This  idea  is  supported  by  their  finding  in  volunteers  that  the  early  morning 
Cortisol  surge  is  associated  with  an  increase  in  both  pulse  frequency  and 
amplitude  of  naturally  occurring  ACTH  secretory  episodes. 

To  explore  the  potential  clinical  applications  of  CRF,  Dr.  Gold  and  his  col- 
leagues conducted  a  series  of  dose-response  and  pharmacokinetic  studies  with 
ovine  and  human  CRF  to  optimize  conditions  for  a  CRF  stimulation  paradigm.   A 
test  using  1  vg/kg  of  ovine  CRF  has  proven  extremely  useful  in  a  variety  of 
clinical  situations.   Most  notably,  the  CRF  stimulation  test  has  helped  to 
clarify  the  pathophysiology  of  hypercortisolism  in  depression  and  Gushing 's 
disease  and  has  proven  to  be  helpful  in  establishing  differential  diagnosis. 
Depressed  patients  showed  a  blunted  ACTH  response  to  exogenous  CRF,  suggesting 
that  the  pituitary  in  these  patients  is  appropriately  restrained  by  the  high 
levels  of  Cortisol.   This  indicated  that  hypercortisolism  in  depression  could 
reflect  a  defect  at  or  above  the  hypothalamus,  resulting  in  the  hypersecretion  of 
CRF,  an  hypothesis  supported  by  the  finding  that  a  continuous  infusion  of  CRF  to 
volunteers  reproduced  the  pattern  and  magnitude  of  hypercortisolism  seen  in 
depression . 

In  contrast  to  depressed  patients,  patients  with  Gushing 's  disease  showed  an 
exaggerated  ACTH  response  to  CRF,  indicating  a  defect  in  glucocorticoid  negative 
feedback  upon  the  pituitary.   Moreover,  in  contrast  to  depressed  patients,  the 
hypothalamic  CRF  neuron  in  patients  with  Gushing 's  disease  seemed  to  be  normally 
suppressed  by  hypercortisolism.   Thus,  all  patients  with  Gushing 's  disease 
treated  effectively  with  a  selective  microadenectomy ,  show  undetectable  ACTH  and 
Cortisol  levels  after  surgery;  they  show  a  normal  ACTH  response  to  CRF  at  this 

12 


time.   This  suggests  that  they  would  not  have  shown  adrenal  insufficiency  in  the 
post-operative  period  had  their  CRF  neuron  not  been  suppressed  by  long-standing 
hypercortisolism.   In  support  of  the  idea  of  a  differential  functioning  of  the 
CRF  neuron  in  depression  and  Gushing 's  disease,  is  Dr.  Gold's  recent  finding  that 
CSF  CRF  is  several-fold  higher  in  depressed  patients  than  in  patients  with 
Cushing's  disease,  whose  levels  fall  well  below  the  normal  range. 

The  clinical  work  of  Dr.  Gold  and  his  colleagues  has  also  proven  helpful  as 
an  aid  in  the  differential  diagnosis  of  other  entities  such  as  Cushing's  disease 
from  ectopic  ACTH  secretion  and  pituitary  from  hypothalamic  adrenal 
insufficiency.   In  work  with  psychiatric  patients  who  carry  diagnoses  other  than 
affective  disturbance.  Dr.  Gold  showed  that  the  ACTH  response  to  CRF  in  patients 
with  anorexia  nervosa  qualitatively  resembled  the  findings  seen  in  depression. 
This  phenomenon,  indicative  of  CRF  hypersecretion  in  anorexia  nervosa,  was 
supported  by  the  finding  of  higher  CSF  CRF  in  anorectic  patients  compared  to 
controls.   Although  this  defect  in  hypothalamic  CRF  secretion  seemed  to  resolve 
in  anorectic  patients  soon  after  correction  of  the  weight  loss,  subtle 
abnormalities  in  hypothalamic-pituitary-adrenal  function  persisted  for  some  time. 
In  another  group.  Dr.  Gold  and  his  colleagues  showed  that  patients  with 
Alzheimer's  disease  showed  significant  reduction  in  the  level  of  CSF  CRF, 
compatible  with  findings  that  the  post-mortem  brain  content  of  CRF  is  reduced  in 
patients  with  this  disorder.   Interestingly,  this  decrease  in  CSF  CRF  is  not 
associated  with  hypof unction  of  the  pituitary-adrenal  axis. 

The  work  of  this  group  has  also  helped  to  clarify  the  pathophysiology  of  the 
hypersecretion  of  growth  hormone  in  patients  with  anorexia  nervosa.   It  was  shown 
that  anorectic  patients  manifest  an  exaggerated  growth  hormone  response  to  growth 
hormone  releasing  hormone,  which  was  negatively  correlated  with  plasma 
somatomedin  levels.   Since  somatomedin  is  a  potent  inhibitor  of  growth  hormone 
secretion,  it  seems  likely  that  increased  growth  hormone  secretion  in  anorectics 
reflects  diminished  hepatic  production  of  somatomedin,  secondary  either  to 
hypercortisolism  or  chronic  inanition. 

Work  is  progressing  on  the  newly  developed  glucocorticoid  receptor  antagon- 
ist, RU486,  which  produces  dose-dependent  increases  in  plasma  ACTH  and 
vasopressin  secretion.   This  compound  should  provide  a  new  tool  for  dissecting 
hypothalamic-pituitary-adrenal  axis  abnormalities  and  the  role  of  excess 
circulating  glucocorticoids  in  the  symptoms  of  a  variety  of  neuropsychiatric 
conditions.   Dr.  Gold's  group  is  utilizing  a  variety  of  animal  models,  including 
a  recently  developed  tethered  system  for  chronic  sampling  in  unrestrained 
non-human  primates,  in  order  to  study  various  endocrine  regulatory  mechanisms. 

Unit  on  Peptides  and  NIMH  Consultation  Liaison  Service  (Dr.  David  Rubinow, 
Chief) 

A  series  of  protocols  and  collaborative  projects  have  been  initiated  in  con- 
junction with  the  Consultation  Liaison  Service.   In  collaboration  with  Dr.  Joffe, 
Dr.  Rubinow  observed  that  endogenomorphic  depression  was  a  frequent  presenting 
symptom  in  patients  with  carcinoma  of  the  pancreas  (6  of  12) ,  but  not  in  patients 
with  gastric  carcinoma  (0  of  9) .   These  data  support  clinical  observations  of  a 
high  incidence  of  depression  in  carcinoma  of  the  pancreas  and  open  the 
possibility  of  studying  underlying  pathophysiological  mechanisms.   Patients  with 
acquired  immune  deficiency  syndrome  (AIDS)  were  found  to  have  both  generalized 
and  selective  cognitive  defects  when  compared  with  appropriately  matched 
controls.   These  findings  are  of  particular  interest  in  relation  to  recent 

13 


reports  of  neuropathological  findings  in  this  disorder.   Interferon  therapy  for 
chronic  active  hepatitis  appeared  to  induce  endogenomorphic  depressions  in  10%  of 
the  30  patients  so  far  treated  with  this  novel  regime;   this  side  effect 
necessitated  termination  of  the  clinical  trial.   Five  other  consultation-liaison 
protocols  are  in  progress  or  near  completion. 

Dr.  Rubinow  has  been  following  a  cohort  of  patients  who  were  self-referred 
for  menstrually-related  mood  disorder,  utilizing  prospective  daily  self-ratings 
on  a  visual  analogue  scale.   Using  this  prospective  assessment,  he  has  found  that 
only  43%  of  215  self-referred  patients  had  clear-cut  mood  disorder  that  occurred 
in  consistent  and  close  association  with  the  pre-menstrual  period.   These  data 
are  of  substantial  import  since  including  more  than  50%  of  patients  who  do  not 
have  menstrually-related  mood  disorder  into  biochemical  studies  and  clinical 
trials  purporting  to  treat  this  disorder  would  obviously  lead  to  erroneous 
conclusions.   The  prospective  criteria  have  now  been  included  into  a  DSM-III-R 
diagnosis  of  premenstrual  dysphoric  disorder.   Basal  endocrine  data  collected 
from  35  women  with  premenstrually-related  mood  disorder  and  ten  controls  revealed 
no  significant  diagnostic  group-related  differences.   However,  blunted  TSH 
responses  to  TRH  were  observed  in  8  of  12  women  with  menstrually-related  mood 
disorder  vs.  one  in  ten  controls.   Plasma  calcium  to  magnesium  ratios  were  signi- 
ficantly reduced  in  two  different  luteal  phase  blood  samples  in  women  with 
menstrually-related  mood  disorder  relative  to  controls.   However,  no  abnormality 
of  dexamethasone  response  or  in  plasma  catecholamine  levels  was  evident  during 
either  phase  in  these  patients. 

Progesterone  and  its  analogues  have  been  reported  by  others  to  be  useful  in 
the  treatment  of  PMS,   However,  in  eight  women  who  completed  a  10-month  double- 
blind,  placebo-controlled  crossover  trial  of  progesterone  and 
medroxyprogesterone,  none  of  the  patients  showed  a  successful  therapeutic 
response.   These  data,  again,  highlight  the  importance  of  prospective  diagnostic 
assessment  and  double-blind  followup  of  women  with  reported  menstrually-related 
mood  disorder,  prior  to  their  entering  controlled  clinical  trials. 

Dr.  Rubinow  has  pioneered  in  the  study  of  the  role  of  somatostatin  in 
depression  and  its  neuroendocrine  abnormalities.   He  has  documented  that 
somatostatin  is  significantly  lower  in  the  CSF  of  depressed  patients  compared  to 
normal  volunteer  controls.   This  state-related  abnormality  returns  toward  normal 
with  clinical  improvement  or  the  switch  into  mania.   Reductions  in  CSF 
somatostatin  have  also  been  demonstrated  by  him  in  Alzheimer's  disease.   These 
data  are  consistent  with  a  large  literature  supporting  this  observation  and  with 
the  well  replicated  findings  that  somatostatin  is  decreased  in  the  cerebral 
cortex  of  patients  with  Alzheimer's  disease  and  is  found  in  the  senile  plaques 
associated  with  this  syndrome. 

In  depressed  patients  who  showed  abnormal  escape  from  dexamethasone  suppres- 
sion, CSF  levels  of  somatostatin  were  significantly  lower  than  those  with  normal 
dexamethasone  responses.   These  data  further  suggest  the  possibility  that  inade- 
quate somatostatin  inhibitory  control  over  the  pituitary-adrenal  axis  may  play 
some  role  in  syndromes  associated  with  pathological  escape  from  dexamethasone 
suppression.   Both  the  anticonvulsant  carbamazepine  and  the  neuroleptic  fluphena- 
zine  produced  decreases  in  CSF  somatostatin. 

Dr.  Rubinow  received  the  A.E.  Bennett  Prize  of  the  Society  of  Biological 
Psychiatry,  May,  1985,   for  his  work  on  CSF  somatostatin  in  neuropsychiatric 
syndromes. 

14 


Unit  on  Behavioral  Pharmacology  (Dr.  Agu  Pert,  Chief) 

Biochemical,  electrophysiological,  and  behavioral  evidence  suggests  that 
nicotine  may  act  in  the  brain  through  specific  nicotine  receptors.   Drs.  Pert  and 
Clarke  have  demonstrated  the  distribution  of  H-nicotine  binding  in  rat  and 
monkey  brain  using  autoradiographic  techniques.   In  the  rhesus  monkey  as  well  as 
rat  brain,  nicotinic  receptors  were  particularly  dense  in  certain  "specific" 
thalamic  nuclei,  the  interpeduncular  nucleus,  the  zona  compacta  of  the  substantia 
nigra  and  layer  III  of  the  cortex,  especially  the  primary  sensory  areas.   Lesion 
studies  revealed  that  the  interpeduncular  nucleus  possesses  both  postsynaptic  and 
presynaptic  nicotinic  receptors,  the  latter  apparently  located  on  cholinergic 
terminals  afferent  to  the  nucleus.   Nicotine  receptors  were  also  found  to  be 
present  on  dopamine  cell  bodies  in  the  substantia  nigra.   Cholinergic  projections 
to  the  zona  compacta  may  originate  from  the  pedunculopontine  nucleus,  since 
injections  of  kainic  acid  into  this  nucleus  were  found  to  excite  these 
dopaminergic  cells  in  the  substantia  nigra  in  a  fashion  similar  to  systemically 
administered  nicotine. 

Opiates  are  known  to  have  an  important  influence  on  dopaminergic  neurotrans- 
mission.  An  attempt  was  made  to  localize  opiate  receptors  in  the  substantia 
nigra,  the  origin  of  dopaminergic  nigro-striatal  system.   Lesion  studies  revealed 
that  opiate  receptors  are  not  localized  on  dopaminergic  cell  bodies.   Since 
lesions  of  the  striato-nigral  and  pallido-nigral  systems  drastically  reduced 
binding  of  ^H-naloxone  in  the  substantia  nigra,  it  appears  that  the  majority  of 
opiate  receptors  in  the  substantia  nigra  are  on  terminals  of  neurons  originating 
from  the  f orebrain .   These  results  suggest  that  opiate  effects  on  dopamine 
function  and  dopamine-linked  behaviors  are  likely  to  be  mediated  by  actions  at 
presynaptic  receptors  localized  on  nigral  afferents. 

3 
With  Dr.  Ostrowski,  a  novel  opiate  ligand  (  H-cyclo-foxy)  has  been  employed 

in  in  vivo  autoradiographic  studies.   Pattern  analysis  of  autoradiographs  showed 

that  the  binding  of  this  ligand  was  virtually  identical  under  in  vivo  and  in 

vitro  conditions.   The  results  of  these  studies  revealed  that  this  compound  binds 

to  the  well-described,  naloxone-sensitive  opiate  receptors  and  suggest  that  it 

will  be  an  excellent  candidate  for  use  in  PET  scanning  in  live  animals  and 

humans. 

Endogenous  opioid  systems  have  been  implicated  in  the  tonic  regulation  of 
the  hypothalamic-pituitary  gonadal  axis.   In  addition  there  is  evidence  to 
suggest  that  there  are  sex  differences  in  the  response  of  animals  to  opiates.   In 
order  to  explore  the  possibility  that  sex  differences  in  opiate  receptors  may 
account  for  some  of  the  differential  effects  of  opiates  in  males  and  females  and 
to  determine  the  extent  of  involvement  of  opiate  receptors  in  reproductive 
cyclicity,  autoradiographic  experiments  were  conducted  with  Dr.  Ostrowski 
assessing  regional  binding  densities  and  pattern  of  ^H-naloxone  and 
^H-ala-D-leu-enkephalin  in  male  and  female  hamsters.   Clear  sex  differences 
emerged  regarding  the  binding  of  ^H-naloxone.   Quantitative  analyses  confirmed 
that  the  sexually  dimorphic  nucleus  of  the  male  showed  denser  binding  than  that 
of  the  estrous  female. 

Likewise,  the  stria  terminalis  in  the  male  and  in  the  diestrous  female  bound 
3H-naloxone  more  densely  than  that  in  the  estrous  female.   Since  binding 
densities  of  diestrous  females  were  more  similar  to  densities  of  males  than 
estrous  females,  it  appears  that  the  endocrine  status  of  the  adult  animal  may 
modulate  opiate  receptors. 


15 


In  vivo  autoradiographic  techniques  were  employed  to  assess  the  activity  of 
endogenous  opioid  systems  during  various  behavioral  and  physiological  manipula- 
tions.  This  procedure  is  based  on  the  principle  that  exogenously  released 
opioids  will  displace  radiolabeled  opiates  that  are  injected  systemically .   Such 
displacement  is  revealed  autoradiographically  by  a  decrease  in  binding  of  the 
labeled  ligand  to  a  brain  section.   Using  this  technique,  Dr,  Pert  and  colleagues 
have  found  that  stress  is  a  very  potent  activator  of  the  endogenous  opioid 
systems  in  many  regions  of  the  brain.   Since  opiates  have  been  shown  to  influence 
mating  behavior,  attempts  were  made  to  evaluate  the  activation  of  endogenous 
opioid  systems  during  copulation.   It  was  found  that  ^H-diprenorphine  binding  was 
substantially  decreased  in  various  hypothalamic  and  other  diencephalic  structures 
of  mated  males,  suggesting  release  of  endogenous  opioids  during  this  behavior. 

Electrical  stimulation  of  the  arcuate  nucleus  (the  origin  of  the  beta-endor- 
phin  system)  also  produced  decreases  in  binding  of  the  exogenously  administered, 
radiolabeled  ligand,  specifically  in  the  terminal  areas  of  this  system;  such 
stimulation  was  also  accompanied  by  a  naloxone  reversible  analgesia.   In  animals 
that  were  kindled  to  seizures  by  amygdaloid  stimulation,  there  was  also  a 
decrease  in  opiate  binding  as  revealed  by  in  vivo  autoradiographic  procedures. 
These  results  suggest  widespread  occupation  of  forebrain  opioid  receptors  by 
endogenous  opiates  released  during  the  ictal  process. 

There  is  evidence  to  suggest  that  calcitonin  levels  may  change  during 
various  phases  of  manic-depressive  disorders.   Dr.  Pert  has  found  that  salmon 
calcitonin  (sCT)  binds  heavily  to  the  nucleus  accumbens.   Injections  of  sCT  into 
this  structure  produce  profound  depression  of  locomotor  output  in  rats.   It  is 
possible  that  the  n.  accumbens  represents  a  site  of  importance  in  the  modulation 
of  psychomotor  activity. 

Drs.  Pert  and  Wheeling  are  presently  using  electrical  stimulation  of  the  rat 
mesencephalon  as  a  model  to  study  the  af fective-integrative  component  of  pain. 
Rats  stimulated  in  this  region  will  readily  learn  to  escape  from  stimulation 
while  humans  have  reported  sensations  of  burning  pain,  nausea,  and 
unpleasantness.   Intraventricular  injections  of  morphine  elevate  aversive  brain 
stimulation  thresholds  while  neurotensin  lowers  them.   This  model  has  potential 
for  evaluating  the  effects  of  novel  compounds  on  the  central  components  of  the 
pain  experience. 

Unit  on  Neurochemistry  (Dr.  Paul  Marangos,  Chief) 

Basic  science  work  with  neuron-specific  enolase  (NSE)  has  now  demonstrated 
clearcut  clinical  relevance.   NSE  levels  are  of  diagnostic  and  prognostic  value 
in  both  oat  cell  lung  cancer  and  pediatric  neuroblastoma  patients.   Exacerbation 
of  the  small  cell  carcinoma  can  be  detected  by  serum  NSE  elevations  prior  to 
evidence  from  chest  x-ray,  such  that  salvage  chemotherapy  can  be  initiated 
earlier  in  this  illness.   CSF  levels  of  NSE  are  highly  elevated  in  patients  with 
brain  metastases.   Two  year  survival  in  pediatric  neuroblastoma  is  predicted  by 
serum  levels  of  NSE  above  100  ng/ml  while  five  year  survival  can  be  expected  when 
serum  levels  are  below  this  value.   Modest  increases  within  NSE  levels  have  been 
detected  in  Alzheimer's  disease.   Electron  microscopy  has  revealed  that  NSE 
levels  are  higher  in  unmyelinated  compared  to  myelinated  neurons  and  are  higher 
in  the  cell  body  and  dendritic  areas.   Purkinje  cells  have  less  NSE  than  other 
neuronal  cell  types.   Work  is  proceeding  in  obtaining  a  cDNA  probe  for  NSE. 


16 


Autoradiographic  studies  have  elucidated  a  differential  distribution  of 
adenosine  uptake  sites  (marked  by  ^H-NBI)  and  adenosine  receptor  binding  sites 
(labeled  by  ^H-CHA  in  brain.   The  two  sites  were  found  in  high  levels  in  the 
caudate  nucleus,  superior  colliculus,  and  thalamus,  suggesting  possible 
adenosinergic  neurotransmission  in  these  areas  of  brain.   Two  areas  high  in 
adenosine  receptors,  the  molecular  layer  of  the  cerebellum  and  hippocampus, 
interestingly,  have  very  low  levels  of  the  uptake  site. 

Both  the  adenosine  uptake  site  and  the  receptor  have  been  solubilized.   They 
remain  pharmacologically  distinct  in  the  soluble  state;  the  adenosine  uptake 
blockers  such  as  dipyridamole,  dilazep,  and  hexobendine  are  several  orders  of 
magnitude  more  potent  in  inhibiting  ^H-NBI  binding  to  the  solubilized  preparation 
when  compared  to  the  receptor  ligands  CHA  and  DPX. 

Dihydropyridine-type  calcium  channel  blockers  such  as  nifedipine, 
nimodipine,  and  nisoldipine  inhibit  binding  of  ^H-NBI  in  both  heart  and  brain. 
These  data  suggest  that  there  is  an  interaction  between  the  voltage-dependent 
calcium  channel  and  adenosine  uptake  mechanisms.   Carbamazepine,  like  caffeine, 
when  chronically  administered  to  rats,  increases  brain  adenosine  receptors, 
suggesting  an  adenosine  antagonist  effect  of  carbamazepine  on  this  measure.   A 
new  series  of  studies  have  confirmed  the  original  observations  and  have  indicated 
that  chronic  carbamazepine  treatment  leads  to  a  persistent  upregulation  of 
adenosine  receptors  which  lasts  for  three  weeks  after  carbamazepine  withdrawal. 
The  relationship  of  carbamazepine ' s  clinical  effects  to  its  relatively  potent 
ability  to  inhibit  adenosine  receptor  binding  remains  to  be  clarified.   Studies 
utilizing  ^H-carbamazepine  are  proceeding  in  an  attempt  to  identify  a  possible 
specific  binding  site  for  carbamazepine. 

A  possible  link  between  adenosine  receptor  binding  and  behavioral  pathology 
has  been  made  with  the  finding  that  Maudsley  reactive  compared  to  non-reactive 
strains  of  rats  show  increased  adenosine  receptor  binding;   there  is  no  signifi- 
cant change  in  central  or  "peripheral-type"  benzodiazepine  receptor  binding  in 
these  animals. 

Drs.  Tamborska  and  Marangos  have  demonstrated  that  there  is  an  increase  in 
the  "peripheral-type"  benzodiazepine  receptor  as  marked  by  increased  binding  of 
Ro5-4864  during  states  of  alcohol  intoxication  and  withdrawal.   This  increase  in 
receptor  number  of  Ro5-4864  binding  sites  is  evident  in  cerebellum,  hippocampus, 
and  cerebral  cortex.   It  persists  for  up  to  seven  days  after  cessation  of  alcohol 
treatment,  raising  the  possibility  that  some  of  the  behavioral  manifestations  of 
alcohol  withdrawal  (i.e,  anxiety,  delirium,  and  convulsions)  could  be  related  to 
this  robust  and  persisting  change  in  "peripheral-type"  benzodiazepine  receptors. 
This  possibility  is  further  supported  by  the  data  discussed  below,  that  the 
anticonvulsant  carbamazepine  appears  to  have  important  physiological  as  well  as 
biochemical  interactions  with  the  "peripheral- type"  binding  site. 

Dr.  Jitendra  Patel  continues  to  focus  efforts  on  the  role  of  protein 
phosphorylation  in  the  regulation  of  neurotransmission.   He  has  established  that 
SlOO  modulated  phosphoprotein  (SMP)  is  a  major  substrate  of  calcium-phospholipid- 
dependent  protein  kinase  (C-kinase) .   He  has  found  that  C-kinase  is  involved  in 
the  modulation  of  receptor  sensitivity,  as  evidenced  by  studies  of  human  chorio- 
gonadotropin-sensitive  adenylate  cyclase-cultured  leydig  cells.   C-Kinase  phos- 
phorylates  a  number  of  membrane  proteins  and  desensitizes  the  adenylate  cyclase. 
These  data  raise  the  possibility  that  plasticity  in  several  receptor  systems  may 
be  regulated  by  a  protein  kinase.   He  has  observed  that  the  neuropeptides  somato- 

17 


statin  and  calcitonin  are  potent  inhibitors  of  calmodulin-dependent  protein  kin- 
ases (a  50k  and  60k  protein) .  A  20k  phosphoprotein  has  been  associated  with  anti- 
tumor drug  resistance. 

Lithium  inhibits  the  breakdown  of  polyphosphoinositide  (IP  )  which  alters  the 
permeability  of  the  plasma  membrane  to  intracellular  calciiam,  resulting  in  intra- 
cellular mobilization  of  calcium.   In  collaboration  with  Dr.  E,  Klein,  Dr.  Patel 
found  that  chronic  lithium  treatment  of  rats  for  three  weeks  increased  the  phos- 
phorylation of  a  64k  phosphoprotein  in  all  brain  areas  examined.   This  54k  protein 
may  be  the  alpha  sub-unit  of  C-kinase. 

Efforts  are  ongoing  to  more  precisely  examine  and  replicate  previous  findings 
of  alterations  in  protein  phosphorylation  that  persist  following  amygdala  kindling. 

Section  on  Psychobiology  (Dr.  Robert  M.  Post,  Acting  Chief) 

Work  co-directed  with  Dr.  Uhde  focuses  on  delineating  the  clinical  and  bio- 
logical predictors  of  response  to  the  anticonvulsant  carbamazepine  in  manic-depres- 
sive illness.   Carbamazepine  (Tegretol),  a  routinely-used  anticonvulsant  which  is 
also  effective  in  the  treatment  of  trigeminal  neuralgia  and  related  pain  syndromes, 
has  been  increasingly  recognized  as  an  alternative  or  an  adjunct  treatment  for 
lithium-non-responsive  patients  with  manic-depressive  illness.   Our  studies,  now 
confirmed  by  four  others  in  the  literature,  using  controlled  methodologies,  indi- 
cate that  carbamazepine  has  a  magnitude  and  time-course  of  clinical  efficacy  in 
acute  mania  similar  to  that  of  neuroleptics.   Moreover,  it  does  not  pose  some  of 
the  problematic  side  effects  that  are  associated  with  neuroleptics,  including  acute 
parkinsonism  or  longer-term  tardive  dyskinesia.   Twelve  of  the  first  19  patients 
had  good  acute  antimanic  responses  to  the  drug.   These  patients  are  distinguished 
by  their  having  a  history  of  more  rapid  cycling  in  the  year  prior  to  admission, 
and  their  presenting  with  more  severe  ratings  of  mania  in  the  period  prior  to  drug 
treatment.   Twenty  of  the  first  37  acutely  depressed  patients  have  shown  at  least 
a  mild  antidepressant  response  to  carbamazepine;  25%  of  these  patients  showed  a 
clinically  robust  response.   Clinical  predictors  include  increased  severity  of 
baseline  depression,  less  chronic  course  with  more  discrete  episodes,  and  good 
acute  response  to  one-night's  sleep  deprivation. 

In  addition  to  this  evidence  of  acute  antimanic  and  acute  antidepressant  re- 
sponse, our  data  and  that  in  the  literature  are  suggestive  that  long-term  or  pro- 
phylactic treatment  with  carbamazepine  may  be  a  useful  alternative  in  lithium  non- 
responsive  patients.   We  have  followed  nine  patients  for  an  average  of  2.7  years 
on  carbamazepine  and  have  found  a  statistically  significant  reduction  in  both  the 
frequency  and  duration  of  manic  and  depressive  episodes  compared  to  the  prior  2.7 
years,  using  a  mirror  image  analysis.   It  appears  that  those  patients  with  rapid 
cycling  manic-depressive  illness  who  are  among  the  least  responsive  to  traditional 
treatment  with  lithium  carbonate,  are  among  those  who  respond  well  to  carbamaze- 
pine.  Pertinent  non-predictors  of  clinical  response  include  mild  EEC  abnormali- 
ties, family  history  of  manic-depressive  illness,  and  history  of  psychosensory 
or  epileptoid  symptoms.   Thus,  even  though  carbamazepine  is  a  useful  anticonvulsant 
agent,  neither  minor  EEC  abnormalities  nor  the  history  of  symptoms  that  have  been 
associated  with  epilepsy  appears  to  be  associated  with  carbamazepine  response. 

We  have  employed  pharmacological  challenges  with  low  doses  of  the  local  anes- 
thetic procaine  in  attempts  to  relatively  selectively  activate  limbic  system 


18 


structures  in  patients  with  manic-depressive  illness,  with  borderline  personality 
disorder,  and  in  normal  volunteer  controls.   In  collaboration  with  Drs.  Coppola 
and  Kellner,  we  have  found  that  procaine  produces  selective  activation  of  fast 
EEC  frequencies  (26-45  Hz)  over  the  temporal  lobes,  consistent  with  evidence  from 
animal  studies  that  local  anesthetics  produce  physiological  activation  of 
temporal  lobe  and  limbic  system  structures.   The  degree  of  dysphoria  induced  in 
the  first  21  subjects  was  highly  correlated  with  this  increase  in  fast  activity 
over  the  temporal  cortex,   Drs.  Kellner,  Kling,  and  Gold  found  that  procaine  was 
associated  with  dose-related  increases  in  ACTH,  Cortisol,  and  prolactin  but  not 
growth  hormone.   Clinically,  the  patients  experienced  a  range  of  sensory  and 
cognitive  distortions  that  are  similar  to  those  achieved  with  electrophysio- 
logical stimulation  of  deep  temporal  structures,  as  reported  in  the  studies  of 
Gloor  and  Halgren  and  associates.   These  included  distortions  and  hallucinations 
in  all  sensory  modalities  and  rare  activation  of  experientially  immediate  memory 
of  such  vividness  that  the  subject  felt  he  was  re-experiencing  the  initial  event 
itself.   Mood  changes  were  variable  and  ranged  from  the  induction  of  euphoria  to 
profound  dysphoria.   In  borderline  patients  studied  by  Drs.  Gardner  and  Cowdry, 
contrary  to  predictions,  the  degree  of  EEG  activation  induced  by  procaine  was  not 
positively  correlated  with  degree  of  clinical  response  to  the  anticonvulsant 
carbamazepine . 

We  have  explored  the  effects  of  carbamazepine  on  a  variety  of  neurotrans- 
mitter, endocrine,  and  peptide  systems  in  both  animals  and  man.   Evidence  is 
mounting  that  noradrenergic  mechanisms  are  important  to  the  anticonvulsant 
properties  of  carbamazepine,  but  the  relationship  of  noradrenergic  mechanisms  to 
the  psychotropic  effects  of  the  drug  remain  to  be  elucidated.   For  example,  we 
have  recently  observed  that  the  alpha-2  antagonist  yohimbine  reverses  the 
anticonvulsant  effects  of  carbamazepine  on  amygdala-kindled  seizures,  suggesting 
that  an  alpha-2  adrenergic  mechanism  is  important  to  carbamazepine ' s 
anticonvulsant  effects.   Similarly,  we  have  completed  a  series  of  electro- 
physiological studies  in  collaboration  with  Dr.  S.R.B.  Weiss,  that  strongly 
indicate  that  carbamazepine  may  interact  with  the  "peripheral-type" 
benzodiazepine  receptor  and  not  with  the  central-type  receptor.   We  have  observed 
that  the  relatively  selective  and  inert  antagonist  for  the  central-type 
benzodiazepine  receptor,  Ro-15-1788,  and  the  inverse  agonist,  beta-carboline 
(B-CCM) ,  both  reverse  the  anticonvulsant  effects  of  the  classical  benzodiazepine 
agonist  diazepam,  but  do  not  reverse  the  anticonvulsant  effects  of  carbamazepine 
on  amygdala-kindled  seizures.   In  contrast,  the  "peripheral-type"  benzodiazepine 
ligand,  Ro5-4864,  reverses  the  anticonvulsant  effects  of  carbamazepine,  but  not 
diazepam.   The  fact  that  this  effect  is  itself  reversed  by  PK-11195,  a  drug  that 
selectively  blocks  the  effects  of  R05-4864  at  the  "peripheral-type"  receptor, 
further  supports  the  proposition  that  this  mechanism  may  be  importantly  involved 
in  carbamazepine' s  actions.   In  addition,  we  have  observed  that  an  active 
metabolite  of  carbamazepine  (carbamazepine-10,ll-epoxide)  is  also  ineffective 
when  used  in  conjunction  with  Ro5-4864. 

These  data  suggest  that  carbamazepine  and  diazepam  act  through  different 
neural  mechanisms  in  the  brain  and  further  support  the  contention  that  the 
"peripheral-type"  benzodiazepine  receptor,  which  has  recently  been  putatively 
linked  to  both  anxiety  and  convulsant  mechanisms  in  animal  studies,  may  be  a 
physiologically  important  receptor  site  in  brain  rather  than  an  inert  acceptor 
site,  as  had  previously  been  thought. 

Considerable  evidence  suggests  that  carbamazepine  does  not  act  through  a 
mechanism  similar  to  that  of  neuroleptics;  it  does  not  block  dopamine  receptors. 

19 


Important  biochemical  candidates  for  carbamazepine ' s  mechanism  of  action,  in 
addition  to  noradrenergic  and  "peripheral-type"  benzodiazepine  mechanisms 
discussed  above,  include  its  ability  to  decrease  GABA  turnover,  increase  plasma 
tryptophan,  potently  interact  with  adenosine  receptors,  inhibit  a  variety  of 
activators  of  adenylate  cyclase,  act  as  a  vasopressin  agonist,  and  decrease  the 
somatostatin  in  the  spinal  fluid. 

A  series  of  other  anticonvulsants  in  addition  to  carbamazepine,  including 
valproic  acid,  clonazepam,  and  related  compounds  have  also  been  reported  to  have 
some  positive  effects  on  patients  with  manic-depressive  illness.   It  is  hoped 
that  by  comparing  carbamazepine ' s  profile  of  biochemical  and  physiological 
effects  with  other  anticonvulsants  which  are  not  effective  in  the  treatment  of 
manic-depressive  illness,  that  the  important  mechanisms  of  psychotropic  action 
may  be  delineated.   Similarly,  by  comparing  and  contrasting  carbamazepine  with 
other  agents  effective  in  the  treatment  of  both  phases  of  manic-depressive 
illness,  including  lithium  carbonate  and  electroconvulsive  treatment,  a  better 
understanding  of  its  important  mechanisms  may  be  derived. 

In  collaboration  with  Dr.  S.R.B.  Weiss,  we  are  seeking  to  elucidate  the  phe- 
nomenology, biochemistry,  and  pharmacology  of  three  different  models  of  long-term 
change  in  behavioral  responsivity .   We  are  examining:  1)  pharmacological  and 
electrophysiological  kindling,  2)  behavioral  sensitization  (the  increased 
behavioral  responsivity  to  psychomotor  stimulant  administration)  and  3)  the 
consequences  of  exposure  to  avoidable  and  unavoidable  stress  (learned 
helplessness) .   We  have  found  that  intracerebral  administration  of  corticotropin 
releasing  factor  (CRF)  induces  late  onset  of  seizures  that  are  behaviorally  and 
electrophysiologically  similar  to  those  produced  by  electrical  kindling  from  the 
amygdala.   CRF  seizures  cross-sensitize  to  amygdala  kindling  in  that  animals  that 
have  experienced  CRF  seizures  kindle  twice  as  fast  as  appropriate  controls. 
Direct  intracerebral  injection  of  CRF  into  amygdala,  hippocampus,  septum,  and 
hypothalamus  has  not  been  sufficient  to  reproduce  the  CRF  seizures  seen  following 
i.c.v.  administration.   Moreover,  lesions  of  amygdala,  hippocampus,  and  olfactory 
bulb  do  not  block  CRF  seizures.   Intracerebroventricular  administration  of  CRF 
also  produces  increases  in  irritable  and  aggressive  behavior  directed  at  other 
laboratory  animals.   As  such,  the  CRF  model  may  be  an  interesting  one  for 
exploring  the  potential  relationship  of  an  endogenously  produced  peptide  which  is 
released  under  stress  conditions  and  might  alter  convulsive  and  aggressive 
responsivity  in  the  rat. 

We  have  found  that  different  pharmacological  agents  are  effective  in 
inhibiting  the  development  of  kindling  as  opposed  to  completed  kindled  seizures. 
For  example,  while  carbamazepine  is  a  potent  anticonvulsant  inhibiting  completed 
kindled  seizures   in  the  rat,  it  is  not  effective  in  inhibiting  the  development 
of  kindling,  as  has  been  reported  in  cat  and  non-human  primate.   Conversely,  pre- 
liminary evidence  suggests  that  carbamazepine  may  inhibit  the  development  of 
lidocaine-induced  kindled  seizures,  but  is  not  effective  once  these  seizures  have 
been  fully  developed. 


20 


ANNUAL  REPORT  OF  THE  CLINICAL  NEUROGENETICS  BRANCH 

National  Institutes  of  Mental  H''  "Ith 

October  1,  1984  -  September  30,  1985 

Elliot  S.  Gershon,  M.D.,  Chief 


The  Clinical  Neurogenetics  Branch  was  officially  formed  last  year,  by 
moving  the  Section  on  Psychogenetics  from  the  Biological  Psychiatry 
Branch,  renaming  it  the  Section  on  Clinical  Genetics,  and  creating  a  new 
Section  on  Biochemical  Genetics,  headed  by  Dr.  Carl  R.  Merril,  who  was 
previously  in  the  Laboratory  of  General  and  Comparative  Biochemistry. 
This  is  the  first  annual  report  of  the  new  branch. 

The  thrust  of  this  branch  is  to  combine  the  most  advanced  disciplines 
within  genetics,  including  molecular  genetics  and  population  genetics, 
with  careful  biological  and  diagnostic  investigation  of  specific  psychiatric 
and  neurologic  disorders.   The  Branch's  investigations  can  be  subsumed 
under  four  investigative  areas.   In  several  of  these  areas,  there  is 
close  integration  and  mutually  reinforcing  complementarity  to  the  efforts 
of  the  two  sections,  so  this  overview  is  not  divided  by  sections.   These 
areas,  and  pertinent  recent  results  from  our  studies,  are  outlined  here. 

1.  Human  gene  mapping.   It  has  been  repeatedly  predicted  that  by  the  end 
of  this  century,  the  entire  human  genome  will  be  mapped  by  linkage  markers. 
These  markers  must  be  identified  one  by  one;  CNG  investigators  have  been 
responsible  for  identification  of  27  new  gene  polymorphisms  by  high 
resolution  2-dimensional  electorphoresis  (2DE)  of  proteins,  and  three  new 
gene  polymorphisms  at  the  DNA  level,  as  restriction  fragment  length  poly- 
morphisms.  Linkage  to  known  markers  has  been  established  for  several  of 
these.   These  and  other  linkage  markers  are  also  tested  for  linkage  to 
several  neuropsych iatric  diseases.   Mapping  of  any  neuropsychiatric 
illness  to  a  single  gene  region  would  constitute  a  major  scientific 
advance. 

2.  Peptides  and  proteins  important  in  the  CNS.   These  investigations  are 

a  necessary  underpinning  of  clinical  studies.   One  of  our  newly  discovered 
polymorphisms  is  for  the  structural  gene  of  neuropeptide  Y,  which  allows 
clinical  investigations  of  this  substance  not  otherwise  possible. 
Peptide  receptor-like  characteristics  have  been  identified  on  adult  skin 
fibroblasts,  for  vasoactive  intestinal  peptide  and  somatostatin. 
Radioimmunoassay  of  a  series  of  CSF  peptides  has  revealed  a  group  of 
peptides  which  appear  to  have  a  common  physiologic  mechanism  controlling 
their  CSF  concentration,   A  map  of  some  300  CSF  proteins  has  been  established 
using  2DE.   Several  of  these  are  polymorphic;  other  proteins  may  be 
produced  by  infectious  CNS  disease. 

3.  Disease  related  physiologic  events.   In  2DE  studies  of  CSF,  two  disease 
associated  proteins  were  found  in  Creutzfeldt-Jakob  disease,  and  two 
other  proteins  were  found  in  several  infectious  CNS  disorders,  and  schizo- 
phrenia, but  not  in  manic-depressives  or  normal  controls.   In  schizophrenia, 
study  of  well  and  ill  sibs  of  schzophrenics  revealed  that  ventricular 
enlargement  is  found  in  ill  but  not  well  sibs,  which  is  consistent  with  a 
genetic  vulnerability  risk  factor.   In  bipolar  affective  disorder,  increased 


21 


sensitivity  to  rapid-eye  movement  sleep  induction  by  infusion  of  a  cholinergic 
agonist  drug  is  being  studied  for  possible  use  as  a  genetic  vulnerability 
indicator.   New  mathematical  models  of  quantitative  characteristics  were 
applied  to  catecholamine  enzyme  activity  data,  revealing  polygenic  components 
to  enzyme  activities  where  single  gene  control  had  already  been  demonstrated. 

4.  Diagnostic  family  studies.   Controlled  family  studies  are  the  basis  of 
clinical  genetic  investigation.   A  novel  use  of  family  studies,  to  validate 
diagnostic  criteria,  has  demonstrated  a  clinical  definition  of  major 
depression  associated  with  familial  illness.   Currently,  family  studies  of 
schizophrenia,  rapid  cycling  manic-depressive  illness,  and  bulimia  are 
ongoing . 

This  brief  overview  demonstrates,  I  think,  the  particular  strength  of  the 
interdisciplinary  approaches  taken  in  this  branch,  and  the  remarkable 
vitality  and  cross-fertilization  of  the  collaborations  within  the  branch. 


SECTION  ON  CLINICAL  GENETICS 
Elliot  S.  Gershon,  M.D.,  Chief 

The  Section  on  Clinical  Genetics  has  as  its  goals:  identification  of 
biological  and  psychological  factors  that  are  genetically  transmitted  in 
major  pyschiatric  disorders,  investigation  of  genes  active  in  the  central 
nervous  system,  and  pharmacologic  and  pharmacogenet ic  studies  of  psychoactive 
agents  in  psychiatric  patients  and  in  normal  controls. 

The  clinical  basis  for  these  investigations  is  systematic  diagnostic 
study  of  patients,  normals,  and  relatives  of  each,  along  with  collection 
of  physiologic  information  and  biologic  specimens  from  them.   These 
specimens  include  most  notably  the  cell  collections  that  have  been 
developed  for  schizophrenic  and  manic-depressive  pedigrees,  with  appropriate 
controls,  and  cerebrospinal  fluid  (CSF)  which  has  been  studied  in  a  large 
series  of  drug-free  patients  and  controls.   Family  diagnostic  studies  of 
schizophrenia,  bulimia,  and  of  rapid-cycling  manic-depressive  illness  are 
currently  in  progress.   A  high-risk  study  of  offspring  of  bipolar  patients 
is  ongoing,  where  people  are  studied  at  an  age  (15-21)  when  mood  disorder 
is  most  likely  to  develop. 

Several  lines  of  investigation  are  pertinent  to  genetics  and  clinical 
neuroscience  in  general,  rather  than  to  specific  diagnostic  entities. 

Genetic  polymorphism  (variation  between  individuals)  can  be  used  to  detect 
chromosomal  linkage  to  illness  or  to  physiologic  functions.   Using  pedigree 
methods,  a  chromosomal  region  adjacent  to  the  polymorphic  site  can  be  studied, 
In  recent  years,  the  method  of  restriction  fragment  polymorphism  (RFLP) 
identification,  using  Southern  blots  of  genomic  DNA,  has  vastly  increased 
the  number  of  polymorphic  markers  in  the  human  genome. 

Several  neuropeptides  are  being  examined  in  our  molecular  genetics  laboratory, 
The  somatostatin  structural  gene  was  demonstrated  to  be  not  linked  to 
manic-depressive  illness,  by  use  of  previously  reported  polymorphisms.   A 
novel  RFLP  was  discovered  using  a  cDNA  probe  for  neuropeptide  Y;  the  less 


22 


frequent  allele  was  found  only  in  several  of  the  manic-denress ives  and 
schizophrenics  studied,  and  not  at  all  in  normals.   Mammalian  calmodulin 
cDNA  clones  were  isolated  from  a  rat  brain  cDNA  library. 

Other  chromosome  regions  of  interest  in  neuropsychiatry  were  examined. 
The  insulin  region  of  chromosome  11  is  being  studied  in  manic-depressive 
pedigrees.   A  novel  RFLP  was  discovered  for  the  complement  gene  C4,  which 
is  part  of  the  HIA  region  on  chromosome  6. 

Mathematical  analyses  of  transmission  and  linkage  in  pedigrees  have 
confirmed  2  new  polymorphisms  on  high  resolution  two-dimensional  electro- 
phoresis (2DE),  and  demonstrated  2  new  linkages  of  2DE  polymorphisms  to 
known  markers.   Other  analytic  methods  assigned  a  mixed  s ingle-locus/poly- 
genic  inheritance  mode  to  erythrocyte  catechol-0-methyltransf erase  and 
plasma  dopamine  beta-hydroxylase  activities;  previously,  only  the  single 
locus  component  to  their  inheritances  was  known. 

Receptor-like  characteristics  observed  on  adult  skin  fibroblasts  include 
specific  and  saturable  binding  of  vasoactive  intestinal  peptide  (VIP),  as 
well  as  VIP  stimulated  release  of  arachadonic  acid.   Somatostatin  ( SRIF) 
shows  specific  binding,  as  well  as  inhibition  of  cGMP  production  in  these 
cells.   Muscarinic  receptor-like  characteristics,  at  this  point,  are 
confined  to  antagonist  binding. 

Investigative  programs  in  schizophrenia  and  affective  disorders  focus  on 
vulnerability  factors,  particularly  heritable  factors,  in  these  disorders. 

In  families  with  more  than  one  sibling  ill  with  schizophrenia,  computed 
tomography  was  performed  on  ill  and  well  siblings.   Ventricular  size  was 
found  to  have  a  familial  component,  and  also  to  be  significantly  larger 
in  the  schizophrenic  siblings  compared  with  their  well  siblings  and  with 
controls.   This  is  compatible  with  increased  ventricular  size  as  a  genetic 
vulnerability  factor,  but  secondary  effects  of  illness  or  treatment  on 
ventricular  size  are  not  ruled  out.   No  ventricular  enlargement  was  seen 
in  a  series  of  affective  disorders  patients. 

In  our  continuing  clinical  CSF  research,  we  have  completed  studies  of 
monoamines  and  their  metabolites,  GABA,  and  13  peptides  in  35  normal 
volunteers  and  25  lithium-treated  euthymic  bipolars  (15  of  whom  also 
provided  samples  in  the  unmedicated  state).   Strong  correlations  were 
found  among  seven  neuropeptides  studied  in  these  subjects:   CRF,  VIP, 
N-POMC  (N-Terminal  fragment  of  pro-opiomelanocortin) ,  ACTH,  B-endorphin, 
B-lipotropin  and  somatostatin.   CSF  levels  of  neuropeptide  Y  and  alpha-MSH 
were  determined  in  anorexic  and  bulimic  patients  and  normal  volunteers. 
No  group  differences  were  found. 

Pharmacogenetic  and  pharmacologic  challenge  studies  are  focusing  on 
responses  that  have  discriminated  affective  disorders  patients  from 
controls  in  our  initial  studies.   Arecoline  induction  of  rapid  eye  movement 
(REM)  sleep  is  a  test  of  sensitivity  of  a  physiological  system  mediated  by 
muscarinic  cholinergic  receptors.   Approximately  60%  of  patients  and  33% 
of  controls,  in  a  new  series,  show  increased  sensitivity  to  arecoline.   If 
our  earlier  finding  of  a  patient-control  difference  is  confirmed,  the 


23 


same  test  will  be  applied  in  our  high-risk  offspring  study. 

SECTION  ON  BIOCHEMICAL  GENETICS 
Carl  R.  Merril,  M.D.,  Chief 

This  section  conducts  research  on  gene  and  gene  product  variations  and 
their  possible  relation  to  the  etiology  of  inherited  disorders.   The 
section  introduced  the  use  of  silver  staining  for  the  visualization  of 
protein  gene  products  separated  from  body  fluids  and  tissues  by  electro- 
phoresis on  polyacry lamide  gels.   These  silver  stains  have  increased 
the  sensitivity  of  detection  of  proteins  100-fold  over  previous  methods. 
Recent  stain  developments  by  the  section  have  resulted  in  rapid  protein 
stains,  less  than  10  minutes,  for  proteins  separated  on  polyacry lamide  gels 
and  nitrocellulose  membranes.   The  section  has  also  participated  in  the 
introduction  of  computerized  quantitative  dens itome trie  analysis  of 
silver-stained  and/or  autoradiographic  images  of  proteins  separated  by 
two  dimensional  gel  electrophoresis. 

The  development  of  these  methodologies  has  permitted  the  examination 
of  protein  gene  products  in  tissues  from  normal  and  diseased  individuals. 
These  studies  have  revealed  both  protein  variations  which  are  inherited 
in  a  mendelian  manner,  polymorphic  proteins,  and  disease-associated 
protein  alterations.   Twenty-seven  independent  polymorphic  protein  loci 
have  been  detected  up  to  now  by  two-dimensional  e lectrophoretic  studies 
of  serum,  erythrocyte,  and  fibroblast  proteins  from  individuals  in  two 
large  pedigrees.   These  protein  variants  were  detected  by  their  altered 
charge  on  2DE  e lect rophoretograms .   The  genetic  basis  of  these  protein 
variants  was  corroborated  by  their  mendelian  inheritance  patterns  in  the 
families  studied  and  by  their  quantitative  gene  dosage  dependence.   These 
protein  polymorphisms  should  prove  useful  in  initial  genetic  screening  of 
families  with  a  known  genetic  disease,  for  the  establishment  of  a  linkage 
to  a  disease  trait  locus. 

Two  disease-associated  proteins  (apparent  pi  5.1  and  5.2,  and  Mr  of 
29kd  and  26kd,  respectively)  have  been  found  in  the  cerebrospinal  fluid 
of  21  patients  (100%)  with  Creutz fe Idt -Jakob  disease  ( CJD) .   Of  18  other 
CNS  diseases  studied  only  spinal  fluid  from  herpes  simplex  encephalitis 
(HSE)  patients  (50%)  displayed  similar  proteins.   Two  other  disease 
associated  proteins  (apparent  pi  5.7  and  5.9,  with  a  Mr  of  40kd  for  both 
proteins)  have  been  observed  in  the  spinal  fluids  of:   CJD  patients  (67%), 
HSE  patients  (32%),  multiple  sclerosis  (13%),  Parkinson's  Disease  (12%),  and 
schizophrenia  (32%),  but  not  in  CSF  of  manic  depressives  or  normals. 
These  proteins  have  not  been  observed  in  CSF  from  manic  depressives  or 
normals.   These  proteins  may  be  of  some  aid  in  the  differential  diagnosis 
of  CNS  disease  and  they  may  also  provide  a  clue  for  the  understanding  of 
the  pathophysiology  of  these  diseases.   The  section  is  currently  purifying 
these  proteins  for  amino  acid  sequence  determination. 

The  section  has  also  begun  to  initiate  an  effort  to  study  the  smallest 
known  genome  in  the  human,  the  mitochondrial  genome.   This  genome  is  only 
I6.5kb  in  length,  it  is  thought  to  be  monoclonal  in  each  individual,  and 
inherited  maternally.   Given  the  importance  of  mitochondrial  metabolism 
for  normal  brain  function,  genetic  variations  in  this  genome  may  play  a 


24 


role  Ln  genetic  diseases  affecting  the  CNS. 

Studies  of  the  mitochondrial  genome,  in  human  brain  tissues  have 
revealed  that  most  of  the  mitochondrial  DNA  is  in  an  oligomeric  form. 
These  oligomers  appear  to  represent  dimeric,  trimeric,  and  tetrameric 
forms  of  the  mitochondrial  DNA.   Previous  studies  of  the  mitochondrial 
genome  in  human  tissues  failed  to  reveal  the  presence  of  a  predominance 
of  oligomeric  forms,  however  these  previous  studies  did  not  involve  human 
postmitotic  tissues,  such  as  the  brain  tissues  examined  in  the  current 
inve  s tig at  ions. 

Lack  of  mtDNA  repair  mechanisms  may  account  for  the  relatively  high 
mutation  rate  found  in  this  genome  when  it  is  comparied  to  the  nuclear  or 
chromosomal  genome.   A  survey  of  the  clinical  literature  demonstrated  a 
number  of  diseases  that  display  non-mendelian  maternal  inheritance  patterns 
that  may  involve  the  mitochondrial  genome.   Studies  of  the  primary 
structures  of  mitochondrial  genomes  from  normal  individuals  and  patients 
with  maternally  inherited  diseases  have  been  initiated.   These  studies 
are  being  performed  in  collaboration  with  the  FBI  Forensic  Science  Research 
Group  to  determine  whether  the  genetically  inherited  variations  in  the 
human  mitochondrial  genome  are  sufficient  to  establish  individuality  in 
forensic  studies. 


25 


Annual  Report  of  the  Clinical  Psychobiology  Branch 

National  Institute  of  Mental  Health 

Thomas  A.  Wehr,  M.D.,  Chief 

October  1,  1984  through  September  30,  1985 

The  Clinical  Psychobiology  Branch  conducts  clinical  and  basic  research 
related  to  affective  disorders  (depression  and  manic-depressive  illness)  and 
sleep  disorders.  The  clinical  research  program  focusses  on  the  role  of 
biological  rhythms  in  depression.  The  inherent  cyclicity  of  affective  illness, 
one  of  its  most  characteristic  features,  has  been  largely  neglected  in 
theories  about  its  causes  and  pathophysiology,  and  has  seldom  been  the 
object  of  clinical  investigation.  The  oscillations  of  various  types  of 
normal  biological  rhythms  are  of  interest  because  they  are  capable  of 
triggering  relapses  and  remissions  in  various  groups  of  patients.  For 
example  depressions  may  recur  annually  in  the  winter  or  menstrually  in 
the  luteal  phase.  Symptoms  of  depression  also  often  exhibit  daily  cycles 
of  intensification  in  the  morning.  Manic-depressive  cycles  spanning 
several  weeks  occur  and  may  have  their  counterpart  in  normal  human 
physiology.  In  effect,  certain  phases  of  normal  human  biological  rhythms 
constitute  risk  factors  that  predispose  to  the  development  of  affective 
episodes.  These  relationships  provide  clues  to  the  causes  of  depression. 
For  example  biological  changes  common  to  winter,  the  premenstrual  period 
and  morning  may  be  more  likely  to  be  triggering  factors  than  changes  that 
do  not  exhibit  these  patterns.  There  are  methodological  advantages  to 
studying  these  cyclic  influences  on  depression,  since  clinical  changes  are 
predictable  and  can  be  studied  prospectively. 

Experimental  manipulations  of  biological  rhythms  are  also  of  interest 
because  they  are  capable  of  altering  the  clinical  state  of  depressives. 
Alterations  in  the  timing  and  duration  of  the  sleep  phase  of  the  daily 
sleep-wake  cycle  can  induce  rapid  remissions  in  depression,  and 
alterations  of  the  light-dark  cycle,  known  to  affect  daily  and  seasonal 
rhythms  in  animals,  are  capable  of  inducing  rapid  remissions  in  a  seasonal 
form  of  affective  disorder.  The  discovery  of  effective  pharmacological 
treatments  for  depression  spawned  a  generation  of  psychobiological 
investigations  of  depression  based  on  the  assumption  that  increasing 
knowledge  of  the  biological  effects  of  these  drugs  would  lead  to  an 
understanding  of  the  causes  of  depression  and  new  more  specific  treatments 
of  depression.  In  contrast  to  drugs,  there  has  been  little  effort  to 
understand  the  mechanisms  of  these  two  non-pharmacological  treatments; 
yet,  there  are  certain  methodological  advantages  to  studying  them. 
Remissions  induced  by  light  and  sleep  perturbations  are  rapid  and  rapidly 
reversible  (drugs  require  2-4  weeks  to  act)  and  are  therefore  highly 
suitable  for  intensive  longitudinal  investigation.  It  is  the  aim  of  this 
branch  to  identify  mechanisms  whereby  spontaneous  oscillations  of  biological 
rhythms  and  perturbations  of  biological  rhythms  induce  clinical  changes 
in  depressed  patients.  In  this  way  we  hope  to  increase  understanding  of 
the  causes  of  depression  and  develop  novel  forms  of  treatment. 


27 


Seasonal ity  Study 
Norman  E.  Rosenthal,  M.D. 

In  previous  years  we  have  described  the  syndrome  of  seasonal  affective 
disorder  (SAD),  a  condition  characterized  by  recurrent  winter  depressions 
alternating  with  periods  of  euthymia  or  hypomania  in  the  spring  and 
summer.  We  have  shown  that  this  condition  responds  to  treatment  with 
bright  (2500  lux)  full-spectrum  light  administered  for  a  total  of  five  to  six 
hours  per  day  either  in  the  morning  and  evening  hours  or  in  the  evening 
hours  alone,  whereas  light  of  ordinary  room  light  intensity,  administered 
at  the  same  times  to  the  same  patients,  is  ineffective.  We  have  previously 
speculated  about  the  mechanism  of  the  development  of  symptoms  in  the 
winter  months  and  of  their  response  to  phototherapy.  The  chemical, 
melatonin,  secreted  by  the  pineal  gland,  has  appeared  to  be  an  attractive 
candidate  as  an  important  neurochemical  mediator  in  the  above  processes 
for  the  following  reasons:  1.  Melatonin  secretion  has  been  shown  to  be  an 
important  neurochemical  mediator  in  a  variety  of  seasonal  rhythms  in 
animals,  which  have  also  been  shown  to  be  influenced  by  daylength 
(photoperiod).  2.  Melatonin  secretion  in  humans  can  be  suppressed  by 
high  intensity  (2500  lux)  light  but  not  by  light  of  ordinary  room  light 
intensity.  For  these  reasons  we  began  to  investigate  the  role  of  melatonin 
in  SAD  in  1984  by  administering  the  substance  orally  to  eight 
patients  with  SAD,  who  had  been  previously  treated  with  phototherapy.  We 
found  that  melatonin  reinduced  some  but  not  all  the  symptoms  of  SAD, 
which  had  responded  to  phototherapy.  This  equivocal  result  left  unresolved 
the  importance  of  melatonin  secretion  in  the  pathophysiology  of  SAD  and 
the  role  of  its  suppression  in  the  response  to  phototherapy. 

This  year  we  added  to  our  knowledge  of  SAD  in  several  ways.  First, 
we  extended  the  description  of  the  syndrome  and  its  response  to  environmental 
light  modifications  in  children  and  adolescents.  Second,  we  learned  more 
about  the  influence  of  the  timing  of  light  administration  on  the  effects  of 
phototherapy.  Finally,  our  studies  over  the  past  year  have  expanded  our 
understanding  of  the  possible  role  of  melatonin  in  the  condition  and  its 
treatment  with  light. 

We  asked  parents  to  rank  order  the  prominence  of  symptoms  in  their 
children  with  SAD  and  found  that  the  commonest  symptoms,  in  order  of 
frequency,  were  irritability,  fatigue,  school  difficulties,  sadness, 
sleep  changes,  decreased  activity,  carbohydrate  craving,  crying  spells, 
anxiety,  social  withdrawal  and  temper  tantrums.  Six  of  the  seven  patients 
were  treated  with  supplementary  bright  full-spectrum  light  in  the  evening 
hours  and,  in  an  uncontrolled  study,  five  of  these  patients  showed  marked 
improvement  in  response  to  treatment.   It  is  important  for  counsellors 
and  therapists  to  recognize  SAD  as  one  of  the  differential  diagnoses  of 
school  difficulties  in  the  fall-winter  semester,  especially  since  the 
condition  is  easily  treatable.  Controlled  trials  of  phototherapy  of 
children  and  adolescents  are  underway  in  collaboration  with  Dr.  William 
Sonis  of  the  Child  Psychiatry  Department  of  the  University  of  Minnesota. 

Two  studies  involved  the  timing  of  light  in  the  treatment  of  patients 
with  SAD.  The  first  study  was  conducted  in  collaboration  with  Dr.  Carla 
Hellekson  in  Fairbanks,  Alaska.  Dr.  Hellekson  treated  6  patients  with 
SAD  with  2  hours  of  bright  light  per  day  for  3  1-week  periods,  separated 

28 


by  2  1-week  withdrawal  periods.  The  light  was  either  given  in  the  morning, 

in  the  evening,  or  divided  between  morning  and  evening  hours.  All  three 

treatment  schedules  were  found  to  be  equally  effective,  suggesting  that 
timing  of  treatment  is  not  critical. 

The  second  light  treatment  study  involved  7  patients  with  SAD,  who 
were  treated  as  inpatients  with  2  1-week  light  schedules,  separated  by  10 
days  of  withdrawal.  Both  schedules  consisted  of  2  3-hour  periods  of 
bright  full-spectrum  light  separated  by  dim  light.  In  the  one  case  the  dim 
light  interval  was  2  hours  (short  skeleton  photoperiod)  and  in  the  other 
the  dim  interval  was  9  hours  (long  skeleton  photoperiod).  In  animal 
studies  the  long  skeleton  photoperiod  has  been  shown  to  produce  summer-type 
behavior  by  means  of  its  effects  on  melatonin.  In  the  short  skeleton 
photoperiod  the  light  exposure  does  not  occur  at  a  time  when  melatonin  is 
being  secreted  and  does  not  produce  summer- type  behavior.  We  thus 
predicted,  based  on  the  animal  literature,  that  the  long  photoperiod 
would  be  an  effective  treatment  for  SAD  whereas  the  short  one  would  not. 
Contrary  to  our  expectations,  both  treatment  schedules  were  effective 
despite  the  fact  that  the  long  photoperiod  suppressed  the  urinary  secretion 
of  the  major  melatonin  metabolite,  6-hydroxymelatonin-sulphate  (6-OH-M-S) 
whereas  the  short  skeleton  photoperiod  did  not.  The  findings  of  this 
study  suggest  that  light  treatment  may  be  effective  even  if  given  during 
the  usual  daylight  hours  (i.e.  the  response  to  phototherapy  is  not 
photoperiodic)  and  that  suppression  of  the  melatonin  may  not  be  the 
mechanism  by  which  phototherapy  produces  its  effects. 

In  our  earlier  studies  we  showed,  in  4  groups  of  patients  with  SAD, 
that  bright  light  has  antidepressant  effects  whereas  ordinary  room  light 
does  not.  The  bright  light  can  suppress  human  melatonin  whereas  the 
ordinary  room  light  cannot.  This  background  information,  as  well  as  the 
widespread  importance  of  melatonin  secretion  as  a  mediator  of  seasonal 
rhythms  in  animals,  led  us  to  study  the  possible  role  of  melatonin  in 
the  pathogenesis  of  the  symptoms  of  SAD  and  of  melatonin  suppression 
in  the  antidepressant  effects  of  phototherapy,  from  a  variety  of  angles. 
We  drew  blood  from  7  SAD  patients  and  7  normal  controls  over  a  48  period 
both  in  summer  and  winter  and  the  samples  are  awaiting  analysis  for 
melatonin  levels.  We  challenged  melatonin  producing  ability  in  10  SAD 
patients  and  10  normal  controls  by  giving  them  a  melatonin  precursor,  5- 
hydroxytryptophan  (5HTP),  orally  and  drawing  blood  over  a  5  hour  period. 
This  strategy  has  resulted  in  melatonin  production  in  sheep  even  when 
5HTP  was  given  during  the  day.  The  results  of  this  study  also  await 
analysis  of  the  melatonin  levels. 

Last  year  we  administered  melatonin  orally  to  8  SAD  patients  who  had 
previously  been  treated  with  phototherapy.  We  predicted  that  melatonin 
would  reinduce  the  symptoms  of  SAD  but  it  did  so  only  to  a  modest  degree. 
This  year  we  treated  18  patients  with  the  beta-adrenergic  blocking  agent, 
atenolol,  in  a  counterbalanced,  double-blind  crossover  study.  Since 
melatonin  is  normally  secreted  as  a  result  of  stimulation  of  beta-adrenergic 
receptors  on  the  pineal  gland,  we  predicted  that  atenolol  would  reverse 
the  symptoms  of  SAD.  Contrary  to  our  expectations,  there  was  no 
statistically  significant  difference  between  the  effects  of  atenolol  and 
of  placebo  with  respect  to  antidepressant  response.  However,  about  5 
patients  appeared  to  do  extremely  well  on  atenolol,  relapsed  when  the 

29 


atenolol  was  discontinued  and  responded  once  again  when  it  was  reinstated. 
The  response  was  maintained  throughout  the  winter  months.  Atenolol  did, 
in  fact,  suppress  the  secretion  of  urinary  6-OH-M-S  significantly. 

In  summary,  the  bulk  of  the  evidence  suggests  that  melatonin  does 
not  play  a  major  role  in  the  pathogenesis  of  SAD  or  the  response  to 
phototherapy.  However,  there  is  a  suggestion  that  it  may  play  some  role  at 
least  in  certain  individuals.  While  the  "melatonin  hypothesis"  has  by  no 
means  been  laid  to  rest,  the  predominantly  negative  results  would  suggest 
that  the  pursuit  of  other  neurochemical  mediators  might  be  more  profitable 
at  this  stage. 

Circadian  Rhythms  and  Affective  Disorders 
David  A.  Sack,  M.D. 

The  possibility  that  circadian  rhythms  are  involved  in  the  pathophy- 
siology of  affective  disorders  was  suggested  by  the  cyclical  nature  of 
recurrences  seen  in  some  manic-depresi ve  patients  and  the  observation 
that  certain  core  depressive  symptoms  are   temporally  distributed  over  the 
course  of  the  day  (for  example,  during  sleep,  episodes  of  awakening  occur 
in  the  early  morning  and  depressed  mood  is  worse  in  the  morning  than  in  the 
evening).  In  addition,  when  normal  subjects  are  placed  in  conditions  where 
all  time  cues  have  been  eliminated,  they  exhibit  changes  in  their  sleep  and 
activity  which  are  similar  to  those  which  occur  in  manic  and  depressed 
patients.  These  naturalistic  observations  led  to  descriptive  investigations 
of  circadian  rhythms  in  patients  with  affective  disorders  and  to  the 
development  of  new  treatments  which  apply  principles  of  circadian  biology 
to  this  population. 

Our  present  research  is  guided  by  two  principle  observations.  First, 
patients  with  depression  show  certain  characteristic  changes  in  their  sleep, 
endocrine  and  neurotransmitters  which  have  been  interpreted  to  reflect  an 
alteration  in  the  underlying  circadian  regulatory  system.  Second,  manipulation 
of  circadian  rhythms  by  altering  the  timing,  amount  or  internal  architecture  of 
sleep  produces  a  marked  reduction  in  the  severity  of  symptoms  in  depressed 
patients.  Our  current  projects  will  better  characterize  the  circadian  system 
in  depression  and  normal  controls,  will  explore  the  therapeutic  mechanism 
of  action  and  clinical  application  of  sleep  deprivation  therapy  and  will 
relate  these  two  areas  to  other  known  aspects  of  the  pathophysiology  of 
depression. 

As  we  noted  in  earlier  reports,  measurements  of  circadian 
rhythms  are  distorted  by  a  variety  of  factors  called  masking.  We  have 
controlled  masking  by  studying  the  rhythms  of  patients  and  normal  controls 
in  conditions  where  sleep,  diet  and  physical  activity  have  been  held 
constant.  Thus  far  we  have  completed  circadian  studies  of  12  patients 
and  7  normal  controls  under  constant  conditions.  Although  much  of  this 
data  remains  to  be  analyzed  the  usefulness  of  this  method  has  already 
been  demonstrated  in  the  analysis  of  neurotransmitter  metabolites.   In 
our  study  both  MHPG  and  HVA  were  examined  every  2  hours  on  a  baseline 
day,  and  a  second  day  of  constant  conditions. 

At  baseline  both  HVA  and  MHPG  showed  significant  daily  variations 

30 


with  the  peak  in  HVA  occurring  close  to  midnight,  and  the  peak  forming  at 
n  p.m.  Constant  conditions  significantly  reduced  the  mean  concentrations 
of  HVA  and  MHPG  but  the  effects  on  the  two  metabolite  rhythms  were 
different.  With  HVA  morning  values  were  generally  lower  whereas  the 
nocturnal  rise  was  largely  uneffected.  The  circadian  fluctuation  in  MHPG 
seen  at  baseline  was  abolished  under  constant  conditions.  There  was  no 
significant  difference  between  patients  and  normals  in  MHPG  or  HVA  on 
either  day. 

Our  study  confirms  the  presence  of  a  circadian  rhythm  in  plasma  HVA  but 
suggests  that  a  previously  reported  rhythm  in  plasma  MHPG  was  probably  the 
result  of  behavioral ly  mediated  masking.  Physical,  diet  or  posture  appear  to 
increase  both  metabolites  above  their  basal  values. 

Another  focus  of  our  circadian  studies  has  been  the  regulation  of 
hormones  whose  secretion  may  be  abnormal  in  depression.  Abnormalities  in 
thyroid  function  have  frequently  been  observed  in  depressed  patients, 
specifically  a  blunted  thyrotropin  response  to  TRH,  an  elevated  TSH  in 
patients  treated  with  lithium,  and  an  antidepressant  response  to  triiodo- 
thyronine when  added  to  antidepressant  medications  for  TSH  in  normals.  A 
circadian  rhythm  has  been  described  which  peaks  late  in  the  evening  shortly 
before  sleep  and  is  partially  suppressed  by  sleep.  We  have  completed  our 
investigation  of  the  circadian  rhythm  of  TSH  in  nine  bipolar  rapid-cycling 
patients  and  9  age  and  sex-matched  controls.  We  have  confirmed  our  preliminary 
finding  that  the  nocturnal  rise  in  TSH  is  diminished  in  the  depressed  patients. 
Sleep  deprivation  increased  the  nocturnal  values  of  TSH  in  both  groups  but  this 
effect  was  significant  only  in  the  normal  controls.  After  sleep  deprivation 
TSH  values  in  the  patient  group  approximated  the  levels  seen  in  controls  at 
baseline.  Cortisol  samples  obtained  simultaneously  during  the  study  failed  to 
reveal  any  patient  normal  differences  effect  of  sleep  deprivation  on  the  Cortisol 
levels  were  minor. 

Thus  far  we  conclude  that  patients  with  rapid-cycling  manic-depressive 
illness  show  reduced  nocturnal  secretion  of  TSH  which  is  not  related  to  Cortisol 
and  is  at  least  partially  reversed  by  sleep  deprivation.  Future  studies  will 
focus  on  the  specificity  of  this  finding  to  this  diagnostic  group  and  will 
explore  the  possibility  that  the  augmentation  of  TSH  at  night  by  sleep 
deprivation  may  mediate  the  therapeutic  effects  of  this  intervention. 

TREATMENT  OF  DEPRESSION  BY  SLEEP  DEPRIVATION 

In  our  earlier  experiments  we  compared  the  therapeutic  effects  of  partial 
sleep  deprivation  occurring  early  in  the  night  (PSD-E)  with  partial  sleep 
deprivation  occurring  late  in  the  night  (PSD-L).  We  found  that  PSD-L  was 
significantly  more  effective  than  PSD-E  and  that  the  therapeutic  effects  of 
PSD  could  be  extended  by  repeating  the  treatment  for  a  second  night.  Improve- 
ment on  PSD-L  was  inversely  correlated  with  the  duration  of  sleep  on  this 
condition.  Our  study  suggested  that  the  timing  of  sleep  is  an  important 
factor  in  the  PSD  response  but  this  finding  was  confounded  by  the  fact  that 
patients  slept  significantly  less  on  the  PSD-L  condition. 

For  this  reason  we  have  initiated  a  replication  study  of  our  earlier 
PSD  experiment.  In  the  present  study,  sleep  times  will  be  precisely  regulated 
on  the  treatment  nights  to  ensure  equal  sleep  periods  on  the  two  PSD 

31 


conditions.  Since,  in  our  previous  study,  positive  responses  were  generally 
associated  with  sleep  times  of  less  than  4  hours  the  duration  of  sleep  on 
treatment  nights  has  been  correspondingly  reduced.  In  addition  we  have  added  a 
longitudinal  trial  of  PSD  in  which  subjects  will  be  treated  with  repeated 
nights  of  PSD  in  order  to  determine  whether  complete  remissions  can  be  achieved 
with  this  treatment.  Circadian,  sleep,  biochemical  and  neuroendocrine  predictors 
of  the  clinical  response  to  sleep  deprivation  will  be  measured  as  part  of  this 
investigation. 

We  continue  to  be  interested  in  the  possible  interactions  between 
circadian  and  pharmacologic  treatments.  We  have  previously  reported  a 
synergistic  interaction  between  phase  advance  of  the  sleep-wake  cycle  and 
other  antidepressant  therapies.  In  our  present  trial  we  will  assess  the 
prophylactic  effects  of  lithium  carbonate  in  patients  who  respond  to  PSD. 
Patients  will  be  randomly  assigned  to  lithium  therapy  or  placebo  and  their  mood 
will  be  observed  weekly  for  a  period  of  up  to  six  months. 

In  addition,  in  a  separate  trial,  patients  who  fail  to  respond  to 
treatment  with  tricyclic  depressants  or  MAOI's  will  be  treated  with  repeated 
nights  of  PSD.  These  studies  will  provide  insight  into  common  mechanisms  of 
action  as  well  as  explore  the  role  of  circadian  treatments  in  a  variety  of 
clinical  problems.  Because  sleep  deprivation  and  other  circadian  treatments 
work  more  quickly,  combining  them  with  conventional  treatments  may  reduce 
morbidity  and  mortality  associated  with  the  major  depressive  disorders. 

THEORETICAL  CONSIDERATIONS 

The  circadian  rhythm  disturbances  seen  in  affective  illness  could  arise 
from  abnormalities  in  (1)  the  mechanism  that  entrains  the  pacemaker  (s) 
to  zeitgebers  (2)  the  pacemaker  itself  (3)  coupling  between  pacemakers  or 
(4)  distal  to  the  pacemaker  in  the  expression  of  its  rhythm.  A  major  goal 
of  our  program  is  to  systematically  test  these  hypotheses  in  our  affective 
disorder  patients. 

An  abnormality  in  the  entrainment  process  could  result  in  rhythms 
becoming  desynchronized  from  one  another,  a  phenomenon  which  has  been 
observed  clinically  in  certain  rapid  cycling  manic-depressive  patients. 
An  abnormal  sensitivity  to  an  entraining  stimulus  could  also  alter  the 
relative  timing  of  two  circadian  oscillators.   In  most  animals,  including 
primates,  light  plays  an  important  role  in  synchronizing  circadian  rhythms. 
Recently  a  similar  role  for  light  has  been  demonstrated  in  humans.  Studies 
performed  in  our  laboratory  and  by  others  have  shown  that  manic-depressive 
patients  are  supersensitive  to  the  effects  of  light  on  the  secretion  of 
melatonin,  a  hormone  which  is  secreted  predominantly  at  night  and  whose 
secretion  is  suppressed  by  light.  This  supersensitivity  appears  to  be  a 
trait  marker  since  recovered  manic  depressives  continue  to  show  this 
abnormality.  It  is  possible  that  these  patients  are  also  supersensitive  to 
the  entraining  effects  of  light. 

Several  methodological  problems  limit  the  interpretation  of  these 
studies.  First,  no  attempt  was  made  to  control  for  the  prior  exposure 
to  light.  Secondly,  since  the  amount  of  light  received  during  the  study 
was  not  precisely  controlled,  differences  in  exposure  could  have  accounted 
for  some  of  the  differences  observed.  We  have  developed  a  ganzfeld  dome 

32 


which  will  enable  us  to  administer  to  subjects  lighting  of  uniform  intensity 
and  to  precisely  manipulate  the  lighting  source  with  respect  to  wavelength 
and  intensity. 

Preliminary  results  from  our  ganzfeld  dome  experiment  suggest  that  the 
threshold  for  light  effects  on  melatonin  is  significantly  lower  than  was  found 
in  previous,  less  well  controlled  studies.  Once  we  have  established  a 
normal  fluence  response  curve  for  light  suppression  of  melatonin,  we  will 
initiate  studies  with  depressed  patients  and  other  diagnostic  groups.  Our 
first  task  is  to  confirm  or  refute  previous  observations,  and  second,  to 
explore  the  neurochemical  and  neurophysiological  basis  for  differences  in  the 
sensitivity  to  light. 

Premenstrual  Syndrome 
Barbara  L.  Parry,  M.D. 

The  symptoms  of  premenstrual  syndrome  (PMS)  consist  of  mood,  cognitive,  and 
behavioral  disturbances  occurring  in  the  premenstrual  phase  of  the  menstrual 
cycle.  They  may  become  severe  enough  to  cause  suicidal  depression  or 
psychosis.  Objective  physiologic  parameters  that  correlate  with  the 
subjective  symptoms  of  PMS  need  to  be  identified  in  order  to  delineate 
this  syndrome  further  and  possibly  to  suggest  better  forms  of  treatment. 
First,  this  study  examined  sleep,  temperature  and  activity  changes  across 
the  menstrual  cycle  in  women  with  moderate  to  severe  premenstrual  syndrome 
and  in  normal  volunteers.  Two  months  of  baseline  activity  recording, 
objective  ratings  and  self-ratings  of  sleep,  mood,  and  energy  were  obtained. 
Subjects  were  then  admitted  to  the  hospital  where  they  underwent  sleep 
EEG  and  temperature  recordings  two  nights  a  week  for  the  duration  of  one 
menstrual  cycle.  Sleep  studies  showed  patients  had  more  Stage  II  sleep 
and  less  REM  sleep  than  normals;  and  that  stage  III  sleep  and  awake  time 
showed  variation  across  the  menstrual  cycle. 

Premenstrual  syndrome  may  represent  a  variant  of  affective  disorder. 
Therefore,  treatment  modalities  found  to  be  effective  in  the  major  affective 
disorders  may  be  useful  in  treating  patients  with  PMS.  For  example,  sleep 
deprivation  which  induces  transient  remissions  in  affective  disorder  may  do  the 
same  in  PMS.  Furthermore,  sleep  deprivation  lowers  prolactin,  and  hyperpro- 
lactinemia  has  been  associated  with  mood  disturbances  in  patients  with  PMS. 
Therefore,  the  effects  of  sleep  deprivation  were  investigated  in  these 
patients.  Prolonged  intense  light  exposure  alleviates  symptoms  in  patients 
with  seasonal  affective  disorder.  Since  symptoms  of  SAD  and  PMS  are  similar, 
prolonged  intense  light  exposure  or  treatment  with  the  beta  blocking  drug, 
atenolol,  which,  like  bright  light,  suppresses  melatonin  was  evaluated  as 
a  possible  treatment  for  PMS.  Phototherapy  and  atenolol  were  effective  in 
one  seasonal  PMS  patient;  these  interventions  were  not  effective  in  treating 
nonseasonal  PMS  patients.  Sleep  deprivation  was  effective  in  eight  of  ten 
unmedicated  PMS  patients  who  underwent  total  sleep  deprivation.  Some  of  the 
patients  who  responded  to  total  sleep  deprivation  also  responded  to  partial 
sleep  deprivation  in  the  second,  but  not  first  half  of  the  night. 

Results  of  sleep  deprivation  and  light  treatment  experiments  may  increase 
our  understanding  of  the  pathophysiological  mechanisms  of  PMS  and  the  relation- 
ship between  PMS  and  affective  disorders. 

33 


Sleep  Studies 

Wallace  B.  Mendelson,  M.D. 

The  Section  on  Sleep  Studies  carries  out  work  of  three  general  types: 

1.  support  for  Branch  projects,  of  which  sleep  studies  are   one  part; 

2.  clinical  studies  initiated  by  the  Sleep  Lab,  which  this  year  have 
focused  on  various  aspects  of  chronic  insomnia;  and 

3.  studies  in  the  basic  science  sleep  laboratory  which  are  primarily 
concerned  with  the  mechanism  of  action  of  hypnotics. 

Support  of  other  projects:  these  studies  are  considered  under  the  sections 
of  the  annual  report  written  by  the  prinicipal  investigators.  Among  these  are 
an  extensive  series  of  recordings  of  the  sleep  of  premenstrual  syndrome  patients 
throughout  the  menstrual  cycle,  as  part  of  a  larger  project  by  Dr.  Barbara 
Parry.  Dr.  David  Sack  has  completed  a  study  of  partial  sleep  deprivation  which 
involved  sleep  recordings.   It  was  found  that  sleep  deprivation  during  the 
second  half  of  the  night  was  an  effective  treatment  for  affective  disorder. 
Sleep  recordings  from  this  study  indicated  that  among  those  patients  who 
responded  there  was  a  significant  inverse  correlation  between  REM  sleep  and 
total  sleep  time  on  the  one  hand  and  clinical  improvement  on  the  other. 
Currently  the  sleep  lab  is  supporting  a  follow-up  study  of  partial  sleep 
deprivation.  The  section  has  also  provided  support  to  Dr.  John  Nurnberger's 
study  of  arecoline  infusions  in  affective  disorder  patients,  studies  of 
carbamazepine  conducted  by  Dr.  Robert  Post,  and  verapamil  administration  to 
schizophrenics  performed  by  Dr.  David  Picar. 

Clinical  studies  of  insomnia:  this  work  has  centered  on  a  special  group 
of  people  with  sleep  d1sorder--individual s  with  a  chronic  complaint  of  poor 
sleep,  in  whom  none  of  the  major  physiologic  disturbances  of  sleep  (e.g.,  sleep 
apnea)  can  be  found.  In  an  earlier  study  there  were  two  major  findings:  the 
first  is  that  although  insomniacs  describe  themselves  as  chronically  sleepy  and 
fatigued  in  contrast  to  controls,  reports  of  their  feelings  at  any  given  moment 
are  similar  to  normal  subjects.  The  second  finding  was  that  although  they  had 
good  ability  to  learn  new  information  (episodic  memory),  they  were  \jery   impaired 
in  their  ability  to  retrieve  and  use  information  already  well  known  (semantic 
memory).  More  recent  work  has  centered  on  exploring  possible  cognitive  deficits 
in  insomniacs  both  when  awake  and  asleep.  Among  the  more  recent  findings  are 
the  observations  that  after  an  experimental  awakening  both  insomniacs  and 
controls  go  back  to  sleep  about  as  soon  as  each  other  and  sleep  an  equal  amount 
afterwards;  the  insomniacs,  however,  retrospectively  estimated  that  their  sleep 
was  only  half  as  long  as  did  the  controls.   It  was  also  observed  that  when 
awakened  from  the  same  EEG-defined  sleep  stage  (eg,  10  minutes  after  the  onset 
of  stage  2),  the  controls  tended  to  report  that  they  had  been  asleep  while  the 
insomniacs  tended  to  say  that  they  had  been  awake.  These  findings  seem  to 
suggest  that  the  experience  of  chronic  insomnia  may  involve  both  cognitive 
deficits  and  a  difficulty  in  accurately  determining  one's  own  state  of 
consciousness.  This  raises  the  possibility  that  cognitive  alterations  known 
to  be  induced  by  most  hypnotics  may  not  be  'side  effects',  but  rather  may  be 
crucial  to  the  way  these  compounds  give  relief  to  insomniacs. 

Basic  science  studies:  the  focus  in  the  animal  laboratory  has  been  on  the 
mechanism  of  action  of  hypnotics.   In  the  past  our  work  has  shown  that  the 
benzodiazepine  (BZ)  receptor  complex  appears  to  mediate  the  sleep-inducing 

34 


properties  of  BZ  hypnotics.  Last  year  we  showed  that  pre-treatment  with  the 
calcium  blocker  nifedipine  given  intraventricularly  will  prevent  the  hypnotic 
action  of  flurazepam.  This  seems  to  suggest  that  the  hypnotic  properties  of  BZ 
involve  alterations  in  calcium  flux.  This  year  we  tested  this  proposition 
further  by  giving  BAY  K  8644,  a  compound  which  enhances  calcium  flow,  and  showed 
that  it  potentiates  the  sleep-inducing  properties  of  flurazepam.  We  have  also 
examined  the  possible  functional  significance  of  the  division  of  BZ  receptors 
into  types  I  and  II,  by  giving  the  type  I  agonist  CL  218,  972,  alone  and  in 
combination  with  flurazepam.  It  as  found  that  the  CL  compound  had  a  potent 
effect  in  increasing  total  sleep  time,  suggesting  that  this  property  is  mediated 
by  type  I  receptors.  Future  work  will  involve  the  interaction  of  CL  with 
calcium  channel  blockers  and  agonists. 

The  laboratory  has  also  continued  searching  for  agents  which  might  prevent 
toxicity  of  large  doses  of  barbiturates.  Last  year  we  showed  that  the  'cage 
convulsant'  compound  IPPO  could  significantly  reduce  mortality  from  overdoses 
of  barbiturates  in  mice,  although  it  had  significant  toxicity  of  its  own.  This 
year  we  have  found  that  a  related  compound,  TBPS,  is  just  as  effective  in 
reducing  the  number  of  deaths,  but  is  much  less  toxic.  Currently,  we  are 
beginning  studies  of  adenosine-related  compounds.  The  general  theme  of  this 
work  is  to  apply  knowledge  of  the  BZ  receptor  complex  gained  from  in  vitro  work 
to  issues  regarding  the  pharmacological  properties  of  hypnotics. 

Biology  of  Melatonin 
Lawrence  Tamarkin 

Melatonin,  a  hormone  secreted  by  the  pineal  gland,  regulates  seasonal 
responses  in  a  variety  of  species.  From  a  variety  of  animal  studies,  a  consensus 
has  now  emerged  that  seasonal  changes  in  daylength  are  transduced  into  a  change 
in  the  duration  of  the  nocturnal  secretion  of  melatonin,  suggesting  that  the 
daily  rhythm  in  plasma  melatonin  conveys  its  information  to  specific  target 
sites  through  a  change  in  the  duration  of  its  nocturnal  secretory  phase  rather 
than  its  amplitude.  The  unraveling  of  how  this  signal  is  processed  intracellularly 
will  provide  us  with  new  insights  into  hormone  action. 

Unfortunately,  the  slowness  of  seasonal  responses  to  melatonin  makes  them 
unsuitable  for  intracellular  analysis  of  mechanism  of  action.  We  have  sought  a 
variety  of  rapid  physiologic  responses  to  melatonin,  and  have  successfully  found 
two  systems  which  will  serve  our  purpose.  One  system  we  have  used  is  an  in  vivo 
response  of  the  hamster  uterus  to  exogenous  melatonin.  Forty  minutes  after  a 
single  injection  of  melatonin  estrogen  uptake  and  estrogen  receptors  in  juvenile 
hamster  uteri  increase.  Curiously,  in  our  most  recent  studies  the  exact  opposite 
response  occurs  in  ovariectomized  adults:  melatonin  acutely  reduces  estrogen 
uptake  and  reduces  the  concentration  of  estrogen  receptors.  This  inhibition  of 
estrogen  action  by  melatonin  was  demonstrated  in  an  in  vivo  uterine  growth  study 
in  which  we  demonstrated  a  significant  inhibition  of  estrogen-stimulated  uterine 
growth  by  melatonin.  Thus,  melatonin's  action  may  vary  as  a  function  of  other 
endocrine  parameters,  such  as  those  associated  with  puberty  and  tumori genesis. 

The  second  system  we  have  investigated  is  based  on  melatonin's  effect  on  a 
hormone  dependent  human  breast  cancer  cell  line.  Melatonin  caused  an  increase 
in  estrogen  receptor  concentration  within  40  minutes  in  these  cells.  We  are 
now  determining  the  effect  of  melatonin  on  cell  growth  using  cells  growing  in 

35 


culture  and  cells  injected  into  athymic  nude  mice.  Preliminary  data  indicate 
that  melatonin  enhances  hormone  stimulated  cell  growth.  The  response  of  the 
cancer  cells  to  melatonin  may  or  may  not  be  tied  to  estrogen  action  since 
melatonin's  effect  on  insulin  stimulated  cells  appears  comparable. 

Although  we  have  observed  effects  of  melatonin  on  estrogen  and  insulin 
stimulated  growth,  we  have  been  unsuccessful  in  demonstrating  a  specific  binding 
protein  for  melatonin.  Our  failure  to  demonstrate  a  melatonin  receptor  and 
the  failure  of  many  other  laboratories  to  reproducibly  describe  a  melatonin 
receptor  has  lead  us  to  ask  new  questions  concerning  melatonin's 
intracellular  mechanism  of  action. 

Our  second  approach  to  these  questions  was  to  determine  if  melatonin  alters 
cyclic  nucleotide  (cAMP  and  cGAMP)  concentrations.  Results  of  these  studies 
were  equivocal.  Most  recently,  we  have  turned  our  attention  to  the  phos- 
phorylation of  intracellular  proteins  by  specific  protein  kinases  as  a  possible 
mechanism  of  action  of  melatonin  in  responses  to  estrogen  or  insulin  stimulation. 
We  have  isolated  cAMP-dependent  protein  kinases  and  preliminary  data  suggest 
that  melatonin  may  alter  the  ratio  of  type  I  to  type  II  concentrations  of 
these  enzymes.  At  this  time  we  are  investigating  other  variables  that  may 
affect  the  concentration  of  these  kinases.  Finally,  melatonin's  action  may 
be  related  to  another  class  of  protein  kinases  (C  kinase)  and  studies  relevant 
to  this  possibility  have  been  initiated.  Apparently,  melatonin  differs  from 
classic  hormones  in  its  mechanism  of  action;  our  preliminary  data  suggest 
further  experiments  that  may  identify  new  mechanisms  of  hormone  action.  Under- 
standing melatonin's  action  in  these  model  systems  may  be  relevant  to  melatonin's 
actions  on  other  target  sites,  such  as  those  in  the  central  nervous  system. 

Although  a  role  for  melatonin  has  now  been  established  in  seasonally 
breeding  species,  its  role  in  man  remains  unclear.  The  daily  secretory  pattern 
in  man  is  similar  to  all  other  mammals  thus  far  studied,  with  low  levels  during 
the  day  and  high  levels  at  night.  However,  continued  controversary  exists 
concerning  the  analysis  of  plasma  melatonin.  We  have  spent  considerable  effort 
re-evaluating  the  radioimmunoassay  for  melatonin.  We  have  found  that  changing 
the  extraction  procedure  has  vastly  improved  our  analysis  and  we  have  re-analyzed 
samples  from  other  laboratories  in  the  US  and  Europe  to  arrive  at  a  better 
consensus  among  research  groups  concerning  plasma  melatonin  levels  in  health 
and  disease. 

A  second  line  of  clinical  investigation  relies  on  the  fact  that  the 
nocturnal  synthesis  of  melatonin  requires  adrenergic  stimulation  to  induce  the 
enzymes  to  convert  pineal  serotonin  to  melatonin.  For  example,  drugs  which 
modify  adrenergic  action  or  serotonergic  availability  may  be  reflected  in 
altered  plasma  levels  of  melatonin.  Monitoring  peripheral  melatonin  could 
provide  a  readily  measurable  index  of  adrenergic  and/or  serotonergic  responses 
to  pharmacologic  interventions.   It  is  our  expectation  that  these  studies  may 
prove  to  be  useful  in  the  evaluation  of  biological  changes  in  mental  illness 
that  may  involve  altered  neurotransmitter  function. 

Antidepressant  Pharmacology  of  the  Rodent  Circadian  System 
Wallace  C.  Duncan,  M.S. 

According  to  the  phase-advance  hypothesis  of  depressive  illness,  the 

36 


circadian  rhythms  of  certain  physiological  processes  (body  temperature, 
Cortisol,  REM  sleep)  occur  abnormally  early  with  respect  to  the  sleep-wake 
cycle.  These  changes  are  accompanied  by  mood  and  severe  sleep  disturbance 
such  as  shortened  sleep  duration,  frequent  nocturnal  arousal s,  shortened 
REM  latency,  and  early  morning  awakenings.  Treatment  strategies  consistent 
with  this  hypothesis  attempt  to  achieve  a  more  normal  phase  relationship 
between  circadian  rhythms  observed  in  pathological  temporal  relationship. 
Two  effective  treatment  paradigms  employed  thus  far  include  a)  a  phase-advance 
of  the  sleep-wake  cycle  and  b)  partial  early  morning  sleep  deprivation. 
Both  treatments  have  demonstrated  utility  in  reducing  depressed  symptoms 
in  some  affectively  ill  patients.  A  third  treatment  strategy  may  include 
the  use  of  chemicals  which  affect  circadian  timekeeping  mechanisms  and 
thus  correct  the  pathological  phase  relationships  observed  in  depression. 
We  have  been  pursuing  this  avenue  of  research  by  studying  the  effects  of 
known  antidepressant  compounds  on  the  circadian  system  in  the  golden  hamster 
(Mesocricetus  auratus).  During  the  past  year  our  accomplishments  in 
this  area  include  the  design,  construction,  and  evaluation  of  a  computer 
controlled  rodent  circadian  rhythm  monitoring  facility  as  well  as  compelling 
preliminary  evidence  of  clorgyline  effects  on  the  circadian  pacemaker. 

The  underlying  mechanism(s)  responsible  for  the  abnormal  phase-advanced 
pattern  of  circadian  rhythms  in  depression  are  currently  unknown.  Among  the 
possible  dysfunctional  mechanisms  are  a)  a  primary  pacemaker  dysfunction 
characterized  by  either:  1)  an  circadian  oscillator  with  a  short  period 
duration  relative  to  normal  or  2)  an  abnormal  phase  response  relationship 
to  an  endogenous  or  exogenous  signal  normally  required  for  proper  phasing 
of  circadian  oscillations  or  b)  a  secondary  or  slave  oscillator  dysfunction 
which  cannot  be  compensated  by  primary  pacemaker  coupling.   In  theory, 
chemical  treatments  which  effect  these  mechanisms  are  valuable  for  two 
reasons.  First,  they  may  prove  to  be  effective  in  treatment  of  circadian 
rhythm  disorders,  including  depression.  For  instance,  the  psychoactive 
compound  lithium  has  been  shown  to  increase  the  period  of, or  phase  delay, 
circadian  rhythms  in  a  number  of  vertebrate  species  including  man. 
Secondly,  the  effects  of  these  compounds  may  provide  relevant  information 
with  respect  to  the  regulation  of  the  circadian  system. 

Previous  studies  in  this  branch  have  suggested  that  certain  antidepressant 
compounds  alter  the  expression  of  circadian  activity  rhythms  in  nocturnal 
rodents.  Two  effects  have  been  documented:  a)  the  irreversible  monoamine 
oxidase  inhibiter  (MAOI)  clorgyline  was  found  to  reduce  the  amplitude  of 
motor  activity  and  phase  delay  the  motor  activity  rhythm  relative  to  the 
light-dark  (LD)  schedule  in  chronically  treated  golden  hamsters  and  b) 
clorgyline  was  observed  to  either  dissociate  m^  increase  the  period  of 
circadian  motor  activity  rhythms  in  chronically  treated  hamsters.  While 
these  studies  suggest  the  effects  of  MAOI  compounds  may  have  input  to  an 
endogenous  pacemaker,  the  results  may  also  be  interpreted  as  reflecting 
chemical  input  to  a  slave  oscillator.  During  the  past  year  we  have  begun 
to  resolve  this  question. 

Two  techniques  are  widely  used  to  explore  the  operational  and  functional 
mechanisms  of  the  circadian  system.  The  first  paradigm  explores  the  autonomous 
functioning  of  the  pacemaker  in  constant  conditions  of  light,  temperature  and 
feeding.  Treatments  which  alter  the  period  of  the  measured  circadian  rhythm 
must  be  operating  in  part,  at  the  level  of  an  autonomous  circadian  oscillator. 

37 


A  second  paradigm  measures  the  magnitude  of  circadian  rhythm  phase  shifting 
due  to  test  pulses  (e.g.  light,  chemicals,  etc.)  administered  at  different 
phases  of  the  circadian  rhythm.  Treatments  which  produce  a  phase  response  curve 
must  be  exerting  phase  control  on  the  circadian  pacemaker  not  on  a  slave 
oscillator.  Similarly,  treatments  which  alter  the  shape  of  a  PRC  to  a  well 
studied  treatment  (e.g.  light)  must  be  altering  the  (light)  input  signal  to  the 
clock.  The  advantages  of  the  phase  response  curve  paradigm  are   1)  it  permits 
elucidation  of  oscillator  mechanisms  by  comparison  of  advance  and  delay  portions 
of  the  curve  2)  it  can  be  used  to  identify  treatments  which  selectively  alter 
pacemaker  versus  slave  oscillations  3)  it  can  be  used  to  identify  chemicals 
which  either  modify  input  signals  to  the  circadian  pacemaker  or  themselves 
provide  input  to  the  circadian  pacemaker.  We  have  recently  designed  and 
constructed  an  automated  rodent  circadian  rhythm  facility  which  dramatically 
facilitates  the  collection  of  data  utilizing  these  techniques.  Experiments  are 
currently  in  progress  using  these  paradigms  in  order  to  identify  possible  sites 
of  antidepressant  drug  treatment  action  with  respect  to  the  circadian  system. 

Two  preliminary  experiments  strongly  indicate  chronic,  systemic  administration 
of  clorgyline  1)  increases  the  period  of  the  circadian  oscillator  in  the 
golden  hamster  and  2)  dramatically  alters  the  waveform  of  motor  activity  in  the 
golden  hamster.  We  interpret  these  results  as  compelling  evidence  for  either 
direct  or  indirect  input  to  the  circadian  pacemaker  by  clorgyline. 

We  are  presently  conducting  experiments  to  replicate  and  extend  the  response 
of  the  circadian  system  to  MAOI's.  Among  the  further  questions  to  be  addressed 
are  1)  What  is  the  anatomical  site  of  clorgylines  actions?  We  plan  to  infuse 
this  compound  in  the  vicinity  of  the  suprachiasmatic  nucleus  of  the  golden 
hamster  to  determine  its  action  on  the  circadian  pacemaker,  2)  What  is  the 
mechanism  of  the  circadian  response  to  chronic  clorgyline  treatment?  The 
response  may  reflect  the  pacemaker's  response  to  non-specific,  altered  amine 
metabolism,  to  specific  alteration  in  one  of  the  several  affected  amines,  or  to 
a  non-specific  effect  of  the  clorgyline  molecule  itself  acting  on  a  gear  of  the 
clock  itself  or  input  pathway  to  the  clock.  We  therefore  plan  to  test  the  effects 
of  additional  type  A  MAOI  in  the  rodent  circadian  system,  as  well  as  specific 
neurotransmitters  to  determine  the  mechanism(s)  of  the  clorgyline  effect. 
3)  What  are  the  sites  of  clorgyline's  actions  according  to  existing  theoretical 
models?  Our  current  evidence  indicates  input  to  a  circadian  pacemaker 
itself;  it  is  also  possible  that  downstream  slave  osillators  are  affected,  and 
that  there  are  possible  feedback  effects  of  the  secondary  oscillator 
to  the  primary  pacemaker.  Determination  of  the  phase-response  curve  to  brief 
light  pulses  in  clorgyline  treated  hamsters  would  enable  us  to  address  this 
question. 

In  summary,  we  have  made  progress  in  resolving  some  of  the  vexing  problems 
involved  in  establishing  constant  environmental  conditions  free  of  time  cues 
in  the  complex  clinical  center  environment.  We  are  currently  utilizing  a 
"state  of  the  art"  rodent  circadian  facility  which  tremendously  accelerates  and 
expands  our  basic  research  capabilities.  In  addition,  recently  completed 
preliminary  experiments  provide  the  strongest  evidence  thus  far  of  antidepressant 
drug  input  to  the  circadian  pacemaker  in  the  hamster.  Work  currently  in  progress 
will  unravel  specific  questions  relating  to  the  mechanisms  of  antidepressant 
activity  on  the  circadian  system. 


38 


ANNUAL  KEPORT  UF  THE  LABORATORY  OF  CLINICAL  SCIENCE 

NATIONAL  INSTITUTE  OF  MENTAL  HEALTH 

October  1,  1984  through  September  30,  1985 

Dennis  L.  Murphy,  M.D.,  Chief 

The  Laboratory  of  Clinical  Science  (LCS)  was  reorganized  in  the  past  year. 
Following  a  transition  period  very  ably  managed  by  Dr.  Seymour  Kety  and 
Dr.  Sanford  Markey,  the  Laboratory  was  reconstituted  by  a  merger  with  the 
Clinical  Neuropharmacology  Branch  and  the  Laboratory  of  Brain  Evolution  and 
Behavior,  with  Dr.  Dennis  Murphy  from  the  Clinical  Neuropharmacology  Branch 
appointed  to  head  the  new  LCS.  Several  LCS  sections  were  renamed;  however  the 
major  changes  were  the  formation  of  a  new  Section  on  Comparative  Studies  of 
Brain  and  Behavior  located  at  the  NIH  Animal  Center  in  Poolesville,  Maryland  and 
a  near  en  bloc  incorporation  of  the  Clinical  Neuropharmacology  Branch  as  a  new 
LCS  section  with  the  same  name.  The  Section  on  Comparative  Studies  of  Brain  and 
Behavior  now  includes  Dr.  Thomas  Insel  (formerly  of  the  Clinical  Neuropharma- 
cology Branch),  and  one  of  NIMH's  most  distinguished  senior  scientists.  Dr.  Paul 
MacLean;  it  also  includes  collaborative  elements  jointly  administered  with  the 
newly  reorganized  Poolesville  operation  of  NICHD's  Laboratory  of  Comparative 
Ethology  headed  by  Dr.  Steven  Suomi .  Dr.  Jack  Calhoun,  formerly  of  the 
Laboratory  of  Brain  Evolution  and  Behavior,  has  moved  his  unit  from  Poolesville 
to  the  Federal  Building  in  Bethesda,  where  he  will  be  completing  work  on  a 
monograph  reporting  the  conclusions  of  his  studies  from  the  last  decade.  The 
original  LCS  sections  headed  by  Drs.  Jacobowitz,  Jimerson,  Markey,  and  Potter 
maintain  their  full  strength  and,  as  indicated  in  the  individual  section 
summaries  indicated  below,  also  maintain  their  continued  high  level  of 
productivity. 

This  Laboratory  is  unusual  within  the  NIMH  intramural  program  because  of 
its  large  size  and  diverse  activities.  It  provides  a  home  for  two  major  analy- 
tical resources  for  the  entire  intramural  program,  the  clinical  monoamine  assay 
laboratory  in  Dr.  Potter's  section,  and  the  analytical  development  group  headed 
by  Dr.  Markey.   It  is  spread  across  three  clinical  research  units  (3-East, 
4-West,  and  6-D)  and  the  outpatient  clinic.  The  clinical  research  units  are 
shared  with  investigators  from  the  Child  Psychiatry  Branch,  the  Clinical  Psycho- 
biology  Branch  (which  operates  the  major  research  program  on  4-West),  and  the 
Laboratory  of  Cerebral  Metabolism.  The  LCS  was  originally  formed  to  assure 
close  juxtaposition  of  investigators  working  in  analytic  methods  development, 
basic  neurochemi stry  and  neuroanatomy,  and  clinical  studies  involving  major 
psychiatric  disorders.  This  goal  remains  active  through  the  leadership  of  the 
various  section  chiefs  in  encouraging  collaborative  and  consultative  relation- 
ships throughout  the  Laboratory  and  the  entire  intramural  program.  Several 
major  areas  of  shared  interests  and  collaborative  projects  across  the  sections 
include  clinical  and  basic  studies  of  brain  peptide  functions  and  localization, 
interactions  between  peptides  and  monoamine  neurotransmitter  systems,  basic  and 
clinical  studies  of  serotonin  influences  on  behavior  and  neuroendocrine  func- 
tions, and  many  projects  involving  the  mechanisms  of  action  of  antidepressant  and 
other  psychoactive  agents.  This  goal  is  also  more  broadly  exemplified  in  the 
active  discussions  following  weekly  presentations  by  Laboratory  staff  members  in 
our  seminar  series.  Hallway  meetings  on  the  second  and  third  floors  and  other 
one-to-one  contacts  involving  shared  use  of  equipment,  supplies  and,  of  course, 
information  provide  daily  opportunities  for  collaborative  contact.  The 

39 


Laboratory,  nonetheless,  nust  be  viewed  as  in  a  process  of  evolution,  and  with 
continued  differentiation  can  be  expected  to  subdivide  in  the  future  into 
snaller,  focused  groups  nore  like  other  NIMH  Intranural  laboratories  and 
branches. 

Section  on  Analytical  Biochenistry 
Sanford  P.  Markey,  Ph.D.,  Chief 

This  section  conducts  research  on  the  developnent  and  application  of 
analytical  instrumentation  to  problems  in  neuropharnacology.  During  the  past 
year,  nuch  of  the  research  effort  has  continued  to  be  in  applications,  but  with 
several  major  new  instrumentation  initiatives. 

The  mechanism  of  action  of  the  neurotoxin  1-methyl -4-phenyl -1,2,3,6- 
tetrahydropyridi  ne  (MPTP)  has  been  a  major  research  interest  in  this  section. 
Primates  and  rodents  show  marked  differences  in  sensitivity  to  the  neurotoxic 
effects  of  MPTP.  In  the  rhesus  monkey,  dopaminergic  neurons  in  the  dorsolateral 
part  of  the  striatum  are  most  vulnerable,  with  a  resulting  extrapyramidal 
syndrome  which  closely  resembles  idiopathic  parkinsonism  in  humans.  Immunocyto- 
chemical  and  biochemical  studies  of  rhesus  monkeys  treated  with  MPTP  by  Dr.  C.C. 
Chiueh  indicated  that  clinical  symptoms  appeared  only  in  animals  with  greater 
than  80%  striatal  dopamine  depletion  and  A9  region  cell  body  loss.  Metabolic 
studies  by  Dr.  J.  Johannessen  have  demonstrated  that  MPTP  is  metabolized  in  brain 
to  the  quaternary  species  MPP"*",  and  that  this  compound  persists  in  high  concen- 
tration in  the  caudate  nucleus  relative  to  other  brain  areas.  Rodents  eliminate 
MPTP  and  MPP"^  much  more  rapidly  than  does  the  monkey.  We  have  sought  to 
explain  the  mechanism  of  MPTP  toxicity  by  the  properties  of  this  metabolite 
MPP+. 

Blocking  the  metabolism  of  MPTP  to  MPP+  prevents  its  toxicity.  Inhibi- 
tors of  monoamine  oxidase  type  B  (pargyline,  deprenyl )  are  most  effective,  as 
measured  by  biochemical  and  histochemi cal  studies  in  both  the  dog  and  mouse.  A 
quantitative  mass  spectrometric  assay  of  MPTP  and  MPP"*"  has  been  developed  and 
utilized  to  determine  that  the  effect  of  MAO-B  inhibitors  is  to  inhibit  MPTP 
metabolism  in  the  mouse  brain.  Dopamine  uptake  inhibitors  such  as  mazindol  also 
appear  to  block  MPTP  neurotoxicity  in  the  mouse,  and  mass  spectrometric  assay  of 
homogenates  indicates  that  this  protection  must  be  due  to  uptake  inhibition 
because  normal  conversion  of  MPTP  to  MPP"^  occurs. 

The  role  of  oxidative  stress  in  the  toxicity  of  MPP"*"  has  been  evaluated 
in  the  rodent  by  Dr.  Johannessen.  We  had  observed  that  MPP"*"  is  a  caustic 
compound,  producing  non-specific  lesions  when  directly  injected  into  the  striatum 
of  mice.  Consequently,  we  have  had  to  study  its  systemic  effects  following  peri- 
pheral injection.  MPP"*"  produces  paraquat-like  lung  lesions  and  results  in 
increased  production  of  oxidized  glutathione,  a  specific  marker  for  oxidative 
stress.  Selenium  deficient  mice  are  significantly  more  susceptible  to  the  lethal 
effects  of  both  MPTP  and  MPP"*",  another  measure  of  the  probable  role  of  oxida- 
tive stress  in  oxidation  sensitive  brain  regions. 

A  new  project  area  has  been  initiated  by  Dr.  Chiueh,  the  determination  of 
dopamine  turnover  in  vivo  by  positron  emission  tomography  (PET).  The  pharmacol- 
ogy of  6-fluoro-l-dopa  has  been  studied  in  rodents,  and  it  appears  to  he  a 
suitable  compound  for  indicating  the  turnover  of  brain  dopamine  when  labeled  with 

40 


ISp  and   visualized  by   PET.     The  degree  of  brain  damage   and  dopamine  turnover 
has   been  assessed  using  this   procedure  in   rhesus  monkeys   lesioned  with  MPTP. 

In   a  project   on  the  metabolism  of  melatonin   in   humans,   a   correlative  study 
on  plasma  melatonin   levels   and  urinary  6-hydroxymelatonin  excretion  was 
completed  for  a  group  of   2.1  women.     This   study   indicated  a  strong  association 
between  integrated  plasma   levels   of  the  hormone  and  urinary   levels   of   its  metabo- 
lite,  validating  urinary   6-hydroxymelatonin  measurements   as   a  useful    approach  to 
assess  pineal    gland  production   of  melatonin   in   humans. 

In  another  new   project,   the  synthesis   of   ^-^C-labeled  norepinephrine  has 
been   initiated  by  Dr.   Weisz,   starting  with   ■'■■^Cs-ring-labeled   guaiacol.     The 
resulting  product  will    be  used  for  metabolism  and  pharmacokinetic  studies,   and  as 
an   internal    standard  for  mass   spectrometric  assays. 

Instrumentation  projects   have  taken  several    new  directions.     A  Fourier- 
transform  mass   spectrometer   (FT/MS)   has   been   acquired   and   is   being  tested  for 
its   applicability  to  neuropeptide  analysis   using   laser  desorption   ionization. 
Planned  studies  will    evaluate  the  possibility  of   isotope  dilution  mass   spec- 
trometry  being  applied  to  qualitative  and  quantitative  determination   of   neuro- 
peptides with  molecular  weights   up  to  3000  daltons  with  minimal    chemical    modifi- 
cations.    The  FT/MS  is   also  being  tested  for  capillary  GC-MS  analyses.     The 
experimental   surface   ionization  mass   spectrometer  has  been   used  to  characterize 
the  distribution   of  ion  energies   for  compounds   evolved  from  various   liquid 
matrices.     The  observed  differences  may  explain  the  empirical    data  which   others 
have  obtained,   and  enable  the  prediction   of  suitable  substrates   and  additives 
for  fast   atom  bombardment   experiments.     Finally,  the  previously  developed 
microwave-powered  chemical    reaction  interface  has   been   applied  to  the  analysis 
of  solid  samples  with   a  probe  inlet  design.     Sulfur  was  quantified  in  microgram 
quantities   of  peptides   of  molecular  weights  from  470  to  69  k  daltons. 

Section  on  Biomedical    Psychiatry 
David  C.   Jimerson,  M.D.,   Chief 

During  the  past  year  the  clinical    studies   in  this   section   have  focused  on 
neurotransmitter,   neuroendocrine,   neuropeptide,   and   related  behavioral    studies 
in  patients  with  the  eating  disorder  syndromes   of  anorexia   nervosa  and  normal 
weight  bulimia   (Drs.   Kaye,  Gwirtsman,   George,   Brewerton,  Obarzanek,   and 
Jimerson).     The  clinical    design   for  studies  with   anorexia   incorporates 
behavioral   and  biological   testing  in  a  low  weight  malnourished  state,   and  at 
various  phases  of   recovery  to  a  normal  weight.     Bulimic  patients   at   normal 
weight  are  studied  immediately   after  admission  to  assess   the  biologic  correlates 
of  chronic   bingeing  and   vomiting,   as  well    as  following  a  medication-free 
abstinence  phase.     A  new  outpatient   clinic  for  pharmacologic  treatment  trials   in 
bulimia  was   opened  this  year. 

Neurotransmitter  studies   in   anorexia  have  extended  our  previous   results   on 
changes   in  norepinephrine  turnover  and  adrenoceptor  function   during  the   course 
of  weight  gain.     Following  stable  weight   restoration  for  at   least   six  months, 
anorexic  patients  were  found  to  have  significantly  decreased   levels   of  norepi- 
nephrine and  its  major  metabolite,   3-methoxy-4-hydroxyphenyl ethylene   glycol 
(MHPG).     Further  studies   are  needed  to  evaluate  whether  this   alteration   in 

41 


norepinephrine  metabolism  reflects   a  vulnerability   factor  to   recurring  episodes 
of  weight   loss,   or  a  compensatory   adaptation  that   assisted  the  subjects   in  this 
study  in  maintaining  stable  weight.     A  study   utilizing  isoproterenol    infusion 
to  evaluate  beta-adrenoceptor   function   in   low  weight   anorexia  and  following 
weight  gain   is   in   progress.     We  have  also  begun   a  follow-up  study  of  the 
serotonin  system  in  anorexia,   utilizing   1-tryptophan   and  m-chlorophenylpipera- 
zine    (in   collaboration  with   Drs.    Mueller   and  Murphy). 

Neuroendocrine  studies   in  anorexia   have  focused  on   the   regulation   of 
adrenal    and  thyroid  hormones.     Over  the  past  year  we  have  demonstratea   increased 
urinary  free  Cortisol    excretion,    resistance  to  dexamethasone-induced  suppression 
of  serum  Cortisol,   and  blunted   corticotropin   responses  to  corticotropin 
releasing  hormone   (CRF)    (in   collaboration  with  Dr.   Gold).      Infusion   studies  with 
thyrotropi n-releasi ng  hormone   demonstrated   blunted   and   delayed  thyroid  stimula- 
ting hormone   (TSH)   responses   in  underweight   anorexics.     The  TSH   response 
remained   delayed   but    returned  to   normal    magnitude   in  weight-recovered  patients. 

Studies   of  peptides   affecting  feeding   behavior  focused   on   cerebrospinal 
fluid   levels   of   pro-opiomelanocortin    (PUMC) -related  peptides.      In  underweight 
anorexic  patients,   there  was   a  significant    reduction   in   beta-endorphi n, 
beta-1 ipotropi n,   ACTH,   and  the   N-terminal    fragment   of   POMC   in   comparison   to 
control    values.     Concentration   of  these  peptides    returned  to   normal    levels 
following  weight    recovery,   suggesting  a  major   influence  of  weight   loss   on   this 
peptide  system. 

Evaluation   of   energy  metabolism  as    reflected   in   body  weight    regulation 
showed  that   bulimic  anorexics,   studied   at   low  weight   or  following  weight    restor- 
ation,   required  significantly   less   caloric   intake  to  maintain   stable  body  weight 
than  anorexics   in  the   restrictor   groups.     Future  studies  will    evaluate  the   rela- 
tionship between  energy  metabolism  and  neurotransmitter  function   in  these 
patients. 

Brain   imaging  studies   have  shown   significant   cortical    atrophy  and  decreases 
in   rates  of  glucose  utilization    (using   •'■°F-2-deoxyglucose)   in   low  weight 
anorexic  patients,  with  a   return  toward  normal    values   following  weight    restora- 
tion.    Data  on   psychological    and  nutritional    correlates   of  these  changes   are 
presently   being  analyzed. 

Neurotransmitter  studies   in   bulimia  demonstrated   large   increases   in   plasma 
norepinephrine  following   binge/vomit   episodes.     When   studied  after  abstinence 
from  bingeing  and   vomiting,   bulimic  patients   had   low  resting  plasma   norepineph- 
rine and  pulse  rates,   increased  heart   rate   response  to   isoproterenol    infusion, 
and  a  tendency  toward  increased  density  of  lymphocyte  beta-adrenoceptors 
(studied  in   collaboration  with  Drs.   Potter  and  Buckholtz).     Further  studies   are 
required  to  evaliiate  whether  these  changes   ^re   long  term  consequences   of   chronic 
bulimic  behavior,   or  whether  they   represent   premorbid   risk   factors   for  bulimia. 
Studies  were   begun   to  evaluate   serotonin   function   in   bulimia   utilizing 
behavioral    and   neuroendocrine   responses  to  the  serotonin  precursor  1-tryptophan 
and  the  serotonin   receptor   agonist  n-chlorophenylpiperazi ne    (in   collaboration 
with  Drs.   Mueller  and  Murphy). 

Neuroendocrine  studies  in  bulimia  demonstrated  normal  urinary  free  Cortisol 
excretion  but  a  substantial  incidence  of  Cortisol  escape  following  dexamethasone 
administration    (studied   in  collaboration  with  Dr.   Gold).     Abnormal    Cortisol 

42 


responses  were  observed   in   bulinic  patients  without  major  depression   as  well    as 
in  those  with  this   additional    diagnosis.     Bulimic  subjects  had  low  levels   of 
triiodothyronine  in   comparison  to  controls,   with   blunted   and   delayed  TSH 
responses  to  TRH.     Elevations   of  serum  amylase  were  observed  consistently   in 
bulimic-anorexic  and  normal   weight   bulimic  subjects.     Longitudinal    studies 
suggested  that  spot  serum  amylase  determinations  might   provide  a   laboratory 
index  of   recent  binge/vomit  episodes   in  bulimic  subjects. 

As  part   of  a  systematic  approach  to  comparative  studies   of  the  psycho- 
biology  of  bulimia  with   other  major  psychiatric  syndromes    (e.g.,   major 
depression  and  anxiety  disorders),  we  evaluated  the  behavioral    response  to 
double-blind  infusion  of   sodium  lactate  in  bulimic  patients   and  controls.      In 
contrast  to  the  results   reported  for  panic  disorder  patients,   bulimic  subjects 
did  not  have  panic  attacks  during  the  lactate  infusion.     Several    studies 
involving  behavioral    ratings  and  psychological   testing  quantified  the  broad 
spectrum  of  psychopathology  observed   in  bulimic  as  well    as   anorexic  subjects, 
and  underscored  the  severity   of  symptoms   of  depression  and   generalized  anxiety 
present   in  many  bulimic  patients.     A  family   interview  study  of   bulimic  patients 
and  their  relatives    (in   collaboration  with  Dr.   Gershon)  was   begun  this  year. 

Laboratory  work   this  year  focused  on   norepinephrine  metabolism  in   relation 
to  clinical    neuroendocrine  studies   and  preclinical   studies   on  mechanisms   of 
antidepressant  drug  activity.      In   a  previous   report  we  demonstrated   increased 
norepinephrine  turnover  in   depressed  patients  with  hypothalamic-pituitary- 
adrenal    axis  dysfunction,   as  manifested  by   resistance  to  dexamethasone-i nduced 
suppression   of  plasma  Cortisol.      Preliminary   results   recently  obtained  in  eating 
disorder  patients   indicate  that   dexamethasone  non-suppression   in  this   population 
is  not  associated  with  increased  norepinephrine  turnover,   suggesting  that  the 
interaction   between  these  two  systems   observed   in  depressed  patients  may   show 
syndromal   specificity.      In  preclinical   studies   of  a  proposed  new  antidepressant 
drug  rolipram,  which   acts   as   a  cyclic-AMP  phosphodiesterase  inhibitor,  we  showed 
that   rolipram  treatment  alone  for  up  to  3  weeks   does   not   decrease  cortical 
beta-adrenoceptor  density   in   rat   cerebral    cortex   (studied   in  collaboration  with 
Dr.   Lozovsky).     Combination   of  rolipram  with  desipramine  did,   however, 
accelerate  the  down-regulation   of  beta-adrenoceptors   observed  with  desipramine 
alone.     This  drug  combination  strategy  merits   further  evaluation  for  its 
possible  utility   in  clinical    studies  with  depressed  patients. 

Section  on  Clinical    Neuropharmacology 
Dennis   L.   Murphy,   M.D.,   Chief 

This  Section's  work   is   directed  towards   increasing  understanding  of  the 
mechanism  of   action  of  drugs   used   in  treating  patients  with   neuropsychiatric 
disorders.     Neurochemical   and  behavioral    studies   in  animals   are  designed  to 
complement  ongoing  studies  of  drug  effects   in  patients   and  normals.     A  special 
focus   is  the  study   of  drugs  with   relative  neurotransmitter  system  specificity, 
including  agents  with  therapeutic  potential    (for  example,   a   series   of  substrate- 
selective  monoamine  oxidase   (MAO)-inhibiting  antidepressants   currently  under 
study).     Other  drugs  with   relatively   specific  effects   are   investigated  as  probes 
of  the  status   of  central    neurotransmitter   receptor  sensitivity  or  neurotrans- 
mitter system  function  to  obtain  possible  evidence  of  psychobiologic  abnormali- 


43 


ties   in  patients  with   psychiatric  disorders   or  more  detailed   infornation   on  the 
central   nervous  system   (CNS)   changes   produced   by  psychotherapeutic  drugs. 

As  part   of   a  systematic  comparison  of   substrate-selective  MAO-i nhi biti ng 
antidepressants,   a  dose-response  study  of  deprenyl    in   depressed  patients  was 
completed  this  year.      Minimal    antidepressant   effects  were   obtained   at   an 
MAO-B-selecti ve  deprenyl   dose  of  10  mg/day,  which   inhibited  platelet  MAO 
activity   greater  than   95%.      With   higher  doses    (30  and   60  mg/day),    reductions   in 
plasma  concentrations   of  the  major  norepinephrine  metabolite,   MHPG,   approached 
those  found   in  our  previous  studies  with   non-selective  MAO-i nhi bitors   or  the 
MAO-A  selective  inhibitor,   clorgyline.     Moreover,   a  markedly   increased  pressor 
sensitivity   to   intravenous  tyramine   occurred  with   the  higher  deprenyl    doses   in   a 
study  conducted  by  Dr.   Sunderland.     These   results   add  further  support  to  our 
hypotheses  that   both   antidepressant   activity   and  the  potentiation   of  tyramine 
sensitivity  are  closely   linked  with  MAO-A  inhibition  and  changes   in  noradrener- 
gic functions. 

In  animal    studies   of   two  new  MAO-A  selective   reversible   inhibitors,   one  of 
these,   cimoxatone,   most  closely   resembled  clorgyline  in   its   effects   on  norepi- 
nephrine and  dopamine  metabolism  in   non-human  primates,  while  another,   amifla- 
mine,   affected  serotonin  metabolism  to  the  same  extent   as   it  did  the  metabolism 
of  the  catecholamines.     Amiflamine,   however,   also  possessed  monoami ne-releasing 
effects,   complicating  its  use  in  studying  the  consequences   of  selective  MAO-A 
inhibition.      In  continuing  studies  of  complex  neuronal    adaptational    changes  that 
occur  during  the  longer-term  administration   of  antidepressant  drugs,   evidence 
was  obtained  that  the  changes   in  alphai,   alpha^,   and  beta-adrenergic   recep- 
tors  following  chronic  clorgyline  treatment   reflect  a   response  to  an   increase  in 
catecholamine  receptor  occupancy,   as  these  receptor  changes  were  prevented  by 
6-hydroxydopamine-induced   lesions   of  the  brain  catecholamine  systems.      In  a 
related   investigation,   Dr.   Aulakh  found  that  desipramine,    like  clorgyline, 
attenuated  the  suppressant  effects   of  clonidine  on  self-stimulation   behavior  in 
rodents.     These  effects   of   both   antidepressants   depended  on   longer-term   (3 
weeks)  treatment  with  the  drugs,   and  were  not   apparent   during  the  first  week  of 
treatment.      The  use  of   the  alpha-adrenergic   agonist,   clonidine,    in  this    reward 
system  behavioral    model   complements  the  neurochemical    studies   in   verifying  the 
importance  of   the  noradrenergic   system  in  the   responses   to  chronic  antidepres- 
sant drug  treatment. 

In   clinical    investigations   similar  to  our  work   over  the  past   several   years 
with  clonidine  as   a  probe  of  the  status   of  central    noradrenergic  function   in 
humans,   we  have  currently  been  emphasizing   investigations   of   serotonin   (5-HT) 
receptor   responses  to  the   b-HT^g   receptor  agonist,   meta-chl orophenylpi pera- 
zine   (m-CPP)   in  normal    subjects.     This   agent  has   not   previously  been  adminis- 
tered to  humans,   although   it   is   a  metabolite  of  the  novel    antidepressant,   traza- 
done,  used  in  patients.      Previous  studies   in   rodents   and  our  investigations   in 
non-human  primates   demonstrated  that  m-CPP  produced  dose-dependent   changes   in 
plasma  prolactin,   Cortisol   and   growth  hormone,   and  provided  a   basis   for  the 
current  phase   I   studies   being  conducted  by  Dr.   Mueller   investigating   neuroendo- 
crine and  other  effects   of  m-CPP  following  various   oral    and   intravenous   doses   in 
normal    volunteers. 

m-CPP   (U.b  mg/kg)  was  found  to  be  wel 1 -tolerated   in  humans,   and   in  a  subse- 
quent placebo-controlled,  double-blind  evaluation   of  the  effects   of  this 
serotonin   receptor  agonist,    statistically   significant  three-fold   increases   in 

4A 


plasma  prolactin  and  two-fold  increases   in  plasma  Cortisol  were  found  in  a  group 
of  14  normal   subjects.     Small    but  statistically-significant   increases   in 
self -rated  activation/euphoria  and  anxiety  and  in  body  temperature  also  followed 
m-CPP  administration.      No  cardiovascular  changes  were  observed.     We  plan  to  move 
on  to  use  this  CNS  serotonin  system  probe  in  investigations   of  patients  with 
psychiatric,   neurologic,   and  other  disorders  thought  to  involve  serotonin  abnor- 
malities,  as  well    as  to  use  it   in   investigations   of  the  possible   involvement   of 
serotonin   changes   in  the  actions   of  psychoactive  drugs. 

In  our  developing  program  of  study  directed  towards   the  affective  elements 
of  Alzheimer's   disease  and  other  dementias,   and  the  associations   among  cognitive 
impairment,   depression,   and  the  functional    status  of  the  brain   neurotransmitter 
systems,   Drs.   Sunderland,  Tariot,   and  Newhouse,   in  collaboration  with  Dr.   Robert 
Cohen  of  the  LCM,  have  been  using  a  series   of  drug  challenge  paradigms   in 
patients  with  early  Alzheimer's  and  aged-matched  groups   of  normals   and  older 
patients  with  depression.      Patients  with  Alzheimer's   disorder  were  found  to  have 
a  marked  cognitive  and  affective  sensitivity  to  the  cholinergic  agonist, 
scopolamine.     Behavioral    responses  to  naloxone  were  found  to  occur  at   a  lower 
dose  compared  to  our  earlier  findings   in  young  normals.     The  effect   of  age  on 
the  response  to  scopolamine  and  naloxone  is   currently  being  examined   in 
controlled  trials.     We  are  also  now  in  the  initial   stages   of  testing  the 
cognitive,   behavioral,   and  neuroendocrine   reactions   of  Alzheimer  patients   and 
controls  to  the  cholinergic  agonists,   arecholine  and  nicotine.      In   our  continued 
efforts  to  explore  the  links  between   cognition  and  affective  state  and   its 
neuropharmacology,  we  are  also  actively  investigating  a  therapeutic  drug 
strategy  with  monoamine  oxidase  inhibitors   in  this  population  of  dementia 
patients. 

The  ongoing  studies  of   adults  with  obsessive-compulsive  disorder  directed 
by  Dr.    Insel   has   progressed  on  several    fronts  this  year.     Data  from  last  year's 
examination  of  the  hypothesis   implicating  serotonin  dysfunction   in  obsessive- 
compulsive  disorder  failed  to  find   important   differences   from  controls   in 
several    indices   of  serotonin  function.     As  a  follow-up  to  our  previous  study   of 
responsiveness  to  pharmacologically-selective  antidepressants,  which   did  not 
suggest  that  zimelidine  equalled  clomipramine's   effects,   desipramine   is   now 
being  compared  to  clomipramine.       Results   so  far  suggest  that  clomipramine 
appears   to  be  more  effective  than  desipramine  in   reducing  obsessional    symptoms, 
although  both  drugs  have  previously  been  shown  to  be  equally  effective  as 
antidepressants.      In  a  new   investigation.  Dr.   Zohar  is   studying  alterations   in 
cerebral   blood  flow  during  exposure  to  obsessional    stimuli.     This   study,   which 
looks  at  autonomic  responses   as  well    as   cerebral    blood  flow,   promises  to  provide 
a  novel    approach  to  the  neurobiology  of  this   disorder.     Finally,   patients  from 
the  first  two  years   of  this   study  are  being  brought  back   for  follow-up 
evaluations  to  study  the  natural    course  of  obsessive-compulsive  disorder  and  the 
long-term  impact   of  drug  treatment   in  a  study  which  will    be  completed   later  this 
year. 

Section   on  Clinical    Pharmacology 
William  Potter,  M.D.,   Chief 

This  Section  was   renamed  this  year  and  its  three  functional    components  were 
formalized  into  three  units:      a  Clinical   Research  Unit  under  the  Chief,   a  Unit 

45 


on  Clinical  Neurochenistry  under  Or.  Ivan  Mefford,  and  a  Unit  on  Preclinical 
Neuropharmacology  under  Dr.  Juan  Saavedra. 

Dr.  Saavedra's  unit  includes  Dr.  Laura  Plunkett,  a  PRAT  Fellow,  and 
Drs.  Shigematsu  and  Kurihara,  both  Fogarty  Visiting  Fellows.  To  date,  quantita- 
tive autoradiographic  methods  involving  inage  analysis  coupled  to  computerized 
micro densitometry  have  been  modified  for  application  to  characterization  of 
angiotensin  II,  somatostatin  and  substance  P  receptors  in  the  central  and 
peripheral  nervous  system  of  the  rat.  This  has  been  achieved  despite  a  maximum 
of  12  hours/week  of  the  necessary  computer  time.  The  delivery  of  a  dedicated 
image  analyzer  to  the  Section  this  fall  will  open  up  these  techniques  for  a  much 
greatly  increased  number  of  projects  and  collaboration. 

Mapping  of  angiotensin  II  receptors  reveals  two  systems:  one  in  the 
circumventricular  organs,  with  access  to  blood-borne  angiotensin  II,  and  another 
inside  the  blood-brain  barrier,  representing  the  endogenous  brain  angiotensin 
system.  Alterations  in  angiotensin  II  and  substance  P  receptors  occur  in 
specific  brainstem  areas  of  the  spontaneously  (genetic)  hypertensive  rat  (SHR). 
These  findings  indicate  a  role  for  both  angiotensin  II  and  substance  P  in 
cardiovascular  regulation  and  probably  in  the  etiology  of  genetic  hypertension. 
The  SHR  strain  possesses  increased  angiotensin  II  receptors  in  the  sympathetic 
ganglia,  the  anterior  pituitary  and  the  adrenal  gland.  These  results  point  to  a 
participation  of  local,  peripheral  angiotensin  systems  in  the  regulation  of 
cardiovascular  function  and  suggests  the  existence  of  multiple  sites  of  close 
biochemical  interactions  between  the  angiotensin  and  the  sympathetic  systems. 
Moreover,  the  autoradiographic  methods  have  been  applied  for  the  first  time  to 
the  quantitation  of  enzyme  kinetics  in  individual  brain  nuclei  of  single  rats. 
Angiotensin  converting  enzyme  has  been  mapped  and  quantitated  throughout  the 
brain,  in  the  sympathetic  ganglia,  pituitary  and  adrenal  gland,  and  other  organs 
of  the  rat.  These  techniques  can  be  applied  to  the  study  of  other  enzymes, 
provided  a  suitable  ligand  is  available. 

During  the  last  year  (prior  to  the  arrival  of  Dr.  Mefford  in  July,  1985), 
the  Unit  on  Clinical  Neurochemistry  has  been  operating  under  the  direction  of 
the  Section  Chief  and  Dr.  Markku  Linnoila,  Chief  of  the  Laboratory  of  Clinical 
Studies,  NIAAA.  This  joint  analytical  laboratory  provides  assays  of  neurotrans- 
mitters to  clinical  investigators  in  both  institutes.  Over  500U  clinical 
samples  in  plasma,  CSF  or  urine  have  been  analyzed  for  the  now  routine  measures 
of  norepinephrine,  MHPG,  VMA,  HVA  and/or  5HIAA.   In  addition,  hundreds  of 
primate  tissue  samples  have  been  analyzed.  Although  the  bulk  of  these  analyses 
have  been  a  natural  outgrowth  of  ongoing  clinical  studies  in  each  of  the  clini- 
cal branches,  a  number  have  represented  special  collaborations  in  which  problems 
of  general  interest  were  approached. 

In  particular,  a  study  designed  by  Drs.  Wehr  and  Sack  of  the  Clinical 
Psychobiology  Branch  generated  circadian  samples  under  normal  ward  conditions 
vs.  24-hour  bed  rest  with  equally  spaced  isocaloric  feedings.  Under  these 
latter  controlled  conditions,  the  apparent  but  highly  variable  plasma  MHPG 
rhythm  noted  by  other  groups  disappears  whereas  a  circadian  plasma  HVA  rhythm 
with  a  nocturnal  peak  clearly  emerges.  This  has  produced  a  new  focus  on  circa- 
dian plasma  HVA,  and  its  biochemical  and  behavioral  correlates.  The  results 
showing  an  absence  of  a  significant  rhythm  in  plasma  MHPG  are  of  considerable 
importance  since  they  provide  strong  evidence  that  a  marked  circadian  pattern  of 


46 


Nt  turnover  in  selected  nuclei  in  rat  brain  (see  below)  which  could  also  occur 
in  discrete  human  brain  areas  is  not  reflected  in  plasma  MHPG. 

The  analytical  laboratory  has  also  developed  assays  sufficiently  sensitive 
enough  for  three  neurotransmitters  and/or  metabolites  in  CSF  and/or  plasma 
(assays  for  urine  are  already  available).  Dr.  Tom  Marshall,  in  an  NIAAA  posi- 
tion, has  developed  HPLC  assays  for  both  serotonin  and  normetanephrine  in  the 
CSF;  Jerry  Oliver  has  developed  a  GC-MS  assay  for  normetanephrine  in  plasma  as 
well  as  CSF;  and  Mark  Stipetic  has  developed  an  HPLC  assay  for  epinephrine  in 
the  CSF.  Plasma  epinephrine  is  beginning  to  be  measured  using  an  assay 
developed  by  Ivan  Mefford.  None  of  these  assays  are  "routine";  several  studies 
are  under  way  to  see  how  these  measurements  can  clarify  long-standing  questions 
on  serotonin  and  norepinephrine  turnover  as  well  as  the  role  of  epinephrine  in 
psychological  stress. 

The  Clinical  Research  Unit  has  continued  its  focus  on  two  major  themes: 
1)  the  role  of  norepinephrine  in  affective  disorders,  and  2)  interactions 
between  the  serotonergic  and  noradrenergic  systems.  Under  the  immediate  super- 
vision of  Dr.  Matthew  Rudorfer,  Drs.  Golden  and  Sherer  evaluated  two  putative 
new  antidepressants,  bupropion  and  s-adenosylmethioni ne  (SAMe)  for  effects  on 
neurotransmitter  systems,  as  well  as  characterizing  the  pretreatment  state  of 
depressed  patients.  Depressed  patients  prior  to  treatment  continue  to  show 
exaggerated  responsivity  of  the  noradrenergic  system.  We  have  extended  this 
observation  by  showing  altered  regulation  of  lymphocyte  beta-adrenergic 
receptors  (Dr.  Neil  Buckholtz)  which  is  only  partly  explained  by  circulating 
levels  of  NE  and  not  at  all  by  levels  of  epinephrine. 

The  antidepressant  bupropion  clearly  reduced  NE  turnover  in  three  patients. 
Initial  studies  with  two  distinct  and  new  serotonin  uptake  inhibitors, 
citalopram  and  fluvoxamine  (used  on  the  NIAAA  research  unit)  also  show  reduction 
of  NE  turnover.  These  findings  add  further  support  to  our  hypothesis  that 
reduction  of  NE  turnover  is  a  necessary  if  not  sufficient  condition  for  antide- 
pressant drug  action.  Results  in  volunteers  with  SAMe  are  less  clear,  although 
both  standing  heart  rate  and  plasma  NE  were  reduced  24  hours  after  seven  daily 
intravenous  doses  in  the  absence  of  significant  NE  turnover  effects  as  measured 
in  urine.  Biochemical  studies  in  patients  should  clarify  the  question;  the 
antidepressant  status  of  SAMe,  however,  is  also  unclear. 

Studies  with  bupropion  also  unexpectedly  revealed  a  close  association  of 
biologically  active  metabolites,  demonstrated  to  be  present  in  brain  by  high  CSF 
concentrations,  with  non-response,  the  induction  of  psychosis  and  elevation  of 
plasma  HVA.   In  addition  to  supporting  the  value  of  plasma  HVA  in  studying 
psychosis,  these  findings  separate  dopaminergic  changes  from  antidepressant 
effects. 

Bupropion  did  not  have  significant  effects  on  serotonin  (5HT)  turnover  as 
measured  by  bHIAA  in  the  CSF.  This  provides  additional  evidence  for  the  primary 
role  of  NE.  Nonetheless,  the  fact  that  a  series  of  selective  5HT  uptake  inhibi- 
tors has  now  been  shown  to  reduce  NE  turnover  indicates  strong  functional 
interdependence  of  these  two  neurotransmitters.   In  a  collaboration  involving 
all  components  of  the  Section,  it  has  been  possible  to  advance  our  understanding 
of  the  functional  interaction  using  a  rat  model.  Circadian  variation  of  bio- 
genic amines  in  discrete  brain  nuclei  was  analyzed  in  terms  of  possible  interac- 
tions with  tailored  applications  of  data  analysis  (Dr.  Hans  Agren).  We  find 

47 


circadian  covariations   of   NE  and   5HT  in  the  locus   coeruleus   and  dorsal    raphe 
nucleus  with  tine  periods   of  high  and   low  association,   the   latter  occurring 
during  the  high  notor  activity   phase   (night).     These  studies   suggest  that   the 
time  of  tissue  sampling  in  man  nay   be  critical   to  assessing   5HT/NE  interactions 
and   give   very  different   pictures   than   integrated  measures. 

An  alternative  approach  to  evaluating   bHT   responsivity   in  man  has   also  been 
followed.      In  parallel    animal    (Dr.   Philippe  Lesieur)   and  hunan   studies,  we  have 
neasured  plasma  prolactin   (PRL)   response  to  intravenous   5HT  uptake  inhibitors. 
In  animals,   clear  dose  and  time  dependent   responses   are  observed,   apparently  via 
a   bHT-mediated   reduction   of  dopamine  turnover  in  the  median  eminence. 
5HT-stimulated   release  of   a  postulated  prolactin   releasing  factor   has   not   been 
ruled  out  by  these  experiments,   but   clearly  does   not   need  to  be  involved. 
Moreover,  the  effects   on   PRL  are  dependent   on  the   rate  of  administration   of  the 
5HT  uptake  inhibitors.     Thus,  with   IV  administration   of  clomipramine  in  man,  we 
showed  acute,   dose-dependent    (in  the   lO-!:^!)  mg   range)   increases   of  PRL.      IV  doses 
of   lU  mg  avoid  both   nausea  and  the  growth   hormone   release   reported  using  other 
paradigms.     We  plan  to  develop  this  test    (with   control    of  plasma   clomipramine 
levels)   as   a  highly  selective   bHT   challenge  with   a   PRL  endpoint. 

Finally,   we  have   studied   covariance  of  the  serotonergic  and   dopaminergic 
systems  with  new  applications   of  statistical    modeling  techniques.     On  the   basis 
of  observed  covariance  of  the  5HT  and  dopamine  metabolites,    5HIAA  and  HVA,   in 
human  CSF  as  well   as  their  covariance  in   discrete  brain  areas   from  rat   and  dog, 
we  have   strong  evidence  for   serotonergic   modulation   of  dopamine   but   not   vice 
versa.     This  fits  with  known  anatomical    projections   in  the  CNS  as  well    as 
behavioral    and   biochemical    function   studies    in   animals   providing  evidence   for 
substantial    direct  serotonergic  modulation   of  dopamine  systems,   and,   at  most, 
indirect  modulation   of   bHT  systems   by  dopamine. 

Section  on  Comparative  Studies   of  Brain  and  Behavior 
Dennis   L.   Murphy,   M.D.,   Acting  Chief 

A  major  focus   of  this   section  this  year  has   been  the  study   of  fear  or 
anxiety   in  various  animal    species.     One  question   recently   addressed  was  which 
brain  areas  are  important   for  the  mediation   of  anxiety.     Using  autoradiographic 
techniques,   Dr.    Insel    has  mapped   receptors   for  the  putative  anxiogenic  peptide, 
corticotropin   releasing  factor.     With  these  maps   and  maps   generated  from  earlier 
studies   of  the  distribution   of  the  benzodiazepine   receptor,   selective  sites  for 
the  microinjection  of  drugs,   peptides   or  antisera  were   identifed.      In  the  first 
application  of  this  technique,   microinjections   into  the  basolateral    nucleus   of 
the  amygdala  produced  a   rapid  and  robust   behavioral    response  to  corticotropin 
releasing  factor  and  to  the  benzodiazepines   at   doses   several-fold   below  those 
traditionally  used  with   intraventricular  or  peripheral    injections.     This  method 
not   only   promises   to  identify  discrete  areas   relevant  to  the  mediation   of 
anxiety  but  also  provides   a  test  of  the  functional   significance  of  the   regional 
distribution  of   receptors   in  brain. 

In  a  developmental    approach  to  anxiety.  Dr.   James  Hill    adapted  his   earlier 
work  with  ultrasonic   vocalizations   in  adult   rodents  to  study  the  distress   calls 


48 


of  the  isolated   rat   pup.     These  distress   calls   are   robust   and   reproducible 
behaviors  that  are  critical   to  mother-infant   attachment,   but   because  they  are 
ultrasonic,  they   have   rarely  been  studied.     There  are   considerable   individual 
and  strain   differences   in  the  number  of  these  calls.     The  pups   of  Maudsley 
reactive   rats,   an   inbred  strain  of  emotional    or  anxious   rats,  emitted  five-fold 
more  calls  than  their  non-reactive,   less   anxious   congeners.     This  finding 
suggests  that   separation  distress   in   infancy  may  be   related  to  anxious   behavior 
in   adulthood.      In  addition,   a  series   of  neuropharmacologic  studies   of  these 
ultrasonic  distress   calls    revealed  that  these  calls   are  exquisitely   sensitive  to 
drugs  that  act  on  the  benzodi azepine-GABA  receptor  system,   but  unresponsive  to 
opiates,   imipramine,   corticotropin   releasing  factor,   and  the  noradrenergic 
toxin,   6-hydroxydopamine.     As  the  benzodiazepine   receptors   in  brain   develop 
rapidly   in  the  first  week  of  life,   these   results   suggest   a   role  for  the   receptor 
system  in  mother-infant   bonding. 

In   related  studies   in  squirrel    monkeys.   Dr.   Paul   Maclean,   in  collaboration 
with  Dr.   John  Newman,   has   demonstrated  the   importance  of  the  cingulate  cortex 
for  the  production  of  the  primate   isolation   call.     Using  selective   lesion 
studies  over  the  past  two  years.   Dr.   Maclean  has   shown  that   ablations  within  the 
thalmo-cingulate  division   of  the  limbic  system   (which   is   phylogenetically 
relatively   recent)   reduces   isolation  calls  without   affecting  other 
vocalizations. 

Studies  of  genetic  strains   of   anxious   animals   have  been  used  to  study 
differences  in  specific  brain   systems.     The  guiding  hypothesis   for  this  work   has 
been  that  behavioral    differences  will    be   reflected  in  morphologic  or   receptor 
sensitivity  differences   in  brain.     Using  Maudsley   reactive  and  non-reactive 
rats,   differences   in  benzodiazepine  and  adenosine  binding  have   been   assessed  by 
both   autoradiographic  and  homogenate  binding  techniques.     Curiously,   the 
differences   in  the  adenosine  system  are  more  marked  that   those  in  the 
benzodiazepine  system.      In  a  study  with  Dr.   Stephen  Suomi ,    rhesus  monkeys 
selected  for  more  distress   or   less  distress   during  brief   separations   in   infancy 
were  studied  for  responsiveness  to  the  putatively  anxiogenic  benzodiazepine 
receptor  inverse  agonist,   B-CCE.     Results   from  these  studies   should  provide  some 
neurobiological    evidence  for  individual    differences   in  the   response  to 
separation   and  novelty. 

Section   on   Histopharmacology 
David  M.   Jacobowitz,   Ph.D.,   Chief 

This   section   continues   its   involvement  with   studies   of  peptides   and 
proteins.      Immunocytochemical    and  neurochemical    characterization  of 
peptide-containing  nerves   in  the  brain  and  periphery  has  been  pursued.     This 
year  we  have  added  studies   on  the  autoradiographic   localization   of  peptide 
receptor  binding  sites   in  the  brain.     We  are  interested   in  the  integration   of 
the  known  localization   of  neurotransmitter-neuromodulatory   nerves   in  the  brain 
with  biochemical    and  behavioral    analyses   at  the  level    of  discrete  central    nuclei 
and  pathways.     This  work   is   focused  on   defining  the  underlying  basis   for 
behavioral    changes   and   improvement   of  our  understanding  and  treatment   of  mental 
disorders. 


49 


We  have  continued  our  ongoing  interest  in  napping  the  localization  of  brain 
neuropeptides  as  antisera  became  available.  Complete  brain  maps  of  four  neuro- 
peptides (atrial  natriuretic  factor,  calcitonin  gene-related  peptides,  galanin, 
melanin  concentrating  hormone)  have  been  prepared.  We  have  shown  for  the  first 
tine  that  the  atrial  natriuretic  factor  (ANF),  found  in  the  atrial  muscle  cells, 
has  a  widespread  distribution  in  the  brain.  ANF  nerve  fibers  and  cell  bodies 
were  observed  in  the  preoptic  area,  hypothalamus,  mesencephalon,  and  pons  of 
rats.  Cell  bodies  were  seen  in  the  organum  vasculosum  of  the  lamina  terminalis, 
in  several  hypothalamic  nuclei  and  in  the  dorsolateral  tegmental  nuclei.  The 
function  of  ANF  in  the  brain  is  unknown.  The  largest  accumulation  of 
ANF-positive  cells  is  contained  in  the  anteroventral  third  ventricle  (AVSV) 
which  is  a  critical  area  of  the  development  and  maintenance  of  experimental 
hypertension,  as  well  as  fluid  and  electrolyte  balance.  Therefore  ANF  was 
microi njected  into  this  brain  region  of  anesthetized  rats.  The  results  of  this 
study  reveal  that  pnol  quantities  of  ANF  produce  substantial  increases  in  both 
blood  pressure  and  heart  rate.  This  provides  evidence  for  a  functional  role  of 
ANF  in  the  AVSV  area  of  the  brain.  We  therefore  suggest  that  ANF  may  play  an 
important  role  in  central  cardiovascular  regulatory  mechanisms  (Skofitsch, 
Sills,  Zamir,  Eskay). 

A  detailed  stereotaxic  map  of  the  brain  distribution  of  calcitonin 
gene-related  peptide  (CGRP)  was  prepared.  A  widespread  distribution  of  imnuno- 
reactive  cells  and  fibers  was  observed  at  all  brain  levels  and  in  numerous  peri- 
pheral organs.  The  most  unique  feature  of  the  CGRP  neuronal  distribution  is  its 
presence  in  motor  neurons  of  the  brain  (III,  IV,  V,  VII,  XII)  and  in  the  ventral 
motor  horn  cells  of  the  spinal  cord.  This  is  the  first  peptide  found  to  coexist 
with  cholinergic  motor  cells  of  the  brain  and  spinal  cord.  The  possibility  of  a 
CGRP-like  peptide  in  the  action  of  acetylcholine  at  the  motor-end  plates  of 
striated  muscle  is  of  great  clinical  and  physiological  significance.  The  quan- 
titative distribution  of  CGKP  was  measured  by  RIA.  The  highest  concentrations 
were  observed  in  the  amygdala  centralis,  caudal  portion  of  the  caudate  putamen, 
nucleus  and  tractus  spinalis  of  the  trigeminal  nerve  and  dorsal  horn  of  the 
spinal  cord.  There  was  a  good  correlation  between  neuronal  density  and  RIA 
values.  To  complement  this  study,  the  autoradiographic  distribution  of 
l^^I-rat  CGRP  receptor  binding  sites  was  demonstrated.  A  comparison  of  the 
distribution  of  CGRP  binding  sites  to  the  localization  of  CGRP  fibers  and 
perikarya  revealed  that  most  of  the  areas  that  contained  CGRP  positive  nerve 
fibers  also  contained  appreciable  binding  sites  with  some  exceptions.  Very 
prominent  receptor  density  binding  sites  were  observed  in  the  nucleus  accunbens, 
amygdala  centralis,  ventral  caudate-putamen,  superior  colliculus,  medial  genicu- 
late body  and  spinal  cord  (Skofitsch). 

The  distribution  of  a  relatively  new  peptide,  galanin  (GA),  was  studied  in 
the  rat  brain.  GA-immunoreacti ve  cell  bodies  and  nerve  fibers  were  widely 
distributed  in  the  CNS.  Cell  bodies  were  observed  in  the  cortex,  preoptic  area, 
hypothalamus,  thalanus,  amygdala,  mesencephalon,  and  hindbrain.  Capsaicin 
sensitive  neurons  have  been  identified  in  the  trigeminal  and  dorsal  root 
ganglia,  in  the  dorsal  horn  of  the  spinal  cord,  substantia  gelatinosa,  the 
nucleus  and  tractus  of  the  spinal  trigeminal  nerve  and  the  nucleus  commis- 
suralis.  Since  it  is  well  documented  that  capsaicin  causes  selective  degenera- 
tion of  primary  sensory  neurons  of  sensory  ganglia,  it  is  suggested  that  GA  in 
capsaicin  sensitive  nerves  represents  a  novel  peptidergic  system  possibly 
involved  in  the  transformation  or  modulation  of  peripheral  nociceptive  impulses 
(Skcfi.sch), 

50 


A  new  peptide,   nelanin   concentrating  hornone   (MCH)  was   found  to  be  present 
in  the   rat  brain  by   immu no cytochemistry.     The  distribution   of  MCH-like  imnuno- 
reactivity   is  unique  and   different   from  the  distribution   of  other  neuropeptides. 
Cell   bodies  are  located  mainly  in  the  medial    forebrain   bundle  and  perifornical 
area.      Nerve  fibers  were   seen  throughout  the  rat   brain   in  all    neocortical    areas, 
the  neostriatum  and  the  amygdala,   in  the  diencephalon   in  most   hypothalamic 
nuclei,   mesencephalon   and  pons-medulla  and  spinal    cord.     The  immunohistochemical 
distribution  was   verified  by  RIA  of  microdissected  brain  areas.     Analysis   of   rat 
brain  extracts   by  HPLC  and  KIA   revealed  that   synthetic  MCH  elutes  more  than   15 
minutes   prior  to  the  MCH-like  immunoreacti ve  material    in  the   rat  brain.     Using 
parallel    displacement  techniques   in  RIA,   synthetic  MCH  and  tissue  extracts 
displaced   125i_jviqh  fpQ,^  the  antiserum  in  a  parallel    dose   response  curve.      It 
is  suggested  that  a  peptide  similar,   but   not   identical,   to  salmon  MCH  is   present 
in  the   rat   brain    (Skofitsch,   Zamir). 

Our  interest   in  the  coexistence  of  peptides   and  neurotransmitters   continues 
and  we  have  revealed  that   vasoactive  intestinal   polypeptide   (VIP)   coexists  with 
acetylcholinesterase   (AChE)   in   cholinergic  nerves   around  cerebral    arteries   at 
the   base  of  the  brain.     We  have  also  discovered  that  these  VIP-AChE  nerves   arise 
from  the  sphenopalatine  ganglion   since  lesions   of  this  ganglion   resulted   in  a 
marked  reduction   of  both   VIP  and  AChE  activity    (Hara). 

Colocalization  of   substance  P   (SP),   corticotropin  releasing  factor   (CRF), 
and  AChE  was   detected  by   retrograde  tracing  and  immunocytochemical    staining  in 
the  nucleus  tegmentalis  dorsalis   lateralis    (ntdl )   projecting  to  the  medial 
frontal   cortex   (MFC),   septum,   and  thalamus   of  the   rat.     These   results   suggest 
that  SP  and  CRF  coexist  within   a  subpopulation   of   ntdl    cholinergic  neurons   that 
project  to  a   number  of  forebrain   regions   including  the  MFC.     Behavioral    studies 
of  the  effects   of  SP,   CRF,   and  the  cholinergic  agonist,   carbachol,  employed 
microinjections  into  the  MFC  of  rats.     SP  and  CRF  did   not  elicit   any  behavioral 
effects  when  administered  alone.     Carbachol   produced  a  stereotyped  motor 
behavior,   consisting  of   rapid  forepaw  treading  while  in  a  upright   posture, 
resembling   "boxing."     SP   increased  carbachol -i nduced   "boxing."     CRF  decreased 
carbachol -induced   "boxing."     One  possible  functional   significance  of  the  coexis- 
tence of  SP,   CRF,   and  acetylcholine,   in  neurons   projecting  to  the  medial    frontal 
cortex  in   rats,   appears  to  be  a  modulatory  potentiation   of  cholinergic   response 
by  SP,   and  a  modulatory   inhibition   of  the  cholinergic   response  by  CRF. 

Using  two-dimensional    gel   electrophoresis    (2DE),  we  have  continued  our 
studies   of  proteins   in  the  CNS.     We  have  shown  that   a  number  of  proteins   are 
altered  in  concentration   in  the  parietal   cortex,   hippocampus   and  cerebellum 
following  either  bilateral    lesioning  of  the  locus  coeruleus  or  neonatal    treat- 
ment with  6-hydroxydopamine.     These   results   suggest  that  the  concentration   of  a 
number  of  different   proteins  may,   under  normal   physiological    conditions,   be 
regulated  in  vivo  by  norepinephrine  in  the  brain   (Heydorn). 

We  have  demonstrated  that   chronic  administration  of  the  type  A  monoamine 
oxidase  inhibitor  clorgyline  to   rats   for  three  weeks   produced  a   change  in  the 
relative  amount  of   five  proteins   in  the  parietal   cortex  but  only   a  single 
protein  in  the  hippocampus.     A  previous   study   indicated  that  the   relative 
concentration  of  three  different   proteins   are  significantly   altered   by  the 
repeated  administration   of  DMI.     These   results  are  consistent  with  the  idea  that 
different  classes   of   antidepressants   alleviate  depression  through  different 
mechanisms   of  action   (Sills,  Heydorn,  Cohen). 

51 


The  subcellular  localization  of  about  5U%  of  the  proteins  visible  on 
two-dimension  gels  generated  using  rat  brain  has  been  determined.  The  crude 
homogenate  of  rat  brains  was  fractionated  into  seven  different  subcellular 
components:  crude  synaptic  vesicles,  cytosol ,  microsomes,  mitochondria,  myelin, 
nucleus  and  synaptic  membranes.  The  proteins  of  each  fraction  were  separated  by 
2DE,  stained  with  silver  and  analyzed  by  computerized  densitometry.  Since  the 
fractionation  procedure  produces  enriched  fractions  but  not  a  total  separation 
of  individual  subcellular  components,  most  proteins  appeared  in  multiple  frac- 
tions. Of  lib  proteins  analyzed,  61  (53%)  were  determined  to  be  present  primar- 
ily in  a  single  fraction.  The  breakdown  by  fraction  was  as  follows:  cytosol 
(17%),  mitochondria  (8%),  microsomes  (8%),  nucleus  (8%),  crude  synaptic  vesicles 
(4%),  myelin  (<  1%),  synaptic  membranes  (<  1%).  This  identification  of  the 
subcellular  localization  of  individual  proteins  will  prove  valuable  in  efforts 
aimed  at  determining  the  function  of  each  protein  in  brain  tissue  (Heydorn). 

The  effect  of  salt  loading  and  water  deprivation  on  the  relative  rate  of 
turnover  of  individual  proteins  within  the  subfornical  organ  of  the  rat  was 
investigated.  For  four  days,  three  groups  of  rats  (control,  2%  NaCl ,  and  water 
deprived)  were  given  an  appropriate  fluid  diet,  with  all  groups  having  free 
access  to  food.  Rats  were  killed  by  decapitation,  the  subfornical  organ  was 
quickly  dissected  out  and  incubated  for  6  hr  in  a  medium  containing  -^^S- 
methionine  and  ■^'^S-cystei  ne.  The  poteins  from  these  organs  were  then 
separated  by  two-dimensional  electrophoresis.  The  gels  were  then  stained, 
photographed,  dried  and  exposed  to  autoradiographic  film.  The  results  show  that 
the  relative  turnover  rate  of  eight  proteins  were  changed  due  to  the  experimen- 
tal manipulations.  Four  of  these  proteins  were  apparently  sensitive  to  both 
salt  loading  and  water  deprivation,  since  both  experimental  manipulations  caused 
a  change  in  the  relative  rate  of  protein  turnover.  Of  the  four  proteins,  three 
were  affected  by  water  deprivation  while  a  single  protein  was  sensitive  to  only 
salt  loading.  These  results  represent  the  first  biochemical  study  of  the  sub- 
fornical organ  in  vitro,  and  provide  information  as  to  the  biochemical  effects 
of  changes  in  fluid  homeostasis  on  the  subfornical  organ  (Heydorn,  Klein). 

Two-dimensional  gel  electrophoresis  with  silver  staining  was  used  to  study 
protein  patterns  in  20  high-grade  human  astrocytomas  (anaplastic  astrocytomas 
and  glioblastomas)  obtained  during  surgery.  Histological  correlates  of  the 
sampled  tissue  were  carefully  established.  There  was  a  general  consistency  in 
the  protein  pattern  from  one  sample  to  the  next,  despite  variations  in  certain 
spot  densities.  When  these  patterns  were  compared  to  those  of  normal  human 
cerebral  cortex,  several  proteins  (most  notably  albumin  and  glial  fibrillary 
acidic  protein)  were  clearly  more  prominent  in  the  tumor  gels,  while  others  were 
comparatively  diminished.  Several  of  the  prominent  protein  spots  including 
albumin,  actin,  alpha  and  beta-tubul i n,  neuron  specific  enolase,  glial  fibril- 
lary acidic  protein  and  glutamic  oxaloacetic  transaminase  have  been  identified. 
A  major  strength  of  this  technique  lies  in  its  capacity  to  semiquantitati vely 
display  a  large  number  of  proteins  simultaneously,  using  just  a  few  milligrams 
of  tissue.   Its  potential  applications  to  diagnosis  and  to  the  study  of  tumor 
biology  are   under  investigation  (Narayan,  Heydorn). 

Using  a  sensitive  and  specific  antiserum,  the  existence  of  two  subforms  of 
soluble  glutamic  oxaloacetic  transaminase  in  both  rat  and  human  brain  has  been 
demonstrated.   In  the  rat,  the  two  proteins  each  have  a  molecular  mass  of  47,000 
daltons.  The  more  abundant  basic  protein  has  an  isoelectric  point  of  5.9  while 
the  sparsely  staining  more  acidic  protein  has  an  isoelectric  point  of  5.8.   In 


52 


the  human  the  two  proteins  visualized  each  have  a  nolecular  mass  of  43, QUO 
daltons.  The  more  abundant  basic  protein  has  an  isoelectric  point  of  5.7  while 
the  sparsely  staining  nore  acidic  protein  has  an  isoelectric  point  of  5.6.  In 
both  rat  and  human,  it  seems  reasonable  to  conclude  that  the  abundant  basic 
protein  that  reacts  with  the  antiserum  is  the  alpha  subform  of  the  enzyme,  while 
the  sparsely  staining  more  acidic  protein  is  the  beta  subform  of  the  enzyme. 
These  results  are  of  interest  for  a  number  of  reasons.  First,  they  establish 
the  existence  of  at  least  two  subforms  of  soluble  glutamic  oxaloacetic  trans- 
aminase in  both  rat  and  human  brain.  Secondly,  they  show  that  this  enzyme  is  a 
major  protein  visible  on  two-dimension  gels  of  rat  and  human  brain,  and  that 
this  enzyme  is  widely  distributed  throughout  the  central  nervous  system. 
Finally,  they  positively  identify  two  more  proteins  visible  on  two-dimension 
gels  generated  using  rat  and  human  brain  tissue  (Heydorn). 


53 


1985  Annual  Report  for  the  Child  Psychiatry  Branch 

National  Institute  of  Mental  Health 

October  1,  1984  -  September  30,  1985 

Judith  L.  Rapoport,  M.  D.,  Chief 


The  Child  Psychiatry  Branch,  formed  in  October  1,  1984,  conducts  research 
on  biological  aspects  of  child  psychiatry.   Response  to  pharmacologic  agents 
are  current  tools  for  this  research,  as  well  as  newer  neuroradiological 
techniques-   Because  research  in  child  psychopathology  is  at  an  early  stage 
of  its  development,  a  great  deal  of  effort  has  gone  into  developing  methodology 
for  clinical  research.   This  includes  standardization  of  attentlonal  tasks, 
ward  ratings,  and  activity  monitoring.   For  children  with  obsessive  compulsive 
disorder,  rating  scales  and  structured  interviews  have  been  devised  and  age 
norms  obtained. 

Several  collaborators  from  three  other  Institutes  and  from  NIMH  have 
participated  in  these  child  psychiatric  studies.   The  NINCDS  (Drs.  Denckla, 
Fedio,  and  Ludlow),  NIA  (Dr.  Stanley  Rapoport  and  staff),  NIAAA  (Dr.  Markku 
Linnoila),  as  well  as  other  branches  within  the  NIMH:   LPP,  LCS,  BPB, 
and  NPB.   In  addition,  our  collaborations  with  other  institutions  are:  Dr. 
Nancy  Andreasen  (University  of  Iowa,  School  of  Medicine),  Dr.  David  Shaffer 
(Columbia  University  School  of  Medicine),  and  Drs.  Michael  Rutter  and  Eric 
Taylor  (The  Maudsley  Hospital,  London,  England),  all  of  whom  are  involved  in 
ongoing  projects. 

The  Anxiety  Disorders  of  Childhood  are  being  studied  with  a  five  year 
follow-up  study  of  children  and  adolescents  with  Obsessive  Compulsive  Disorder, 
as  well  as  an  epidemiological  study  of  this  disorder  in  a  high  school 
population.   Children  with  generalized  anxiety  disorder  are  also  being  studied 
with  caffeine  challenges  to  see  if  there  is  differential  sensitivity  to 
dietary  caffeine  compared  with  normal  controls. 

Our  focus  has  been  the  hyperactive  child,  that  is  children  with  the  DSM 
III  diagnosis  of  Attention  Deficit  Disorder  with  Hyperactivity.   Ongoing 
research  has  systematically  investigated  monoamine  dysfunction  in  hyperactive 
children  utilizing  relatively  specific  neuropharmacological  probes.   While  it 
is  probable  that  multiple  neurotransmitter  systems  are  involved,  there  is 
increasing  evidence  for  noradrenergic  dysfunction  and/or  noradrenergic  med- 
ication of  stimulant  drug  effects.   New  neuroradiological  and  electrophys- 
iological brain  imaging  techniques  are  being  used  increasingly.   In  one  study, 
fathers  of  hyperactive  boys  who  were  themselves  hyperactive  as  children,  are 
being  examined  with  Positron  Emission  Tomography  (PET).   Children  with  specific 
developmental  disabilities  are  a  particularly  appropriate  group  for  such 
studies,  as  their  disabilities  suggest  specific  neurological  localization. 
A  new  series  of  studies  has  been  Initiated  in  which  children  and  adults 
having  pervasive  and  specific  developmental  disabilities  are  examined  with  a 
variety  of  brain  imaging  techniques  including:  NMR,  cortical  evoked  potential, 
and  EEC  tomographic  mapping.   For  an  adult  dyslexic  group,  NMR,  cerebral 
blood  flow  (xenon  inhalation),  CT  scan  and  electrophysiological  mapping  are 
being  compared.   The  strength  of  these  studies  is  that  several  imaging  techni- 
ques are  available  at  the  National  Institute  of  Mental  Health,  and  can  be 
compared  in  a  single  sample . 


55 


ANNUAL  REPORT  OF  THE  CLINICAL  NEUROSCIENCE  BRANCH 
National  Institute  of  Mental  Health 
October  1,  1984  -  September  30,  1985 

Steven  M.  Paul,  M.D.,  Chief 

Introduction 

The  Clinical  Neuroscience  Branch  conducts  an  interdisciplinary  research 
program  in  the  neurosciences  with  a  major  emphasis  on  etiology,  pathophysiology, 
and  treatment  of  the  major  neuropsychiatric  disorders.  The  Branch  consists  of 
four  sections,  1)  the  Section  on  Preclinical  Studies  (Steven  M.  Paul,  M.D., 
Chief),  2)  the  Section  on  Brain  Biochemistry,  (Candace  B.  Pert,  Ph.D.,  Chief), 
3)  the  Section  on  Molecular  Pharmacology  (Steven  M.  Paul,  M.D.,  Acting  Chief), 
and  4)  the  Section  on  Clinical  Studies  (David  Pickar,  M.D.,  Chief).  In 
addition,  two  units  have  been  formed  in  the  Section  on  Molecular  Pharmacology: 
1)  the  Unit  on  Behavioral  Pharmacology  (Jacqueline  N.  Crawley,  Ph.D.,  Chief); 
and  2)  the  Unit  on  Histochemistry  and  Electrophysiology  (Drs.  Daniel  Hommer, 
M.D.,  and  Lana  Skirboll,  Ph.D.;  Chief).  The  current  research  focus  of  the 
members  of  the  Branch  are  quite  diverse  and  although  an  attempt  is  made  to 
emphasize  studies  related  to  the  basic  pathological  (or  pathophysiological) 
processes  underlying  neuropsychiatric  disease,  individual  investigators  are 
encouraged  to  work  on  any  problem  related  to  the  basic  mechanisms  of  brain 
function.  In  general,  the  major  areas  of  investigation  involve  characterizing 
critical  aspects  of  synaptic  transmission  including  the  various  neurotransmitter 
and  neuromodulator  substances  themselves  as  well  as  their  biosynthetic  and 
metabolic  enzymes. 

Over  the  past  12  months  the  personnel  in  the  Branch  has  remained  rather 
stable  and  the  major  new  scientific  initiatives  of  the  previous  year  including 
those  in  neuroimmunology,  molecular  neurobiology,  and  behavioral  pharmacology 
have  been  extended.  A  brief  summary  of  the  various  research  projects  and  some 
of  the  results  to  date,  follows. 

SECTION  ON  PRECLINICAL  STUDIES 

Steven  M.  Paul  ,  M.D.,  Chief 

Members  of  the  Section  on  Preclinical  Studies  continue  to  study  the 
mechanisms  of  action  of  important  psychotropic  agents.  The  principal  drugs 
under  investigation  include  the  minor  tranquilizers,  such  as  the  benzodiazepines 
and  barbiturates,  as  well  as  the  tricyclic  antidepressants,  and  various 
psychomotor  stimulants,  including  amphetamine  and  methyl pheni date.  Over  the 
past  year  our  work  on  the  benzodiazepine/GABA  receptor  complex  has  continued 
with  a  primary  emphasis  on  studies  measuring  GABA-stimulated  chloride  (CI") 
flux  in  a  subcellular  brain  preparation,  the  synaptoneurosome.  During  the 
previous  year.  Dr.  Rochelle  Schwartz  succeeded  in  developing  a  reliable  method 
for  measuring  GABA  receptor  and  barbiturate  receptor-mediated  CI"  efflux  and 
uptake.  Using  this  method.  Dr.  Schwartz  has  shown  that  pentobarbital  produces  a 
concentration-dependent  increase  in  the  efflux  or  uptake  of  radioactive 
chloride,  (36ci-)  from  preloaded  synaptoneurosomes;  and  the  effects  of 
barbiturates  are  reversed  by  the  CI"  channel  antagonist  picrotoxin.  A  good 
correlation  was  observed  between  the  potencies  of  a  series  of  barbiturates  in 


57 


stimulating  36^]-  efflux  from  preloaded  synaptoneuromes  and  their 
anesthetic  potencies  in  mice.  Furthermore,  their  potencies  in  stimulating 
CI"  eflux  were  also  highly  correlated  with  their  ability  to  al losterical ly 
enhance  the  binding  of  [3h]  benzodiazepines  to  the  benzodiazepine  receptor. 
In  addition  to  barbiturates,  GABA  receptor  agonists  such  as  muscimol,  THIP, 
and  GABA  itself  also  increase  36  ci-  efflux,  and  this  effect  is  reversed 
by  the  GABA  receptor  antagonist,  bicuculline.  A  series  of  experiments  has 
established  the  validity  of  this  method  for  measuring  the  functional  activity  of 
the  GABA-gated  ion  channel.  During  this  past  year,  a  number  of  experiments  on 
the  sensitization  and  desensitization  of  this  receptor  complex  have  been  carried 
out.  Using  radioligand  binding  studies  it  was  shown  that  the  acute  exposure  of 
rats  to  a  stressful  environmental  stimulus  (i.e.  ambient  swim  stress)  results 
in  a  marked  enhancement  of  the  ability  of  various  halides  (including  chloride 
and  iodide)  to  stimulate  specific  [^H]  flunitrazepam  binding  to  subsequently 
prepared  brain  membranes.  Moreover,  the  specific  binding  of 
35s-t-butylbicyclophosphorothionate  (TBPS)  a  ligand  that  binds  to  the 
picrotoxin  recognition  site  on  the  chloride  ionophore  is  greatly  augmented  in 
membranes  prepared  from  "stressed"  animals.  Taken  together,  these  studies 
suggested  that  a  functional  activity  of  the  GABA-gated  ion  channel  is  altered 
following  brief  exposure  to  "stress".  Subsequent,  studies  using  the  36^]- 
flux  method  demonstrated  that,  indeed,  the  ability  of  GABA  to  stimulate  chloride 
flux  was  markedly  enhanced  in  animals  exposed  to  "stress".  Moreover,  animals 
habituated  to  stress  by  repeated  handling  over  the  preceding  two  weeks 
demonstrated  a  marked  decrease  in  sensitivity  of  the  GABA-gated  channel  (i.e. 
right  "shift"  of  the  dose  response  curve  for  muscimol).  The  mechanisms  for  the 
rapid  "sensitization"  and  "desensitization"  of  the  GABA  receptor  complex  are 
unknown  and  are  currently  being  pursued.  Using  this  method  we  have  also 
demonstrated  that  very  brief  exposure  to  various  agonists  (i.e.  GABA,  muscimol 
or  pentobarbital)  produces  a  rapid  desensitization  of  the  receptor,  which  is 
similar  to  what  has  been  reported  in  electrophysiological  experiments  with  these 
compounds.  Thus,  this  method  should  prove  useful  for  exploring  the  mechanisms 
of  desensitization  of  this  ion-gated  receptor  channel. 

Recently,  Dr.  Peter  Suzdak  has  shown  that  ethanol ,  an  important 
psychotropic  agent  which  shows  cross-tolerance  and  cross-dependence  with 
benzodiazepines,  and  barbiturates  also  stimulates  CI"  flux  in  a  picrotoxin  and 
bicucull  ine-sensitive  fashion.  The  effects  of  ethanol  in  stimulating 
36ci-  uptake  occurred  at  concentrations  that  were  well  within  those 
observed  during  pharmacologically-relevant  administration  of  this  compound 
either  to  animals  or  man.   In  addition,  at  sub-intoxicating  concentrations 
ethanol  markedly  potentiated  the  ability  of  both  muscimol  and  pentobarbital  to 
stimulate  GABA-mediated  CI"  flux.  These  data  suggest  that  ethanol  acts  like  a 
barbiturate  in  having  a  biphasic  action  on  GABA  receptor  mediated  CI" 
conductance.  Taken  together  with  many  previous  studies  implicating  GABAergic 
transmission  in  the  major  action  of  ethanol  our  results  suggest  that  at  least 
some  of  the  major  pharmacological  actions  of  ethanol  occur  via  the  GABA  receptor 
complex.  More  recently.  Dr.  Suzdak  has  studied  a  series  of  alcohols  of  varying 
chain  length  and  has  observed  a  good  correlation  between  their  intoxication 
potencies  and  their  potencies  in  stimulating  36^1-  uptake  in  a 
picrotoxin-sensitive  fashion.  A  series  of  experiments  have  been  initiated  on 
the  role  of  membrane  phospholipids  in  the  desensitization  and  sensitization  of 
the  GABA  receptor  complex,  including  the  possible  activation  of  phosphol ipase 
A2  in  the  desensitization  of  the  GABA  receptor. 

58 


In  another  series  of  studies  exploring  the  possible  modifications  of  the 
GABA  receptor  complex  by  stress-related  hormones,  Dr.  Dorota  Majewska  has  been 
pursuing  the  interaction  of  naturally-occurring  steroid  hormones  with  the 
receptor  complex.  Over  the  past  12  months  she  and  her  colleagues  have  shown 
that  the  natural ly-occuring  metabolites  of  progesterone 
(3a,5a-dihydroprogesterone)  and  deoxycorticosterone 
(3a-tetrahydrodeoxycorticosterone)  are  rather  potent  ligands  of  the 
GABA/barbiturate  receptor  complex.  Both  of  these  steroids:  displace 
[35s]-TBPS  binding  with  ICsq's  of  100-200  nM,  stimulate  36  ci- 
uptake  into  synaptoneurosomes,  allosterically  activate  [^H]  flunitrazepam 
binding  and  in  electrophysiological  studies  (carried  out  in  collaboration  with 
Dr.  Neil  Harrison  and  Jeffrey  Barker)  stimulate  CI"  conductance  and  potentiate 
GABA-mediated  Cl~  conductance  in  primary  cultures  of  hippocampal  and  spinal 
cord  neurons.  These  data  suggest  that  these  naturally-occurring  (ring  A 
reduced)  steroids  act  as  barbiturate-like  ligands  of  the  GABA  receptor  complex, 
perhaps  released  during  stress  to  modulate  GABA  mediated  synaptic  events. 

Using  a  recently  cloned  cDNA  probe  for  glutamic  acid  decarboxylase  (GAD) 
the  biosynthetic  enzyme  for  GABA,  Drs.  John  Thomas,  Yong  Sik  Kim,  and  Peter 
Suzdak  have  been  studying  the  turnover  of  this  enzyme  under  a  variety  of 
pharmacological  and  physiological  conditions.  To  date,  the  methods  for  carrying 
out  dot  blot  and  Northern  blot  hybridization  experiments  using  this  cDNA  probe 
have  been  refined  and  these  experiments  are  in  progress. 

The  peripheral  benzodiazepine  receptor  (which  does  not  appear  to  be 
structurally  related  to  central  benzodiazepine  receptors)  has  been  further 
characterized  in  our  laboratory  over  this  past  year.  In  collaboration  with  Drs. 
Pert,  Ruff  and  Weber  (the  Section  on  Brain  Biochemistry)  we  have  demonstrated 
that  the  peripheral  benzodiazepine  receptor  ligand  Ro5-4864  is  a  potent 
chemotactic  agent  in  human  macrophages.  Moreover,  the  effects  of  Ro5-4864  in 
stimulating  macrophage  chemotaxis  are  reversed  by  the  selective  peripheral 
benzodiazepine  receptor  antagonist  PK-11195.  Other  benzodiazepines  that  bind  to 
the  peripheral  benzodiazepine  receptor,  such  as  diazepam,  also  stimulate 
chemotaxis  in  a  PK-11195-sensitive  fashion.  The  ability  of  peripheral 
benzodiazepine  receptor  agonists  to  stimulate  macrophage  chemotaxis  are 
reminiscent  of  a  variety  of  other  neuropeptides  and  have  prompted  further 
studies  on  the  purification  of  a  substance(s)  from  a  variety  of  tissues  that 
inhibits  radioligand  binding  to  the  peripheral  benzodiazepine  receptor. 
Previous  studies  in  our  laboratory  have  shown  that  acidified  methanol  or 
trichloroacetic  acid  extracts  of  peripheral  tissues  contains  a  substance  which 

nhibits  [^H]  R05-4864  binding  to  peripheral  benzodiazepine  receptors.  Over 
the  past  year  considerable  effort  in  purifying  this  high  molecular  weight  (viz. 
14  to  15  Kd)  protein  has  resulted  in  the  isolation  of  an  almost  pure  protein 
which  potently  displaces  radioligand  binding  to  peripheral  benzodiazepine 
receptors  as  well  as  to  voltage-dependent  calcium  channels.  A  novel  technique 
for  rapidly  purifying  this  protein  has  been  developed  and  has  facilitated  its 
biochemical  and  pharmacological  characterization.  Recent  studies  by  Drs. 
Charles  Mantione,  Gordon  Bolger,  and  Mark  Goldman,  have  revealed  that  this 
material  called  antral  in  mimics  the  iontropic  actions  of  the  calcium  channel 
antagonists  in  the  guinea  pig  atrium.  In  addition,  the  effect  of  antral  in  in 
reducing  isomeric  contraction  of  the  guinea  pig  atrium  was  reversed  by  the 
peripheral  benzodiazepine  receptor  antagonist  PK-11195  as  well  as  the 
voltage-dependent  channel  agonist  BAY  k864.  Our  data  suggest;  1)  the  presence 
of  naturally-occurring  proteins  or  peptides  that  regulate  voltage-dependent 

59 


calcium  channels  and  peripheral  benzodiazepine  receptors,  and  2)  that  the 
voltage-dependent  calcium  channel  and  peripheral  benzodiazepine  receptor  are 
functionally  and  structurally  coupled.  The  latter  hypothesis  is  also  supported 
by  studies  from  several  other  laboratories.  Current  work  on  antralin  includes 
determining  its  amino  acid  composition  and  partial  sequence.  These  data  will 
hopefully  allow  us  to  screen  several  cDNA  libraries  and  determine  the  entire 
structure  of  this  protein  as  well  as  study  its  possible  role  in  various 
physiological  states. 

This  year   has  also  seen  an  extension  of  a  project  initiated  several  years 
ago  on  the  specific  binding  of  [3H](+)-amphetamine  to  brain  membranes. 
Previous  research  in  our  laboratory  indicated  that  the  [3H](+)-amphetamine 
binding  site  may  be  involved  in  the  anorectic  properties  of  a  series  of 
phenyl  ethyl  amine  derivatives  including  amphetamine,  mazindol,  and 
parachloramphetamine.  In  a  confirmation  of  this  hypothesis.  Dr.  Itzchak  Angel 
has  succeeded  in  labelling  these  sites  with  a  slightly  higher  affinity  ligand 
[3h]  mazindol.  The  characteristics  of  the  [-^H]  mazindol  binding  site  are 
virtually  identical  to  those  of  the  [3h]  amphetamine  binding  site.  Moreover, 
in  studying  the  displacement  potencies  of  a  series  of  anorectic 
phenyl  ethyl  amines.  Dr.  Angel  found  a  highly  significant  correlation  (r  =  .87, 
p  <  0.01)  between  their  IC50  values  and  their  potencies  in  decreasing  food 
intake  in  food-deprived  rats.  Dr.  Angel  has  also  observed  that  the  density  of 
these  binding  sites  is  rapidly  altered  during  periods  of  food  deprivation  and 
refeeding  in  rats  and  that  these  changes  are  highly  correlated  to  circulating 
blood  glucose  concentratioin.  In  related  studies.  Dr.  Angel  using  selective 
neurotoxins  has  shown  that  specific  lesions  of  the  serotonergic  system  in  the 
hypothalamus  (using  5,7-dihydroxytryptamine) ,  as  opposed  to  lesions  of  the 
noradrenergic  system  with  6-hydroxydopamine,  results  in  a  significant  increase 
in  the  density  of  [^h]  mazindol/C^H]  amphetamine  binding  sites.  These 
studies  indicate  that  serotonin  may  be  involved  in  tonically  inhibiting  the 
number  of  these  binding  sites  in  hypothalamus.  Dr.  Angel  has  also  pursued  his 
previous  studies  on  the  glucostatic  regulation  of  these  sites  in  vitro.  He  has 
shown  that  the  [3h  mazindol /amphetamine  binding  site  is  stimulated  in 
hypothalamic  slices  by  physiological  concentrations  of  glucose  and  that  this 
effect  is  highly  correlated  to  changes  in  Na'^'K"''  ATPase  activity.  The 
glucostatic  regulation  of  these  sites  are   altered  (subsensitive)  in  the 
genetically  obese  mouse.  We  are  currently  attempting  to  identify  the  exact 
nature  of  the  [3h]  mazindol  binding  site.  Recent  evidence  suggests  that  these 
sites  are   localized  to  a  metabolic  enzyme  that  is  highly  concentrated  in  brain 
membranes. 

SECTION  ON  CLINICAL  STUDIES 

David  Pickar,  M.D.,  Chief 

Research  on  the  neurobiology  of  schizophrenia  continues  to  be  a  major 
effort  of  the  Section  on  Clinical  Studies.  Our  previously  reported  finding  of  a 
neuroleptic-induced  decrease  in  the  levels  of  plasma  homovanillic  acid  (HVA)  in 
schizophrenic  patients  has  now  been  replicated  in  a  larger  series  of  patients 
and  the  results  further  suggest  that  changes  in  the  levels  of  plasma  HVA  may  be 
predictive  of  neuroleptic  response  in  individual  patients.  Studies  of  the 
diurnal  rhythm  in  plasma  HVA  by  Dr.  Allen  Doran  and  colleagues  have  revealed 
differences  between  schizophrenic  and  normal  subjects.  In  other  studies  the 


60 


effects  of  neuroleptics  on  positive  and  negative  symptoms  of  schizophrenia  are 
being  examined  and  preliminary  data  has  indicated  the  therapeutic  effectiveness 
of  the  novel  triazolobenzodiazepine,  alprazolam,  as  an  adjunct  to  neuroleptics 
in  treating  some  schizophrenic  patients.  Further  work  in  these  areas  is  in 
progress. 

Data  from  a  large  cross-sectional  study  of  depressed  patients  have 
continued  to  be  analyzed  and  prepared  for  publication.  We  have  observed 
hyperactivity  of  the  peripheral  sympathetic  nervous  system  in  patients  with 
melancholia  and  have  associated  this  finding  with  hyperactivity  of  the 
hypothalamic-pituitary-adrenal  axis.  An  interesting  interaction  between  the 
occurrence  of  prior  life  events  and  CSF  amine  metabolites  has  been  observed.  In 
a  new  but  related  area  of  research,  an  experimental  paradigm  for  studying 
"learned  helplessness"  in  humans  has  been  developed  by  Dr.  Alan  Breier. 
Preliminary  data  have  indicated  that  transient  "inescapable"  noise  administered 
to  normal  subjects  results  in  a  greater  HPA  axis  response  and  corresponding  mood 
changes  than  a  similar  "dose"  of  "escapable"  noise  stimulation.  This  model  of 
"learned  helplessness"  may  prove  useful  in  studying  the  stress-related  biologic 
diathesis  of  depressive  illness. 

Three  additional  research  areas  are  being  pursued.  Studies  of  the 
endogenous  opioid  system  (EOS)  have  focused  on  two  selected  areas:  EOS 
interaction  with  eating  behavior  and  regulation  of  the  HPA  axis.  A  recent 
finding  showing  naloxone-induced  decreases  in  caloric  intake  by  obese  subjects 
extends  our  earlier  findings  in  normals.  Enhanced  Cortisol  response  to  naloxone 
in  schizophrenic  patients  has  also  been  found,  consistent  with  the  longstanding 
hypothesis  of  enhanced  EOS  activity  in  schizophrenic  patients.  In  a  series  of 
experiments  coordinated  by  Dr.  Daniel  Hommer,  saccadic  eye  velocity  is  used  as  a 
physiologic  measure  of  benzodiazepine  receptor  sensitivity.  In  addition  to  a 
diazepam  dose  response  study,  the  ability  of  specific  and  non-specific 
antagonists  to  block  the  action  of  benzodiazepines  has  been  examined.  Finally, 
Dr.  Rex  Cowdry  has  completed  the  data  collection  phase  of  an  outpatient  study  of 
patients  with  borderline  personality  disorder  including  prospective,  "blind" 
treatment  trials  with  one  of  the  following  agents:  an  MAO  inhibitor 
tranylcypromine,  the  neuroleptic  trifluoperazine,  the  triazolobenzodiazepine, 
alprazolam  and  the  anticonvulsant  carbamazepine.  Therapeutic  effects  of 
carbamazepine  on  symptoms  of  behavioral  dyscontrol  have  been  observed. 

SECTION  ON  MOLECULAR  PHARMACOLOGY 


Steven  M.  Paul,  M.D.;  Acting  Chief 


The  Section  on  Molecular  Pharmacology  consists  of  two  independent  units, 
the  Unit  on  Histochemistry  and  Neurophysiology,  (Drs.  Lana  Skirboll  and  Daniel 
Hommer,  Chiefs)  and  the  Unit  on  Behavioral  Pharmacology  (Dr.  Jacqueline  N. 
Crawley,  Chief).  This  past  year  Dr.  Skirboll  has  continued  her 
immunohistochemical  studies  combined  with  retrograde  tracing  to  examine  the 
innervation  of  several  brainstem  nuclei.  In  several  brainstem  nuclei  it  is 
apparent  that  more  than  one  neurotransmitter  coexists  in  the  same  cell  body. 
Dr.  Skirboll 's  work  has  defined  the  various  neurotransmitter  substances  in  a 
single  cell  body  and  she  has  begun  to  ascertain  the  physiological  significance 
of  coexistence  in  this  area  of  brain.  In  other  studies.  Dr.  Skirboll  and 
colleagues  have  been  studying  the  paraventricular  nucleus  (PVN)  of  the 

61 


hypothalamus  and  especially  with  respect  to  innervating  transmitters  present  in 
the  afferents  to  this  area.     Dr.  Skirboll  and  her  colleagues  have  shown  that  a 
dense  epinephrine-containing  innervation  of  the  parvocell ular  subdivision  of  the 
PVN  is  involved  in  the  regulation  of  corticotropin  releasing  factor-containing 
neurons  in  this  area.  She  has  confirmed  using  retrograde  staining  with 
fluorescent  dyes  that  the  adrenalin  cells  of  the  PVN  originate  in  the  CI  and  C2 
areas  of  the  medulla  oblongata.  These  studies  are  important  in  that  they  define 
the  various  neurotransmitter  candidates  that  are  involved  in  the  release  of  CRF 
and  in  the  regulation  of  the  hypothalamic  pituitary  adrenal  axis.  In  a  \/ery 
recent  series  of  experiments.  Dr.  Skirboll  has  been  exploring  the  possibility 
that  dynorphin  and  GABA  coexist  in  the  same  neuron  in  a  projection  from  the 
caudate  nucleus  to  the  substantia  nigra  zona  reticulata.  The  descending  pathway 
from  the  striatum  to  the  substantia  nigra  has  been  well  known  to  be  an  important 
regulator  of  dopamine  containing  cell  bodies  in  the  substantia  nigra  and  recent 
evidence  from  electrophysiological  experiments  suggest  that  dynorphin 
iontophoretically  applied  to  nigral  neurons  may  alter  or  effect  the  inhibitory 
action  of  GABA  itself. 

Drs.  Hommer  and  Skirboll  along  with  Drs.  Robert  Drugan  and  Jacqueline 
Crawley  have  been  studying  the  effects  of  "stress"  on  the  sensitivity  of  GABA 
receptors,  localized  to  the  substantia  nigra  zona  reticulata.  To  date,  it 
appears  that  subjecting  animals  to  inescapable  aversive  tail  shock  results  in  a 
marked  supersensitive  response  to  intravenously  administered  muscimol  and  to 
iontophoretically  applied  GABA.  Animals  subjected  to  an  identical  shock 
exposure  (but  with  control  over  terminating  shock)  did  not  develop  this 
supersensitive  response  to  muscimol  or  GABA.  Thus,  uncontrollable  stress  lead 
to  an  increased  sensitivity  of  GABA  receptors  localized  on  the  substantia  nigra, 
zona  reticulata  neurons.  These  studies  are  among  the  first  to  attempt  to  study 
the  electrophysiological  consequences  of  uncontrollable  stress  versus 
controllable  stress.  A  variety  of  other  related  projects  concerning  the  effects 
of  anxiogenic  compounds  such  as  caffeine,  diazepam  binding  inhibitor  (DBI),  and 
nicotine  are  also  being  pursued  in  Drs.  Hommer  and  Skirboll 's  unit. 

In  addition  to  these  more  recent  studies,  Drs.  Skirboll  and  Hommer  have 
continued  their  work  on  cholecystokinin  (CCK)-dopamine  interaction  in  the 
substantia  nigra.  They  have  extended  their  previous  results  demonstrating  that 
ceruletide  and  sulfated  CCK-8  potentiate  the  effects  of  the  dopamine  agonist, 
apomorphine  on  DA  neurons  in  the  medial  substantia  nigra.  More  recently  they 
have  found  that  unsul fated  CCK-8  and  CCK-4  possess  a  similar  ability  to 
potentiate  apomorphine  induced  inhibition  in  this  area.  The  relative  potencies 
of  various  peptide  fragments  in  potentiating  apomorphine  induced  inhibition 
correlated  \/ery   well  with  their  affinity  for  brain  CCK  receptors.   In  contrast, 
only  CCK-8  sulfate  produced  an  excitatory  effect  on  DA  neurons,  suggesting  a 
relationship  with  the  peripheral  type  of  CCK  receptor.  Thus,  Drs.  Hommer  and 
Skirboll  have  confirmed  the  significance  of  the  brain-specific  CCK  receptor  as 
characterized  by  radioligand  binding  studies. 

Dr.  Jacqueline  N.  Crawley,  Chief,  Unit  on  Behavioral  Pharmacology  has  also 
been  studying  the  behavioral  consequences  of  coexisting  neurotransmitters.  She 
has  extended  her  previous  work  on  the  ability  of  cholecystokinin  to  potentiate 
dopamine-induced  hyperlocomotion  when  applied  discretely  in  the  nucleus 
accumbens  (as  opposed  to  the  striatum).  She  has  been  testing  a  variety  of 
putative  CCK  receptor  antagonists  using  her  behavioral  model  in  the  hope  of 

62 


identifying  a  substance  that  might  block  the  co-modulation  of  dopaminergic 
receptor  sensitivity  by  CCK.  More  recently,  in  collaboration  with  Dr.  David 
Jacobowitz,  she  has  demonstrated  a  coexisting  pathway  containing  substance  P, 
CRF  and  acetylcholine  which  projects  from  the  dorsal  lateral  tegmentum  to  the 
prefrontal  cortex  in  the  rat.  The  cholinergic  agonist  carbachol  when 
microinjected  into  the  prefrontal  cortex  induces  a  profound  stereotyped  mode  of 
behavior  called  "boxing",  and  this  carbachol-induced  behavior  was  potentiated  by 
substance  P.  More  recent  work  by  Drs.  Mendelson  and  Crawley  suggest  that  this 
"boxing"  type  of  behavior  is  due  to  the  local  formation  of  a  epileptogenic  foci 
in  the  cortex. 

In  addition  to  her  work  on  the  cholecystokinin-dopamine  interactions  in  the 
nucleus  accumbens.  Dr.  Crawley  has  been  working  on  several  other  projects  over 
the  past  year.  As  an  extension  of  her  previous  work  on  animal  models  of 
"anxiety"  she  has  developed  techniques  to  anatomically  localize  the  anxiolytic 
actions  of  benzodiazepines  as  well  as  GABAergic  agonists  such  as  muscimol.  To 
date,  she  and  Dr.  Drugan  have  observed  that  low  doses  of  muscimol  injected  into 
the  septal  region,  (an  area  that  projects  to  the  hippocampus  and  has  been 
previously  proposed  by  Jeffrey  Gray  as  being  in  the  limbic  circuitry  meeting  the 
anxiolytic  actions  of  benzodiazepines)  results  in  an  anticonflict  action  in  the 
Vogel  conflict  test.  Other  neuroanatomic  sites  of  purported  anxiolytic  activity 
are  being  tested  to  decipher  whether  the  anticonflict  effects  of  muscimol  in  the 
septal  region  are  specific  for  this  area. 

SECTION  ON  BRAIN  BIOCHEMISTRY 

Candace  B.  Pert,  Ph.D.,  Chief 

Members  of  the  Section  on  Brain  Biochemistry  have  been  involved  in  a  number 
of  studies  of  the  shared  neurotransmitter/neuromodulator  receptors  that  are 
common  to  both  neurons  and  cells  of  the  immune  system.  Drs.  Pert  and  Ruff  have 
been  using  the  human  monocyte  as  a  model  system  of  signal  peptide-induced 
chemotaxis  and  have  demonstrated  that  a  number  of  neuropeptides,  including 
beta-endorphin,  substance  P,  bombesin  and  cholecystokinin  stimulate  chemotaxis. 
In  addition,  Drs.  Ruff,  Pert,  Weber,  and  Paul  have  shown  that  peripheral 
benzodiazepine  receptor  agonists  are  also  potent  chemotractants.  Radioligand 
binding  studies  on  these  cells  have  directly  demonstrated  the  presence  of 
receptors  for  both  the  neuropeptides  and  benzodiazepines,  and  this  work  suggests 
that  cells  of  the  immune  system  are  affected  by  many  of  the  same  mediators  as 
those  in  nervous  system  and  immunological  network.  In  a  related  area  of  work, 
Drs.  Pert  and  Ruff  have  shown  that  human  alveolar  macrophages  store  and  secrete 
the  neuropeptide,  bombesin.  This  work  supports  their  major  hypothesis  published 
last  year  that  small  cell  lung  carcinoma  may  arise  as  an  abnormality  in  the 
hemopoietic  cell  when  the  normal  macrophage  mediated  repair  of  lung  tissues  is 
deranged  by  continuous  heavy  smoking,  rather  than  arising  from  lung  epithelial 
cells.  Dr.  Pert's  work  on  neuroimmunology  has  extended  into  a  number  of 
different  areas  including  the  mapping  of  receptor  sites  in  CNS  tissue  previously 
thought  to  be  localized  only  to  cells  of  the  immune  system.  Along  with  Drs. 
Richard  Weber  and  John  Knight,  Dr.  Pert  is  also  pursuing  the  hypothesis  that 
schizophrenia  may  have  an  autoimmune  component.  They  have  been  developing  a 
sensitive  assay  to  detect  antibodies  directed  against  human  brain  antigens  and 
have  found  such  antibodies  in  sera  of  schizophrenic  patients  as  well  as 
controls.  Studies  are  now  designed  to  ascertain  whether  these  antibodies  are 

63 


more  frequent  in  schizophrenic  patients  and  to  characterize  the  specific 
antigens  that  these  antibodies  are  directed  against. 

Another  major  area  of  investigation  by  Dr.  Pert's  group  over  the  past  year 
concerns  the  distribution  and  characteristics  of  opiate  receptors  in  various 
species.  In  collaboration  with  Drs.  Weber,  Zipser,  Fraser  and  Venter,  she  has 
succeeded  in  cross-linking  the  opiate  receptor  with  an  iodinated  derivative  of 
beta-endorphin.  These  studies  will  undoubtedly  aid  in  the  purification  of  the 
opiate  receptor  and  in  determining  the  presence  of  one  or  more  receptor 
subtypes.  To  date,  the  iodinated  cross-linked  products  in  Tetrahymena,  leach 
CNS  and  rat  brain  membranes  (carried  out  with  both  Type  I  and  Type  2  incubation 
conditions)  appear  indistinguishable  when  subsequently  visualized  on  SDS  Page 
gels;  with  the  major  cross-linking  products  having  molecular  weights  of  58  and 
100  kd.  These  experiments  demonstrate  that  significant  similarities  between 
opiate  receptors  from  mammals,  invertebrates,  and  a  unicellular  organisms.  In  a 
clinical  application  of  Dr.  Pert's  work  on  the  opiate  receptor  she  and  Drs.  Rice 
and  Burke  have  succeeded  this  past  year  in  developing  a  fluorinated  opiate 
receptor  antagonist  for  use  in  visualizing  the  opiate  receptor  using  positron 
emission  tomography  (PET).  This  opiate  probe  (3-acetylcyclofoxy)  has  been 
studied  in  rodents  and  subhuman  primates  and  clinical  studies  are   anticipated  in 
the  ensuing  year.  The  availablity  of  this  probe  should  provide  a  useful  tool 
for  studying  opiate  receptors  in  both  normal  individuals  and  those  with  various 
neuropsychiatric  conditions. 


64 


Annual  Report  of  the  Laboratory  of  Neuropsychology 

National  Institute  of  Mental  Health 

Mortimer  Mishkin,  Ph.D.,  Chief 

October  1,  1984  to  September  30,  1985 

Two  cortical  visual  pathways 

Cortical  tissue  essential  for  visual  perception  extends  far  beyond  the  primary 
visual  area,  striate  cortex,  to  include  not  only  the  prestriate  regions  of  the 
occipital  lobe  but  also  large  portions  of  the  temporal  and  parietal  lobes. 
Converging  evidence  from  neurobehavioral,  neuroanatomical,  and 
neurophysiological  studies  indicates  that  these  extrastriate  regions  contain 
numerous  areas  that  can  be  distinguished  both  structurally  and  functionally. 
Moreover,  the  multiple  visual  areas  appear  to  be  organized  hierarchically  into 
two  separate  cortical  visual  pathways,  each  having  the  striate  cortex  as  the 
source  of  its  initial  input. 

The  first  pathway  consists  of  an  occipitotemporal  projection  system.   This 
pathway,  which  courses  ventrally  to  interconnect  the  striate,  prestriate,  and 
inferior  temporal  areas,  is  crucial  for  the  visual  identification  of  objects. 
Links  between  this  occipitotemporal  pathway  and  limbic  structures  in  the 
temporal  lobe  as  well  as  ventral  portions  of  the  prefrontal  lobe  appear  to 
make  possible  the  cognitive  association  of  visual  objects  with  other  events, 
such  as  emotions  and  motor  acts.   The  other  pathway  consists  of  an 
occipitoparietal  projection  system.   This  pathway,  which  courses  dorsally  to 
interconnect  the  striate,  prestriate,  and  inferior  parietal  areas,  is  critical 
instead  for  the  visual  localization  of  objects.   Links  between  the 
occipitoparietal  pathway  and  both  dorsal  limbic  and  dorsal  prefrontal  cortex 
enable  the  cognitive  construction  of  spatial  maps,  as  well  as  the  visual 
guidance  of  motor  acts  that  may  have  been  triggered  initially  by  activity  in 
the  occipitotemporal  pathway.   In  contrast  to  the  occipitotemporal  pathway, 
which  remains  modality-specific  throughout  its  course,  the  later  stations  in 
the  occipitoparietal  pathway  appear  to  receive  convergent  input  from  other 
modalities  and  so  constitute  polysensory  areas. 

We  have  now  mapped  the  full  extent  of  the  two  cortical  pathways  combined 
through  application  of  the  [2--'-'^C]  deoxyglucose  method.   By  comparing  a 
visually  deafferented  and  a  normal  hemisphere  in  the  same  monkey,  we  have 
found  that  the  entire  visual  system  can  be  outlined  on  the  basis  of 
differential  hemispheric  glucose  utilization  during  visual  stimulation. 
Reduced  glucose  utilization  in  the  deafferented  as  compared  with  the  normal 
hemisphere  was  seen  cortically  throughout  the  entire  expanse  of  the  two  visual 
pathways  described  above.   In  addition,  the  limits  of  visually  related  tissue 
were  found  to  encompass  a  larger  cortical  territory  than  is  generally 
recognized  as  being  related  to  vision,  including  the  fundus  and  part  of  the 
upper  bank  of  the  intraparietal  sulcus,  the  posterior  part  of  the 
parahippocampal  gyrus,  and  the  lateral  bank  of  the  rhinal  sulcus.   Finally, 
tissue  related  to  vision  could  be  followed  subcortically  into  parts  of  the 
limbic  and  striatal  systems. 


65 


Anatomy  of  the  occipitotemporal  pathway 

To  trace  the  flow  of  visual  information  through  the  large  expanse  of  visual 
cortex  revealed  by  the  metabolic  mapping  studies,  we  have  undertaken  a  series 
of  anatomical  studies  using  both  anterograde  and  retrograde  tracing 
techniques,  such  as  autoradiography  and  horseradish  peroxidase  histochemistry, 
in  combination  with  electrophysiological  recording.   Our  goal  in  these  studies 
is  to  identify  the  multiple  visual  areas  that  comprise  this  cortex,  delineate 
their  topographic  organization,  and  explore  the  complex  circuitry  of  their 
interconnections.   To  establish  the  direction  of  flow  of  information  through 
these  cortical  visual  areas,  we  have  examined  the  laminar  pattern  of  their 
interconnections.    Thus,  "forward"  projections,  i.e.  projections  directed 
forward  in  the  brain  away  from  the  striate  cortex,  are  characterized  by 
connections  that  orginate  in  the  supragranular  layers  of  one  area  and 
terminate  in  granular  layer  4  and  the  lower  part  of  layer  3  of  another.   iiy 
contrast,  "backward"  projections,  or  projections  directed  backward  in  the 
brain  toward  the  striate  cortex,  are  characterized  by  connections  that 
originate  in  the  inf ragranular  layers  of  one  area  and  terminate  in  layers 
exclusive  of  granular  layer  4  of  another.   Determination  of  the  laminar 
patterns  of  the  interconnections  among  areas  thus  permits  an  analysis  of  the 
hierarchical  arrangement  of  the  entire  system. 

Our  results  indicate  that  the  occipitotemporal  pathway  begins  with  the  striate 
projection  to  the  second  and  third  visual  areas,  V2  and  V3,  which  in  turn 
project  to  area  V4.   These  three  prestriate  areas  are  arranged  in  adjacent 
belts  that  nearly  surround  the  striate  cortex,  and,  like  the  striate  cortex, 
each  belt  contains  a  representation  of  the  contralateral  visual  field.   Area 
V2  corresponds  to  cytoarchitectonic  area  OB,  while  V3  and  V4  together 
correspond  to  area  OA,  exclusive  of  its  dorsal  part.   The  major  output  of  V4 
is  to  areas  TEO  and  TE  in  the  inferior  temporal  (IT)  cortex.   Physiological 
studies  have  shown  that,  unlike  the  visual  areas  that  precede  it,  IT  cortex 
has  no  discernible  visuotopic  organization.   Rather,  IT  neurons  have  very 
large  receptive  fields  that  nearly  always  include  the  center  of  gaze  and 
frequently  cross  the  vertical  meridian  into  the  ipsilateral  visual  field. 
Interestingly,  IT  cortex  appears  to  receive  a  very  complex  pattern  of 
projections  from  V4,  with  a  single  site  in  V4  projecting  to  several  separate 
IT  fields  that  interdigitate  with  the  multiple  projection  fields  of  other 
sites  in  V4.   Thus,  there  may  be  a  mosaic  of  visual  areas  within  the  temporal 
lobe,  and  we  are  currently  investigating  this  possibility.   As  described 
later,  our  results  on  the  occipitoparietal  pathway  suggest  that  there  may  be  a 
mosaic  of  visual  areas  within  the  parietal  lobe  also.   Area  TE  m  IT  cortex 
appears  to  be  the  last  exclusively  visual  area  in  the  occipitotemporal  pathway 
for  object  recognition,  as  its  cortical  outputs  are  to  areas  in  the  temporal 
and  frontal  lobes  that  are  probably  multimodal  in  function. 

Although  an  occipitotemporal  pathway  consisting  of  multiple  visual  areas  has 
now  been  demonstrated  in  a  variety  of  monkey  species,  suggesting  a  common 
primate  plan,  this  notion  has  not  yet  been  applied  to  the  visual  system  in 
man.   Indeed,  concepts  regarding  the  organization  of  the  auman  visual  system 
have  not  progressed  much  since  the  turn  of  the  century.   For  example,  many 
complex  visual  syndromes  in  man,  including  visual  agnosia,  prosopagonosia,  and 
loss  of  recent  visual  memory,  are  still  being  attributed  in  the  clinical 
literature  to  lesions  of  the  inferior  longitudinal  fasciculus  CiLF),  commonly 
considered  to  be  a  long  association  fiber  bundle  interconnecting  the  occipital 

66 


and  temporal  lobes.   A  long  fiber  bundle  of  this  type  has  never  been  seen  in 
any  of  our  axonal  transport  studies  in  the  monkey,  however;  and,  indeed,  in 
blunt  dissections  of  both  human  and  monkey  brains  that  we  performed  recently 
to  reexamine  this  question,  we  found  that  the  only  long  fiber  bundle  common  to 
both  the  occipital  and  temporal  lobes  is  the  geniculostriate  pathway.   Our 
axonal  transport  experiments  in  the  monkey  indicate  that  the  pathway  from 
occipital  to  temporal  cortex  in  the  monkey  consists  of  a  series  of  U  fibers 
which  course  beneath  the  cortical  mantle  to  connect  adjacent  regions  in 
striate,  prestriate,  and  inferior  temporal  cortex.   By  implication,  the 
occipital  and  temporal  lobes  in  humans  are  similarly  connected  by  a  series  of 
U  fibers,  and,  therefore,  the  various  clinical  syndromes  that  are  still  being 
attributed  to  ILF  lesions  are  probably  due  instead  to  interruption  of  these 
short  U-fiber  connections  at  different  points  along  a  multisynaptic 
occipitotemporal  pathway.   Further  study  of  human  brain  tissue  is  being 
planned,  with  the  goal  of  identifying  architectonically  all  the  stations  in 
the  human  visual  system  that  are  homologous  to  those  that  have  now  been 
identified  in  the  monkey. 

The  occipitotemporal  pathway  and  the  analysis  of  color,  contour,  and  shape 

Area  V4.  Now  that  we  know  the  location,  visuotopic  organization,  and 
connections  of  many  of  the  extrastriate  areas  in  the  occipitotemporal  pathway, 
we  can  trace  the  transformation  of  visual  information  through  them  at  the 
single  neuron  level.   We  began  our  physiological  analyses  in  area  V4,  which 
plays  a  crucial  role  in  the  relay  of  visual  information  into  the  temporal 
lobe.   We  found  that,  as  in  striate  cortex,  some  neurons  in  V4  are  sensitive 
to  object  contours  and  low  spatial  frequencies,  whereas  others  are  more 
sensitive  to  textures  and  high  spatial  frequencies.   Thus,  the  data  on  V4 
suggest  that  within  each  visual  area  of  the  occipitotemporal  pathway,  the 
contours  of  object  surfaces  and  the  textures  of  object  surfaces  may  be 
processed  by  separate  populations  of  neurons.   Surface  color  also  appears  to 
be  processed  by  neurons  within  each  of  these  visual  areas.   The  results  argue 
against  the  prevalent  view  that  each  area  of  the  occipitotemporal  pathway 
processes  a  different  aspect  of  an  object  separately,  such  as  color  in  V4  and 
depth  in  V2,  and  suggest  instead  that  the  different  features  of  an  object  are 
processed  in  parallel  within  each  area.   At  the  same  time,  the  results  are 
consistent  with  our  anatomical  evidence  indicating  that  the  occipitotemporal 
pathway  is  organized  as  a  serial  hierarchy. 

A  hierarchical  model  predicts  that  the  product  of  visual  processing  will 
become  progressively  more  complex  at  each  successive  stage  in  the  pathway.   So 
far,  our  results  in  V4  support  this  prediction.   In  addition  to  sensitivity  to 
the  contour,  texture,  and  color  of  a  stimulus,  many  V4  neurons  respond  to  a 
stimulus  only  if  it  stands  out  from  its  background  on  the  basis  of  a 
difference  in  color  or  form.   This  responsivity  to  stimulus  differences  in  the 
receptive  field  is  due  to  a  unique  receptive-field  structure  of  the  neurons  in 
V4:  a  small  excitatory  receptive  field  surrounded  by  a  large,  silent, 
suppressive  zone.   The  surround  zone  is  silent  in  that  stimulation  of  it  alone 
is  without  effect,  but  it  is  also  suppressive  in  that  it  has  properties 
antagonistic  to  those  of  the  excitatory  field  and,  hence,  can  suppress  the 
response  to  an  excitatory-field  stimulus  if  the  surround  is  stimulated  in  the 
same  way.   Thus,  V4  neurons  may  play  a  role  in  separating  figure  from  ground, 
a  fundamental  requirement  for  object  perception. 


67 


Recently,  we  discovered  chat  the  suppressive  surrounds  of  V4  neuronal 
receptive  fields  extend  far  across  the  vertical  midline  into  the  opposite 
visual  hemifield  and  that  this  suppression  is  eliminated  by  section  of  the 
corpus  callosum.   Thus,  the  commissural  inputs  to  V4  appear  to  be  largely 
suppressive,  in  contrast  to  the  commissural  inputs  to  the  inferior  temporal 
cortex,  which  are  known  to  be  excitatory.   These  physiological  results  provide 
an  explanation  for  a  puzzling  fact,  namely,  that  despite  heavy 
interconnections  across  the  callosum,  prestriate  neurons,  unlike  neurons  in 
the  inferior  temporal  cortex,  have  excitatory  receptive  fields  that  are 
contralateral  only. 

Our  findings  that  the  corpus  callosum  provides  a  pathway  for  conveying 
inhibitory  influences  between  the  prestriate  cortices  of  the  two  hemispheres 
but  excitatory  influences  between  the  two  inferior  temporal  cortices  also 
resolves  a  paradox  that  had  arisen  from  our  2-deoxyglucose  studies.   The 
results  of  these  metabolic  experiments  provided  clear  evidence  of  commissural 
activation  of  the  inferior  temporal  cortex  but,  surprisingly,  not  of  any  part 
of  the  visual  system  posterior  to  it.   If  the  commissural  input  to  posterior 
visual  cortex  is  largely  inhibitory,  however,  the  functional  consequences 
might  well  not  be  reflected  in  glucose  metabolism. 

Inferior  temporal  cortex.  The  higher-order  cortical  visual  stations  for  the 
analysis  of  object  properties  are  located  in  the  inferior  temporal  (IT) 
cortex.   We  surveyed  the  response  of  IT  neurons  to  a  large  variety  of  stimuli, 
both  simple  and  complex,  and  found  that,  like  neurons  in  prestriate  area  V4, 
most  IT  neurons  give  at  least  a  small  response  to  many  stimuli  even  though 
they  respond  better  to  some  stimuli  than  to  others.   Presumably,  therefore, 
the  neural  representation  of  objects  in  IT  cortex  is  reflected  in  the  pattern 
of  activity  across  a  population  of  cells  and  not  in  the  activity  of  individual 
cells  that  respond  only  to  specific  objects. 

In  our  survey  of  IT  neuronal  responses,  we  found  that  many  cells  seemed  more 
sensitive  to  the  overall  shape  of  stimuli  than  to  the  location  and  quality  of 
individual  edges  and  contours.   Therefore,  in  a  subsequent  study  we  examined 
how  IT  cortex  might  extract  information  about  the  overall  shape  of  an  object 
from  information  about  its  boundary.   We  adopted  a  method  of  representing 
shapes  in  terms  of  shape  descriptors  that  have  been  used  in  computer 
pattern-recognition  systems.   The  results  were  very  encouraging  in  that  over 
half  the  cells  tested  in  IT  cortex  were  tuned  to  different  shape  descriptors. 
For  two-thirds  of  the  tuned  cells,  the  shape  of  the  tuning  curve  remained 
invariant  over  changes  in  the  size  of  the  stimulus  and  in  its  position  on  the 
retina.   These  results  support  the  possibility  that  the  visual  system,  and 
inferior  temporal  cortex  in  particular,  use  periodic  shape  descriptors  in 
classifying  objects. 

Although  it  is  an  assumption  of  most  current  electrophysiological  studies  that 
neurons  code  objects  on  the  basis  of  firing  rate,  it  is  possible  that  neurons 
use  a  more  complex  temporal  code.   To  examine  this  possibility  we  have 
developed  a  new  set  of  analytic  techniques  to  quantify  the  temporal  modulation 
of  the  neuronal  spike  train.   The  techniques  were  applied  to  recordings  from 
IT  neurons  of  awake  monkeys  that  were  presented  with  set  of  128  mathematically 
related,  two-dimensional  black  and  white  patterns.   The  patterns  formed  a 


68 


complete  basis  set;  that  is,  any  imaginable  2-dimensional  stimulus  could  in 
principle  be  represented  by  some  combination  of  the  patterns.   The  results 
suggest  that  IT  neurons  fall  into  two  classes  based  on  the  temporal  patterns 
of  their  stimulus-evoked  activity.   The  results  also  show  that  three  to  four 
parameters  are  needed  to  quantify  this  temporal  pattern,  and  the  parameters 
vary  independently.   Finally,  by  regarding  the  neuron  as  a  communication 
channel  that  transmits  information,  we  were  able  to  apply  the  formalism  of 
Shannon's  information  theory  to  assess  the  amount  of  information  neurons  could 
convey  if  they  were  using  a  count  code  (the  number  of  action  potentials),  and 
the  amount  they  could  convey  if  they  were  using  a  temporal  code  made  up  of 
three  basic  temporal  parameters  found  in  the  spike  trains.   This  analysis 
showed  that  the  amount  of  information  transmitted  via  the  temporal  code  was 
twice  as  great  as  that  tramsmitted  via  the  count  code.   Further,  the  analysis 
showed  that  the  number  of  action  potentials  is  actually  uncorrelated  with  the 
amount  of  information  transmitted.   Therefore,  the  common  assumption  that  the 
spike  count  is  a  direct  measure  of  information  about  the  stimulus  may  well  be 
incorrect.   The  multidimensional  nature  of  the  stimulus-dependent  temporal 
modulation  suggests  that  there  is  no  single  optimal  stimulus  for  a  neuron  and 
that,  rather,  each  neuron  transmits  useful  information  about  a  wide  range  of 
stimuli  using  a  multidimensional  code  which  is  multiplexed  in  the  spike 
train.   If  this  proposal  is  correct,  then  the  temporal  code  must  be  broken 
before  the  information  transmitted  by  individual  neurons  can  be  understood. 

The  occipitotemporal  pathway  and  selective  attention 

Our  retinas  are  constantly  bombarded  by  a  welter  of  shapes,  colors,  and 
textures.   Since  we  are  aware  of  only  a  small  amount  of  this  information  at 
any  one  moment,  most  of  it  must  be  filtered  out  centrally.   Yet,  this 
filtering  cannot  easily  be  explained  by  the  known  properties  of  the  visual 
system.   At  each  successive  stage  along  the  pathway  from  the  striate  cortex 
into  the  temporal  lobe  there  is  an  increase  in  receptive  field  size.   Many 
different  stimuli  will  typically  fall  within  these  large  receptive  fields,  and 
thus,  paradoxically,  more  rather  than  less  information  appears  to  be  processed 
by  single  neurons  at  each  successive  stage.   How  then  does  the  visual  system 
limit  processing  of  unwanted  stimuli?   The  results  of  our  single-neuron 
recording  experiments  in  visual  cortex  of  trained  monkeys  indicate  that 
unwanted  information  is  filtered  from  the  receptive  fields  of  neurons  in 
extrastriate  cortex  as  a  result  of  selective  attention. 

We  trained  monkeys  to  maintain  fixation  on  a  target  while  performing  a  task 
that  required  them  to  attend  selectively  to  stimuli  presented  at  one  visual 
field  location.   Irrelevant  stimuli  were  simultaneously  presented  at  a 
location  outside  the  monkey's  focus  of  attention.   When  stimuli  at  both  the 
attended  and  ignored  locations  were  simultaneously  present  within  the 
receptive  field  of  a  cell  in  either  area  V4  or  the  inferior  temporal  cortex, 
we  found  that  the  cell  responded  to  stimuli  at  the  attended  location.   For 
example,  if  the  cell  was  selective  for  red  stimuli,  it  would  respond  well  if  a 
red  stimulus  appeared  at  an  attended  location  but  poorly  or  not  at  all  if  a 
red  stimulus  appeared  at  an  ignored  location  even  though  it  was  still  in  the 
receptive  field.   Thus,  we  have  shown  for  the  first  time  that  the  processing 
of  unwanted  visual  stimuli  in  extrastriate  cortex  can  be  blocked  as  a  result 
of  selective  attention.   Our  most  recent  evidence  indicates  that  attention  can 
also  be  directed  to  a  specific  sensory  dimension  of  a  complex  stimulus,  such 
as  its  texture  or  shape,  and  that  the  neuronal  processing  of  irrelevant 

69 


information  may  thereby  be  reduced  even  further.   We  propose  that  it  is  these 
extrastriate  neural  mechanisms  for  selective  attention  that  enable  us  to 
identify  and  remember  the  properties  of  a  particular  stimulus  out  of  the  many 
that  may  be  acting  on  the  retina  at  any  given  moment. 

Although  we  have  found  that  the  inputs  to  cells  in  V4  and  ttie  inferior 
temporal  cortex  are  gated  by  attention,  we  have  not  yet  identified  the  source 
of  the  gate.   Our  anatomical  experiments  on  the  occipitotemporal  pathway 
indicate  that  the  major  source  of  visual  inputs  to  V4  is  area  V2,  yet  we  have 
not  found  any  effects  of  attention  in  either  the  latter  area  or  the  primary 
visual  cortex.   The  same  anatomical  studies  suggest,  however,  that  the 
pulvinar  could  be  the  source  of  the  attentional  gating  process.   The  pulvinar 
receives  visuotopically  organized  projections  from  all  of  the  known  visual 
cortical  areas,  including  VI,  V2,  and  V4,  and  there  is  a  similar  visuotopic 
arrangement  of  pulvinar  projections  back  to  the  cortex.   Thus,  area  V4 
receives  from  the  visual  areas  behind  it  two  inputs  that  are  in  perfect 
visuotopic  register,  one  direct,  or  corticocortical ,  and  the  other  indirect, 
relayed  through  the  pulvinar.   If  the  pulvinar  were  also  the  recipient  of 
signals  from  outside  the  visual  system  (e.g.  signals  related  to  instructions, 
sets,  etc.),  then  this  thalamic  nucleus  and  its  projections  could  provide  a 
mechanism  by  which  such  nonvisual  signals  could  acquire  visual  field 
specification,  that  is,  a  mechanism  that  determines  where  in  the  field  the 
animal  will  attend. 

The  occipitoparietal  pathway  for  spatial  vision 

Our  results  indicate  that  the  occipitoparietal  pathway  begins  with  striate, 
V2,  and  V3  projections  to  visual  area  MT,  which  is  located  in  the  caudal 
portion  of  the  superior  temporal  sulcus,  mainly  within  the  dorsolateral 
portion  of  cytoarchitectonic  area  OA.   MT  projects  to  four  additional  areas 
located  in  the  intraparietal  and  superior  temporal  sulci,  and  these  project  in 
turn  to  the  inferior  parietal  lobule,  area  PG.   Thus,  although  MT  receives 
inputs  from  areas  that  participate  in  the  occipitotemporal  pathway,  its 
outputs  appear  to  be  directed  mainly  into  the  parietal  lobe. 

Our  physiological  experiments  along  the  occipitoparietal  pathway  indicate  that 
MT  and  the  areas  to  which  it  projects  may  constitute  a  cortical  system  for 
motion  analysis.   In  MT,  which  is  known  to  be  insensitive  to  both  the  form  and 
color  of  a  stimulus  but  highly  sensitive  to  the  direction  of  stimulus  motion, 
we  have  found  an  elaborate  columnar  system  for  direction  of  motion,  comparable 
to  the  columnar  system  for  orientation  in  striate  cortex  discovered  by  Hubel 
and  Wiesel.   Within  a  vertical  column  in  MT,  all  cells  respond  to  the  same 
direction  of  motion.   Across  the  columns,  the  preferred  direction  of  motion 
changes  systematically,  resulting  in  a  clock-like  representation  of  direction. 

Direction-of  motion-information  analyzed  within  MT  appears  to  be  further 
elaborated  in  subsequent  areas  of  the  occipitoparietal  pathway.   At  each 
successive  stage,  neurons  integrate  motion  information  over  increasingly  large 
portions  of  the  visual  field,  respond  selectively  to  more  complex  types  of 
motion,  and  respond  to  inputs  from  additional  sensory  modalities.   In 
addition,  these  areas  may  become  increasingly  involved  in  visuomotor  control, 
inasmuch  as  lesions  along  the  occipitoparietal  pathway  cause  impairment  not 
only  in  visuospatial  perception  but  also  in  eye  movements  and  in  visually 
guided  hand  movements. 

70 


Inferior  temporal  cortex  and  visual  recognition 

Stimulus  equivalence  across  retinal  translation  is  the  phenomenon  whereby  a 
stimulus  is  recognized  as  the  same  regardless  of  its  retinal  location.   Since 
nearly  two-thirds  of  inferior  temporal  neurons  receive  converging  visual 
information  not  only  from  their  own  hemisphere  but  also  from  the  opposite 
hemisphere  via  the  forebrain  commissures,  we  have  proposed  that  these  neurons 
provide  the  mechanism  for  stimulus  equivalence  across  the  vertical  meridian. 
Support  for  this  hypothesis  was  obtained  from  a  study  in  which  monkeys  with 
bilateral  inferior  temporal  ablations  combined  with  section  of  the  optic 
chiasm  were  trained  first  with  one  eye  and  then  with  the  other  eye  on  a  series 
of  visual  pattern  discriminations.   Unlike  control  monkeys  (with  either 
inferior  temporal  lesions  alone  or  chiasmatic  section  alone),  who  performed 
immediately  with  the  second  eye  any  discrimination  that  they  had  learned  with 
the  first,  the  experimental  monkeys  had  to  learn  each  discrimination  anew  with 
the  second  eye.   Since  inferior  temporal  neurons  have  not  only  bilateral  but 
also  large  receptive  fields,  they  could  of  course  be  responsible  for  stimulus 
equivalence  across  any  retinal  translation,  within  as  well  as  between  the 
hemifields.   We  are  currently  testing  this  more  general  version  of  the 
hypothesis  by  training  monkeys  to  discriminate  a  pair  of  patterns  in  one 
quadrant  of  a  visual  hemifield  and  then  testing  them  for  transfer  to  the  other 
quadrant.   If  our  hypothesis  is  correct,  transfer  should  be  impaired  in  this 
situation  following  inferior  temporal  lesions  alone. 

The  spatial  convergence  on  single  cells  in  area  TE  provides  a  mechanism  that 
is  essential  for  all  visual  learning  under  normal  viewing  conditions.   It 
insures  that  the  same  central  visual  cells  will  be  activated  from  one  exposure 
of  the  stimulus  to  the  next  despite  fluctuations  in  fixation,  distance, 
direction,  and  angle  of  regard,  fluctuations  which  necessarily  lead  to 
activation  of  different  populations  of  striate  and  prestriate  cells  on  the 
successive  presentations.   But  spatial  convergence  alone  is  insufficient  to 
insure  reactivation  of  the  same  central  visual  cells  unless  it  is  associated 
with  a  mechanism  for  temporal  convergence.   That  is,  a  given  stimulus  must 
excite  the  same  visual  neurons  on  a  second  occasion  as  on  the  first  if  there 
is  to  be  any  summation  of  the  effects  of  experience  or  training.   We  now  have 
evidence  that  the  inferior  temporal  cortex  does  indeed  contain  a  mechanism  for 
temporal  convergence,  as  indicated  by  the  following  experiments  on  visual 
recognition. 

Trained  monkeys  that  are  shown  an  object  once  will  demonstrate  that  they 
recognize  it  as  familiar  several  minutes  later  by  avoiding  it  in  favor  of 
another  object  that  is  completely  novel.   Thus,  somewhere  in  the  visual  system 
the  single  presentation  of  a  complex  stimulus  leaves  a  trace  against  which  a 
subsequent  presentation  of  that  same  stimulus  can  be  matched.   If  it  does 
match,  i.e.  if  the  original  neural  trace  is  reactivated,  there  is  immediate 
recognition  of  familiarity.   The  area  in  which  the  neural  trace  is  first 
established  appears  to  be  area  TE,  since  removals  here  but  not  elsewhere  in 
the  visual  system  abolish  the  animal's  ability  to  recognize  an  object  that  it 
has  seen  once  just  a  few  seconds  before.   Apparently,  area  TE  contains  the 
traces  laid  down  by  previous  viewing,  and  these  serve  as  stored 
representations  against  which  incoming  stimuli  are  constantly  being  compared. 
In  the  process,  old  traces  may  either  decay  or  be  renewed  or  even  refined, 
while  new  traces  are  added  to  the  store.   To  study  these  postulated  visual 
traces  at  the  single-unit  level,  we  are  investigating  whether  a  neuron  that  at 

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first  responds  equally  to  two  similar  stimuli  can  be  induced  to  respond 
differently  to  them  by  training  the  animal  to  attend  to  their  physical 
differences. 

A  cortico-limbo-thalamic  circuit  and  recognition  memory 

In  the  process  of  investigating  the  role  of  other  temporal-lobe  structures  in 
object  recognition,  we  obtained  a  result  that  is  particularly  exciting  because 
it  may  help  to  solve  a  long-standing  puzzle  concerning  the  neuropathology 
underlying  the  syndrome  of  global  amnesia  in  man.   This  syndrome,  which  is 
characterized  by  a  profound  inability  to  remember  new  experiences,  has  been 
attributed  in  the  clinical  literature  to  destruction  of  the  hippocampus.   Yet, 
attempts  to  duplicate  this  syndrome  in  animals  by  removal  of  the  hippocampus 
have  largely  failed.   We  have  found  in  our  studies,  however,  that  if  damage  to 
the  hippocampus  is  combined  with  damage  to  the  amygdala  then  a  profound 
recognition  loss  ensues,  a  loss  that  is  even  greater  than  that  produced  by 
lesions  of  inferior  temporal  cortex. 

Before  the  implications  of  this  finding  are  considered,  it  should  be  noted 
that  there  was  an  alternative  explanation  for  the  severe  effects  of  the 
combined  lesion  that  needed  investigation.   That  is,  neither  the  amygdalectomy 
nor  hippocampectomy  had  included  all  of  the  entorhinal  cortex,  whereas  the 
combined  lesion  did.   The  results  were  therefore  consistent  with  the 
possibility  that  the  severe  recognition  loss  after  the  combined  lesion  was 
actually  due  to  destruction  of  the  entorhinal  cortex.   A  test  of  this 
possibility  has  now  shown  that  even  when  total  entorhinal  ablation  is  combined 
with  hippocampectomy  the  recognition  impairment  is  no  greater  than  after 
hippocampectomy  alone,  whereas  when  total  entorhinal  ablation  is  combined  with 
amygdalectomy  the  impairment  is  just  as  severe  as  it  is  after  combined  removal 
of  the  amygdala  and  hippocampus.   In  short,  the  entorhinal  lesion  appears  to 
be  equivalent  to  a  hippocampectomy  in  this  situation,  presumably  because  it 
disconnects  the  hippocampus  from  the  inferior  temporal  visual  cortex.   The 
results  thus  support  our  earlier  conclusion  that  combined  damage  to  the 
amygdaloid  and  hippocampal  systems  is  necessary  to  produce  a  profound 
recognition  impairment  in  monkeys.   That  discovery  has  not  only  opened  up  a 
new  possibility  for  resolving  the  discrepancy  between  clinical  and  animal 
findings  but  has  also  led  to  new  insights  into  the  neural  mechanisms  of  memory. 

According  to  our  present  model,  object  recognition  depends  on  a 
cortico-limbo-thalamic  pathway,  activation  of  which  lead  to  the  storage  in 
cortex  of  the  trace  or  representation  of  the  stimulus  that  gave  rise  to  the 
activation.   In  the  case  of  vision,  as  already  indicated,  the  cortical  tissue 
in  which  the  storage  is  presumed  to  take  place  is  area  TK.   However,  as  will 
be  described  in  a  later  section  of  this  report,  other  sensory  modalities 
appear  to  be  organized  neurally  along  lines  similar  to  those  of  the  visual 
modality,  and  each  is  presumed  to  be  represented  by  a  final  cortical 
processing  station  located  in  the  anterior  temporo-insular  region  with 
functions  analogous  to  those  postulated  for  area  TE.   We  know  that  each  of 
these  final  stations  in  the  sensory  system  of  the  various  modalities  projects 
both  to  the  amygdala  and,  via  relays  in  the  perirhinal  and  entorhinal  cortex, 
to  the  hippocampus.   Furthermore,  each  of  the  limbic  structures  projects  to 
the  adjacent  parts  of  the  medial  thalamus,  the  amygdala  via  the  amygdalofugal 
pathways  to  the  magnocellular  portion  of  n.  medialis  dorsalis  (MDmc),  and  the 
hippocampus  via  the  fornix  to  the  anterior  nuclei  (Ant  N).   Finally,  we  have 

72 


shown  that,  in  vision,  one-trial  object  recognition  is  severely  impaired  not 
only  by  area  TE  lesions,  or  combined  araygdalo-hippocampal  lesions,  but  also  by 
disconnection  of  area  TE  from  the  amygdalo-hippocampal  complex,  disconnection 
of  the  amygdalo-hippocampal  complex  from  their  thalamic  targets,  or  combined 
destruction  of  these  thalamic  targets. 

Since  the  limbo-thalaraic  pathways  are  so  critical  for  recognition  memory,  we 
have  been  investigating  whether  their  prefrontal  projections  might  also  be 
involved  in  this  function.   So  far  we  have  determined  that  removal  of 
ventromedial  prefrontal  cortex  (the  target  of  both  MDmc  and  Ant  N)  yields  a 
severe  recognition  loss,  whereas  removal  of  either  dorsolateral  or  inferior 
prefrontal  cortex  (both  targets  of  the  parvocellular  division  of  MD)  produces 
little  impairment.   The  discovery  of  a  possible  functional  relationship 
between  the  ventromedial  prefrontal  cortex  and  the  limbic  system  provides  an 
important  new  clue  to  the  organization  of  prefrontal  cortex.   That  is,  for 
certain  memory  functions,  the  dorsolateral  prefrontal  cortex  may  depend  on 
interaction  with  the  ventromedial  prefrontal  region  in  much  the  same  way  that 
anterior  temporo-insular  cortex  depends  on  interaction  with  the  medial 
temporal  region.   We  are  currently  testing  whether,  within  the  ventromedial 
prefrontal  region,  the  orbital  cortex  (to  which  MDmc  projects)  and  the 
adjacent  anterior  cingulate  cortex  (to  which  Ant  N  projects)  contribute 
equally  to  recognition  memory,  just  as  we  had  found  to  be  the  case  for  all  the 
earlier  segments  of  the  amygdaloid  and  hippocampal  systems. 

Our  experimental  evidence  that  combined  damage  to  these  two  limbo-thalamic 
systems  is  neces.sary  to  produce  a  disorder  in  monkeys  resembling  the  syndrome 
of  global  amnesia  in  man  is  consistent  with  most  of  the  neuropathological 
evidence  available  on  amnesic  patients,  including  patients  with  temporal  lobe 
resections  and  those  with  Korsakoff's  disease.   One  piece  of  clinical  evidence 
does  not  seem  to  fit  this  view,  however,  and  supports  instead  the  view  that 
damage  to  the  hippocampal  formation  alone  is  sufficient  to  produce  the 
syndrome.   The  evidence  comes  from  amnesic  patients  with  diseases  of  the 
posterior  cerebral  artery,  which  is  known  to  provide  the  blood  supply  of  the 
hippocampus  but  not  the  amygdala.   From  the  few  clinical  studies  that  included 
neuropathological  reports,  however,  it  appears  that  although  the  infarction 
always  includes  the  posterior  part  of  the  hippocampal  formation,  it  may  also 
invade  the  inferior  temporal  cortex,  stria  terminalis,  thalamus,  or 
combinations  of  these,  raising  the  possibility  that  hippocampal  damage  alone 
is  not  responsible  for  the  memory  loss.   To  examine  this  issue  experimentally, 
we  have  initiated  a  study  on  the  effects  of  occluding  the  posterior  cerebral 
artery  in  the  monkey.   Our  preliminary  findings  indicate  that  such  occlusion 
will  produce  substantial  visual  recognition  loss  in  some  monkeys  but  not  all. 
Correlation  of  these  findings  with  the  neuropathology  may  thus  resolve  the 
question  regarding  the  locus  of  damage  responsible  for  the  amnesia  in  patients 
with  occlusions  of  the  posterior  cerebral  artery. 

The  cholinergic  system  and  recognition  memory 

In  the  neural  model  that  we  have  proposed  to  account  for  recognition  memory,  a 
stimulus  leaves  a  lasting  trace  in  the  sensory  modality's  higher  order 
processing  stations  whenever  that  stimulus  activates  the 

cortico-limbo-thalamic  pathway  described  above.   This  hypothetical  circuit  is 
incomplete,  however,  because  it  does  not  specify  the  link  through  which  the 
limbic  and  thalamic  structures  reactivate  the  sensory  cortical  areas  that 

73 


activated  them.   One  possible  candidate  for  this  missing  link  is  the  basal 
forebrain  cholinergic  system.   Two  major  lines  of  evidence  support  this  view. 
First,  cholinergic  agonists  and  antagonists  have  long  been  known  to  influence 
many  forms  of  memory  in  many  species,  and  in  a  recently  completed  study  of  our 
own  we  found  that  the  same  holds  true  for  recognition  memory  in  the  monkey. 
Second,  recent  neuropathological  studies  in  patients  with  Alzheimer's  disease, 
who  often  show  a  marked  memory  loss  as  one  of  their  earliest  symptoms, 
revealed  selected  cell  loss  in  both  the  nucleus  basalis  of  Meynert  (nbM) ,  the 
major  source  of  cholinergic  input  to  the  cerebral  cortex  and  amygdala,  and  the 
nuclei  of  the  diagonal  band  of  Broca  and  medial  septum  (dbB/ms),  the  major 
sources  of  cholinergic  input  to  the  hippocampus.   This  cell  loss  is 
accompanied  by  markedly  decreased  cortical  and  limbic  levels  of  choline 
acetyltransferase  and  acetylcholinesterase,  enzymes  involved  in  the  synthesis 
and  metabolism  of  acetylcholine. 

In  recent  experiments  performed  in  collaboration  with  investigators  from  The 
Johns  Hopkins  Medical  School,  we  have  produced  a  recognition  memory  impairment 
in  monkeys  by  damaging  these  basal  forebrain  areas  with  a  neurotoxin. 
Histological  and  neurochemical  analyses  indicate  that  the  animal  with  the  most 
severe  memory  impairment  had  nearly  complete  destruction  of  the  basal 
forebrain  system  as  well  as  a  60-90%  loss  of  choline  acetyltranferase  (ChAT) 
activity  across  most  of  the  cortex.   However,  there  was  much  less  loss  of  ChAT 
activity  in  area  TE,  the  cortical  sector  that  is  so  critical  for  visual 
recognition  memory.   The  results  suggest  the  interesting  possibility  chat  the 
visual  recognition  impairment  in  the  animals  with  the  combined  lesions 
resulted  from  cholinergic  denervation  not  of  the  cortex  but  of  the  limbic 
system.   This  possibility  would  also  account  for  the  finding  that  only 
combined  damage  of  nbM  and  dbB/ms  yielded  im.pairment,  since  only  such  combined 
damage  would  result  in  cholinergic  denervation  of  both  the  amygdala  and 
hippocampus. 

In  conjunction  with  these  experimental  manipulations  of  the  basal  forebrain 
cholinergic  system,  the  goal  of  which  is  a  better  understanding  of  the  nature 
and  neurobiological  basis  of  the  cognitive  losses  associated  with  Alzheimer's 
disease,  we  have  begun  to  examine  the  cognitive  losses  associated  with  normal 
aging  in  the  monkey.   Like  the  neurotoxic-lesion  study,  this  one  also  is  being 
performed  in  collaboration  with  investigators  from  The  Johns  Hopkins  Medical 
School.   Our  initial  results  have  demonstrated  a  moderate  but  systematic 
decline  in  the  recognition  memory  of  monkeys  from  early  adulthood  (3-6  yrs), 
through  middle  age  (14-17  yrs),  to  old  age  (17-30  yrs).   Since  we  know  much  of 
the  neuroanatomical  substrate  for  this  form  of  memory,  we  will  be  able  to 
examine  that  substrate  for  any  evidence  of  increasing  pathology  with  age. 

In  studies  aimed  at  exploring  the  anatomical  relations  among  the 
limbo-thalamic  and  basal  forebrain  components  of  the  postulated  memory 
circuit,  we  have  found  that  large  midline  thalamic  injections  of  [-^Hj  amino 
acids  lead  to  intense  labeling  of  the  nucleus  basalis.   Smaller  injections  are 
now  being  made  to  reveal  the  precise  nuclear  origins  of  this  thalamic  basal 
projection  to  the  forebrain,  with  the  goal  of  determining  whether  the  anatomy 
is  consistent  with  the  postulated  memory  circuit. 

In  a  related  study,  we  have  now  mapped  the  distributions  of  nicotinic  and 
muscarinic  cholinergic  receptors  in  the  adult  monkey  brain.   Both  types  of 
receptors  are  found  in  all  cortical  areas,  but  the  muscarinic  receptors  are 

74 


more  widely  distributed  across  the  layers  of  a  given  field  and  have  a  wider 
variety  of  laminar  labeling  patterns  across  fields  than  nicotinic  receptors. 
Nicotinic  receptors  are  found  predominantly  in  the  deep  part  of  layer  3,  where 
incoming  thalamic  and  corticocortical  afferents  terminate  densely,  in  keeping 
with  the  pattern  characterizing  the  "forward"  projections  of  the  sensory 
processing  pathways.   Muscarinic  receptors,  by  contrast,  are  commonly  found 
most  densely  in  the  upper  and  lower  layers  of  the  cortex,  a  pattern  that 
suggests  a  muscarinic  role  in  the  central  modulation  of  sensory  processing  via 
the  "backward"  projections  of  those  same  pathways.   The  receptor  distribution 
patterns  thus  appear  to  fit  the  notion  that  the  mnemonic  effects  of  the 
anticholinergic  agent  scopolamine  are  exerted  through  a  blockade  of  the 
central  modulating  system. 

Neurochemical  studies  of  cognitive  functions 

We  have  begun  to  extend  our  receptor  localization  studies  to  other 
neurochemical  systems  that  are  thought  to  modulate  cortical  activity.   For 
example,  opiates  are  known  to  alter  learning  ability  under  a  variety  of 
circumstances,  and  indeed  we  had  found  earlier  that  cortical  regions  in  the 
monkey  that  are  critical  for  learning  and  memory  have  high  levels  of  opiate 
receptors.   The  pathways  and  mechanisms  that  underlie  the  action  of  opiates, 
however,  are  still  unknown.   Sections  from  the  brains  of  monkeys  that  had 
received  unilateral  amygdalectomies  thirty  days  prior  to  sacrifice  showed 
increased  density  of  opiate  receptors  in  anterior  insular  and  orbitof rental 
areas  ipsilateral  to  the  lesion.   We  interpret  this  finding  as  evidence  of 
denervation  supersensitivity  following  destruction  of  an  opioid  projection  to 
these  cortical  areas  from  the  amygdala. 

In  collaborative  studies  with  investigators  at  Northwestern  University,  we 
have  also  obtained  evidence  that  opiates  regulate  the  rate  of  phosphorylation 
of  a  specific  protein  that  has  been  related  to  learning.   A  strong  correlation 
has  been  found  in  the  monkey  between  the  level  of  opiate  receptors  in  a 
cortical  area  and  the  in  vitro  rate  of  phosphate  incorporation  into  a  51  Kd, 
4.5  PI  protein  extracted  from  that  area.   This  51  Kd  protein  from  monkey  brain 
is  similar  to  a  48  Kd  protein  from  rat  brain  that  the  investigators  at 
Northwestern  had  shown  increases  its  rate  of  phosphate  incorporation  during 
memory  formation.   Moreover,  both  the  level  of  opiate  receptors  in  a  given 
area  and  the  rate  of  phophorylation  of  the  51  Kd  protein  in  that  area  are 
correlated  with  the  area's  position  in  the  cortical  sensory  processing 
hierarchy.   For  example,  within  the  visual  system,  the  levels  of  both  opiate 
receptors  and  phosphorylation  of  this  protein  are  highest  in  temporal 
allocortex,  next  highest  in  temporal  neocortex,  lower  in  prestriate  cortex, 
and  lowest  in  striate  cortex.   Thus,  the  phosphorylation  rate  of  the  51 
Kdprotein  may,  like  the  level  of  opiate  receptors,  reflect  the  level  of 
plasticity  within  a  given  cortical  area. 

Amygdala,  hippocampus,  and  associative  memory 

According  to  our  neural  model,  once  the  trace  or  representation  of  a  stimulus 
has  been  stored  in  the  higher-order  processing  stations  of  any  given  modality, 
that  stored  trace  can  enter  into  association  with  the  stored  traces  of  other 
stimuli  and  other  events,  thereby  providing  the  stimulus  with  associative 
meaning.   As  has  been  indicated,  the  amygdala  and  hippocampus,  as  well  as 
their  separate  efferent  pathways  and  separate  thalamic  targets,  make 

75 


approximately  equal  contributions  to  recognition  memory,  presumably  reflecting 
their  roughly  equal  contribution  to  the  cortical  storage  of  stimulus  traces. 
In  the  case  of  associative  memory,  however,  our  results  indicate  tuat  the 
amygdala  and  hippocampus  make  very  different  contributions. 

In  one  experiment,  monkeys  were  trained  preoperatively  on  a  visual  recognition 
task  and,  separately,  on  a  tactual  recognition  task,  with  the  same  set  of 
objects  comprising  the  stimuli  for  both  modalities.   One  group  of  monkeys  then 
received  amygdalectomies  and  the  other,  hippocampectomies,  after  which  both 
were  retrained  on  the  intramodal  memory  tasks  to  a  high  level  of  performance. 
When  tested  later  for  their  ability  to  perform  the  recognition  task  across 
modalities,  i.e.  to  choose  between  two  objects  visually  after  one  had  been 
presented  as  a  tactile  sample,  the  hippocampectomized  monkeys  continued  to 
perform  at  a  high  level,  whereas  the  performance  of  the  the  amygdalectomized 
monkeys  fell  to  chance. 

Nearly  the  opposite  results  were  obtained  in  a  second  study  that  tested  the 
ability  of  monkeys  to  remember  the  spatial  location  of  visual  objects.   In 
this  task,  the  animal  was  required  to  remember  on  the  test  trial  where  on  a 
three-well  tray  each  of  two  different  objects  had  been  presented  on  the 
acquisition  trial.   In  this  case,  monkeys  givert  amygdalectomy  were  able  to 
regain  the  level  of  performance  they  had  achieved  preoperatively,  whereas 
those  given  hippocampectomy  failed  to  rise  above  chance. 

The  results  of  these  two  complementary  experiments  indicate  that,  although 
both  the  amygdala  and  hippocampus  are  critical  for  certain  forms  of 
associative  memory,  their  roles  are  totally  different.   Many  further  analyses 
along  the  lines  of  these  experiments  will  of  course  be  necessary  before  the 
selective  associative  memory  functions  of  the  amygdala  and  hippocampus  can  be 
identified  with  confidence.   For  example,  the  association  of  an  object  with  an 
affective  state,  such  as  fear  or  pleasure,  appears  to  depend  much  more  heavily 
on  the  amygdala  than  on  the  hippocampus.   New  support  for  this  view  has  been 
obtained  in  an  experiment  showing  that  one-trial  object-reward  association  is 
impaired  more  by  amygdaloid  than  by  hippocampal  lesions,  although  neither 
deficit  approaches  in  severity  the  one  produced  by  the  combined  removal  of 
these  two  structures.   By  contrast,  because  of  the  contribution  to  spatial 
memory  that  is  made  by  the  hippocampus,  the  association  of  objects  with 
spatially  directed  motor  acts  could  depend  more  heavily  on  the  hippocampus 
than  on  the  amygdala.   Studies  to  examine  this  possibility  are  being  planned. 

CorticQ-limbic  pathways  in  somesthesis  and  audition 

The  work  described  above  elucidating  a  cortico-limbo-thalamic  system  for 
visual  perception  and  memory  has  led  to  the  search  for  analogous  systems  in 
other  modalities.   In  fact,  analogous  anatomical  pathways  have  been 
tentatively  identified  in  all  of  the  sensory  modalities,  but  because  of  a 
recent  finding  that  combined  amygdalo-hippocampal  lesions  produce  severe 
recognition  loss  not  only  in  vision  but  also  in  somesthesis,  particular 
attention  has  been  paid  to  the  pathway  in  this  modality.   Also,  anatomical 
studies  on  the  somatosensory  system  have  been  supplemented  with 
electrophysiological  studies  in  order  to  permit  functional  comparisons  with 
the  memory  circuit  in  vision. 


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Injections  of  cell  markers  were  made  into  the  hand  representation  of 
physiologically  identified  cortical  fields  lying  in  or  near  the  lateral  sulcus 
of  the  monkey  and  the  results  integrated  with  earlier  findings  from  injections 
in  postcentral  cortex.   Applying  principles  worked  out  in  the  visual  system 
regarding  direction  of  information  flow  based  on  analysis  of  laminar 
projection  patterns,  we  have  determined  that  the  direction  of  flow  of 
somatosensory  information  is  as  follows:   from  the  primary  sensory  area  (SI) 
to  the  superior  parietal  lobule  (area  PE),  from  area  PE  to  the  retroinsular 
and  rostral  inferior  parietal  cortex,  from  these  and  SI  to  the  secondary 
sensory  area  (SII),  and  from  SII  and  retroinsular  cortex  to  the  insula.   We 
also  confirmed  previously  reported  projections  from  the  insula  directly  to  the 
amygdala  and  indirectly  to  the  hippocampus  via  the  perirhinal  and  entorhinal 
cortex.   These  data  thus  demonstrate  a  somatosensory  pathway  that  runs 
principally  from  SI  through  SII  to  the  insula  and  from  there  to  the  limbic 
structures  of  the  temporal  lobe. 

In  the  course  of  working  out  this  ventrally  directed  cortico-limbic  pathway 
for  touch,  we  simultaneously  resolved  a  long-standing  difficulty  for  the 
functional  aspect  of  our  hypothesis,  which  postulates  that  SI  and  SII  process 
tactile  information  in  sequence.   It  was  supposed  earlier  that  SI  and  SII  were 
both  projection  zones  of  the  primary  somatosensory  relay  nucleus  in  the 
thalamus,  n.  ventralis  posterolateralis,  implying  that  the  two  cortical  areas 
received  and  processed  tactile  information  in  parallel.   Our  new  evidence  has 
revealed,  however,  that  SII  receives  its  major  thalamic  afferents  not,  like 
SI,  from  the  primary  relay  nucleus  but  from  n.  ventralis  posteroinferior. 
This  is  a  pattern  of  projections  that  is  consistent  with  a  sequential 
corticocortical  pathway.   As  a  further  test  of  the  proposal,  we  are  currently 
examining  the  effects  of  ablation  of  SI  on  the  somesthetic  responsivity  of 
neurons  in  SII.   In  support  of  the  notion  of  sequential  processing,  we  have 
found  that  the  vast  majority  of  SII  neurons  are  entirely  unresponsive  to 
somatic  stimulation  after  ablation  of  SI,  and  of  the  few  units  that  remain 
responsive,  fewer  than  1%  are  activated  by  light  tactile  stimulation.   These 
experiments  are  currently  being  replicated  and  extended  to  include  recordings 
from  the  insula. 

Since  the  insula  has  turned  out  to  be  an  important  link  in  the  cortico-limbic 
pathway  for  touch,  we  have  begun  to  examine  the  properties  of  insular  neurons 
in  awake  normal  monkeys.   So  far,  about  250  neurons  in  the  granular  field  of 
the  insula  have  been  examined,  and,  of  these,  68%  responded  to  innocuous 
somatic  stimulation,  whereas  none  responded  to  stimulation  in  any  other 
sensory  modality.   In  addition  to  their  modality  specificity,  the  most 
prominent  characteristic  of  insular  neurons  was  their  large  and  often 
bilateral  receptive  fields.   The  properties  of  granular  insular  neurons  in 
touch  thus  resemble  those  of  area-TE  neurons  in  vision,  lending  further 
support  to  the  proposal  that  the  cortico-limbic  pathways  in  the  two  modalities 
are  organized  in  a  parallel  fashion.   One  of  the  most  critical  tests  of  this 
proposal,  however,  remains  to  be  performed,  namely,  a  test  of  whether  insular 
removals  will  impair  tactile  memory  just  as  inferior  temporal  removals  impair 
visual  memory. 

Unlike  visual  and  tactual  recognition,  auditory  recognition  has  proven 
extremely  difficult  to  demonstrate  in  monkeys.   However,  after  several  failed 
attempts,  we  believe  that  we  now  have  a  testing  paradigm  that  will  elicit 


77 


auditory  recognition  reliably.   The  task  requires  delayed  matching  of  short 
segments  of  tape-recorded,  trial-unique  sounds,  such  as  noises,  tunes,  calls, 
sound-effects,  etc.   One  of  these  sounds  is  played  as  the  sample  through  an 
overhead  speaker,  and  then,  after  a  short  delay,  this  one  and  another  are 
played  in  alternation  through  two  widely  separated  speakers  that  serve  as  the 
covers  of  the  food  wells.   Two  monkeys  have  now  been  successfully  trained  in 
the  new  paradigm  and  both  have  shown  an  ability  to  recognize  sounds  after 
intervals  of  20-30  seconds.   These  and  similarly  trained  animals  will  be 
prepared  with  limbic  lesions,  and  thus  the  evidence  should  soon  be  available 
as  to  whether  the  neural  substrates  of  memory  identified  in  the  visual  and 
tactual  modalities  serve  the  auditory  modality  as  well. 

Nonlimbic  structures  and  habit  formation 

On  all  of  the  memory  tasks  that  have  been  described,  the  deficits  are 
especially  severe  when  removals  of  the  amygdala  and  hippocampus  are  combined. 
Yet,  even  the  combined  limbic  lesion  does  not  affect  all  forms  of  learning  and 
retention.   For  example,  despite  their  rapid  forgetting  in  one-trial  object 
recognition,  animals  with  the  combined  limbic  lesions  have  no  difficulty 
learning  object  discriminations,  at  least  in  the  standard  situation  where 
trials  are  repeated  3-4  times  per  minute.   In  an  attempt  to  resolve  this 
discrepancy  between  rapid  forgetting  and  successful  learning,  we  tested 
whether  object  discrimination  learning  would  be  prevented  in  animals  with 
limbic  lesions  if  intertrial  intervals  exceeded  the  putative  memory  span. 
Surprisingly,  animals  with  the  combined  amygdalo-hippocampal  lesions  learned 
to  discriminate  a  long  list  of  object  pairs  even  though  the  list  was  presented 
only  once  every  24  hours.   Thus,  although  the  operated  animals  have  an 
extremely  short  memory  span,  they  can  retain  and  accumulate  information  gained 
from  single  discrimination  learning  trials  separated  by  24-hour  intervals. 

Soon  after  discovering  this  phenomenon  in  tests  with  objects,  we  found  that 
the  same  dissociation,  i.e.  impaired  recognition  but  spared  discrimination 
learning  despite  24-hour  intertrial  intervals,  holds  for  pictorial  stimuli  as 
well.   This  paradoxical  success  in  the  presence  of  severe  memory  loss  implies 
the  existence  of  a  powerful  learning  and  retention  mechanism  outside  the 
limbic  structures  of  the  temporal  lobe. 

We  have  since  performed  additional  experiments  to  characterize  further  the 
essential  difference  in  function  between  the  limbic  and  nonlimbic  mechanisms. 
Our  results  suggest  that  the  limbic  system  is  critical  for  high  levels  of 
retention  of  object-reward  associations  after  a  single  acquisition  trial  with 
short  lists  of  objects,  or  after  two  or  three  repetitions  with  long  lists  of 
objects  but  short  intertrial  intervals.   With  greater  repetition,  however, 
retention  of  object-reward  associations  can  be  mediated  in  the  absence  of  the 
amygdala  and  hippocampus,  and  the  retention  appears  to  be  independent  of  both 
list  length  and  delay.   To  distinguish  this  form  of  retention  from  memory,  we 
have  labelled  it  'habit  formation'.   Further  investigation  of  this  mechanismof 
habit  formation  as  well  as  elucidation  of  its  neural  substrate  have  become 
important  goals  of  our  research.   For  example,  recent  evidence  regarding  the 
effects  of  damage  to  area  TE  indicates  that  this  cortical  region  is  important 
not  only  for  the  limbic  memory  system  but  also  for  the  nonlimbic  habit 
system.   Our  aim  now  is  to  identify  the  subcortical  structures  belonging  to 
the  habit  system,  with  our  initial  target  being  the  corpus  striatum. 


78 


Ontogenetic  development  of  memory  and  habit  formation 

On  the  evidence  that  in  the  adult  monkey  there  may  be  two  relatively 
independent  systems  for  learning  and  retention,  we  recently  initiated  a  series 
of  studies  to  assess  the  development  of  these  two  systems  in  infant  monkeys. 
Results  thus  far  indicate  that  visual  recognition  memory,  as  measured  by 
performance  on  delayed  nonmatching-to-sample,  is  absent  in  infants  younger 
than  four  months  of  age  and  does  not  reach  adult  levels  of  proficiency  even  at 
one  year.   This  slow  ontogenetic  development  of  recognition  memory  was  shown 
even  more  strikingly  with  longer  delays  and  lists.   In  sharp  contrast,  when 
3-month-old  infant  monkeys  were  trained  on  object  discrimination  habits,  they 
performed  as  well  as  adult  monkeys  in  both  acquisition  and  retention,  even 
though  intertrial  intervals  lasted  24  hours.   These  results  strongly  suggest 
that  the  two  systems  of  retention  that  were  found  to  be  relatively  independent 
in  the  adult  monkey  are  also  developmentally  dissociable.   Indeed,  they 
provide  evidence  that  the  mysterious  phenomenon  of  infantile  amnesia  could  be 
due  to  the  absence  of  a  functional  memory  system  in  early  childhood. 

On  the  basis  of  this  evidence,  we  have  begun  to  prepare  monkeys  with  neonatal 
removal  of  the  limbic  system  (i.e.  combined  amygdalo-hippocampal  removals)  in 
an  attempt  to  see  how  cognitive,  emotional,  and  social  behaviors  develop  in 
animals  whose  amnesia  might  persist  from  infancy  through  adulthood.   Animals 
with  neonatal  removals  of  area  TE  are  serving  as  controls.   The  results  so  far 
indicate  that,  at  two  and  six  months  of  age,  monkeys  with  neonatal  limbic 
lesions  display  abnormal  social  behavior,  whereas  the  operated  controls  are 
essentially  unimpaired  relative  to  normal  infants.   At  three  months  of  age, 
neonatal  ablation  of  area  TE  leads  to  a  transient  impairment  in  habit 
formation  (compared  to  permanent  impairment  seen  with  the  same  lesion  in 
adults),  whereas  limbic  lesions  in  both  infants  and  adults  leave  habit 
formation  intact.   Interestingly,  data  from  both  the  normal  and  the  operated 
infants  suggest  that  ontogenetic  development  of  the  habit  system  is  sexually 
dimorphic,  this  system  maturing  earlier  in  females  than  in  males.   This  sexual 
dimorphism  seems  to  be  dependent  on  the  presence  of  high  testosterone  level  in 
male  infants  before  and  shortly  after  birth,  since  we  have  now  found  that 
orchiectomy  in  male  infants  actually  speeds  their  rate  of  habit  formation.   At 
ten  months  of  age,  the  infants  with  limbic  lesions  are  severely  impaired  in 
memory  formation,  whereas  the  operated  controls  show  significant  functional 
sparing  of  the  system  (compared  to  the  effects  of  TE  lesions  in  adults).   Our 
tentative  conclusion  is  not  only  that  early  and  late  brain  damage  have 
different  consequences  but  also  that  the  degree  and  even  the  direction  of  the 
difference  depends  on  whether  the  locus  of  injury  is  cortical  or  subcortical, 
the  task  measures  habit  or  memory  formation,  and  the  subject  is  male  or 
female. 

Our  plan  is  to  follow  the  psychological  development  of  our  neonatally  operated 
animals  into  adulthood.   The  results  of  the  study  will  help  to  evaluate  two 
provocative  proposals  from  the  clinical  literature,  namely,  (a)  that  early 
dysfunction  of  the  limbo-thalamic  memory  system  could  be  one  cause  of 
childhood  autism,  a  syndrome  characterized  by  dramatic  social  and  emotional 
disturbances  not  seen  in  adults  with  the  same  neuropathology,  and  (b)  that  the 
reason  a  pure  case  of  amnesia  like  the  one  seen  in  adults  has  never  been 
reported  in  a  child  is  that  the  clinical  picture  of  an  amnesic  child,  being 
overlaid  with  autism,  is  entirely  different  from  the  clnical  picture  of  an 
amnesic  adult. 

79 


In  addition  to  the  development  of  an  animal  model  of  childhood  autism,  we  have 
initiated  studies  to  determine  more  precisely  the  development  of  memory 
functions  in  infant  monkeys.   Our  first  attempt  was  to  determine  whether  the 
visual  learning  measured  by  preferential-viewing  tasks,  which  have  revealed 
the  presence  of  visual  recognition  ability  in  two-month-old  human  infants,  is 
mediated  by  the  memory  system  or  the  habit  system.   The  results  of  the  study 
indicate  that  visual  recognition  measured  by  this  method  develops  in  rhesus 
monkeys  by  at  least  15  days  of  age;  yet,  despite  being  such  a  primitive 
process,  it  is  markedly  impaired  by  either  early  or  late  limbic-system 
damage.   On  the  other  hand,  early  damage  to  area  IE  leads  to  marked  sparing  of 
this  memory  function,  even  though  late  damage  essentially  eliminates  it.   It 
is  clear  from  these  new  findings  that  all  limbic-dependent  memory  abilities 
are  not  delayed  in  development  as  we  had  earlier  presumed.   A  hint  as  to  why 
some  may  develop  later  than  others  has  come  from  our  concurrent 
neurobiological  studies. 

To  complement  the  developmental  behavioral  experiments,  we  have  been  using  the 
2-deoxyglucose  method  to  trace  the  development  of  the  visual  system 
metabolically.   A  series  of  monkeys  with  one  hemisphere  visually  deafferented 
have  been  studied  at  various  ages  ranging  from  two  days  to  six  months.   We 
found  that,  in  all  cortical  visual  areas  of  the  intact  hemisphere,  glucose 
utilization  was  lowest  in  the  youngest  subjects,  peaked  at  four  months,  and 
then  declined  in  the  six-month-old  subject  to  levels  found  in  adults.   As  a 
consequence,  for  each  cortical  area,  the  relative  difference  between  the 
intact  and  deafferented  hemispheres  was  smallest  in  the  youngest  animals  and 
approached  the  differences  seen  in  adults  only  in  the  four-month-old  subject. 
The  finding  that  adult  levels  of  metabolic  activity  are  not  reached  until 
about  four  months  of  age  may  help  explain  why  the  visual  recognition  ability 
measured  by  delayed  nonmatching-to-sample  does  not  develop  until  about  this 
time.   The  recognition  ability  measured  with  preferential  viewing,  by 
contrast,  although  clearly  dependent  on  a  functional  limbic  system,  may  be 
less  dependent  than  delayed  nonmatching  on  the  developing  visual  cortex. 

Additional  evidence  that  limbic  and  cortical  systems  may  differ  in  rate  of 
development  has  been  provided  by  a  new  study  on  the  neonatal  distribution  of 
opiate  receptors  in  the  rhesus  monkey.   A  comparison  between  a  newborn  and 
adult  brains  revealed  that  the  adult  pattern  of  opiate-receptor  binding  is 
present  at  birth  in  allocortical  areas  and  the  hippocampal  formation.   These 
primitive  areas  showed  conspicuously  higher  levels  of  opiate  binding  than  the 
neocortex  at  both  ages.   In  the  neocortex,  by  contrast,  marked  differences 
were  evident  at  the  two  ages.   Whereas  the  adult  brain  was  characterized  by 
areal-specific  laminar  patterns  in  primary  sensory,  motor,  premotor,  and 
dorsolateral  prefrontal  areas,  the  newborn  brain  was  not.   In  the  newborn 
brain,  only  the  primary  visual  cortex  had  a  distinct  laminar  pattern  of 
labelling;  in  all  other  neocortical  areas  the  inf ragranular  layers 
showedrelatively  high  levels  of  opiate-receptor  binding.   Further,  in  the 
adult  brain,  polysensory  areas  (e.g.  PG  and  TF)  showed  greater  labeling 
density  than  modality-specific  sensory  areas,  but  this  density  difference  was 
not  apparent  in  the  newborn  brain.   At  the  subcortical  level,  the  density  and 
pattern  of  opiate  receptors  in  the  infant  and  adult  brains  were  similar,  with 
a  patchy  mosaic  of  labeling  in  the  striatum,  high  levels  of  labeling  in 
certain  amygdaloid  and  thalamic  nuclei,  and  an  absence  of  labeling  in  the 
mamillary  bodies.   The  findings  suggest  that  the  distribution  of  opiate 
receptors  in  the  macaque  brain  is  adult-like  at  birth  in  limbic  and  striatal 

80 


structures  but  is  not  yet  fully  developed  in  neocortical  areas. 
Summary 

Through  combined  use  of  behavioral  and  neurobiological  methods,  we  are 
beginning  to  discover  some  of  the  general  principles  along  which  the  primate 
forebrain  is  organized  to  serve  memory  and  other  cognitive  processes.   (1) 
Each  primary  projection  area  in  the  cortex  seems  to  be  the  source  of  two 
multisynaptic  corticocortical  pathways.   Both  pathways  are  composed  of  several 
cortical  areas  that  are  arranged  hierarchically,  one  pathway  being  directed 
dorsally  to  the  frontal  motor  system,  the  other  ventrally  to  the  temporal 
limbic  system.   Before  reaching  the  motor  system,  the  dorsal  pathways  from  the 
several  modalities  converge  in  polysensory  areas,  which  are  critical  for 
sensory  attention,  spatial  perception,  and  motor  guidance.   The  ventral 
pathways,  by  contrast,  remain  modality  specific  throughout  their  course  and 
are  important  instead  for  stimulus  recognition  and  stimulus  meaning,  and 
ultimately  for  triggering  the  motor  response.   (2)  Stimulus  recognition 
depends  not  only  on  stimulus  processing  along  the  ventral  cortical  pathway  but 
also  on  storage  of  a  central  representation  of  that  stimulus  in  the  ventral 
pathway's  last  station,  located  in  the  anterior  temporo-insular  region.   This 
region  projects  directly  to  the  amygdala  and  indirectly  to  the  hippocampus, 
and  these  two  limbic  structures  project  in  turn  to  the  medial  thalamus,  with 
further  connections  to  ventromedial  prefrontal  cortex.   All  three  of  these 
regions  (limbic,  thalamic,  and  prefrontal)  project  to  the  basal  forebrain 
cholinergic  system,  which  innervates  the  entire  cortical  mantle.   Storage  of 
the  central  representation  of  a  stimulus  occurs  only  if  this 

cortico-limbo-thalamo-prefronto-basal  forebrain  pathway  is  activated.   Damage 
to  this  pathway  results  in  recognition  failure,  a  core  symptom  of  amnesia. 
(3)  Once  the  central  representation  of  a  stimulus  has  been  stored,  it  can 
enter  into  association  with  the  stored  central  representations  of  other 
stimuli  and  other  events,  thereby  providing  the  stimulus  with  meaning.   Thus, 
stimulus-stimulus  associations  appear  to  depend  on  cortico-amygdalo-cortical 
interactions,  while  stimulus-affect  associations  probably  depend  largely  on 
cortico-amygdalo-hypothalamic  interactions.   By  contrast,  stimulus-place 
associations  seem  to  depend  on  cortico-hippocampal  interaction;  in  this  case 
the  hippocampus  may  act  as  the  site  of  converging  information  from  both  the 
ventral  'stimulus  recognition'  pathway  and  the  dorsal  'spatial  perception' 
pathway,  the  latter  involving  projections  from  the  polysensory  areas  through 
the  cingulate  gyrus  to  the  hippocampus.   Finally,  stimulus-act  associations 
could  depend  on  interaction,  via  the  limbic  system,  between  the  ventral  and 
dorsal  pathways  within  the  lateral  prefrontal  cortex,  which  interacts  in  turn 
with  the  motor  system.   (4)  Destruction  of  the  limbic  portion  of  the  memory 
system  does  not  affect  all  forms  of  learning  and  retention,  however.   At  least 
one  form,  which  has  been  labeled  habit  formation,  remains  nearly  intact, 
presumably  reflecting  the  operation  of  a  powerful  cortico-nonlimbic  mechanism 
for  retention  of  stimulus-response  connections.   Judged  by  the  evidence  from 
our  behavioral  and  developmental  neurobiological  studies,  this 
cortico-nonlimbic  habit  system  appears  to  mature  considerably  earlier  than  the 
cortico-limbic  memory  system. 


81 


Acknowledgement:   This  report  was  prepared  with  the  assistance  of  T.G.  Aignerj 
J.  Bachevalier,  R.  Desimone,  D.P.  Friedman,  D.M.  Kowalska,  K.A.  Macko-Walsh, 
E.A.  Murray,  T.P.  Pons,  B.J.  Richmond,  R.J.  Schneider,  H.  Spitzer,  and  L.G. 
Ungerleider.   Technical  and  secretarial  staff:   H.T.  Crawford,  L.I.  Dorsey, 
T.W.  Galkin,  L.J.  Gannon,  C.  Hagger,  M.A.  McCaffrey,  J.  Koran,  J.B.  O'Neill, 
J.K.  Parkinson,  R.R.  Phillips,  J.N.  Sewell,  L.P.  Stokes,  and  D.L.  Walker;  and 
R.C.  Burt,  N.  Klein,  and  M.  Powers. 


82 


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Desimone,  R. ,  Schein,  S.J.,  Albright,  T.D.   Neural  mechanisms  for  form,  color 
and  motion  analysis  in  prestriate  cortex  of  the  macaque.   In  C.  Chagas, 
R.Gattass,  and  C.  Gross  (eds.):   Pattern  Recognition  Mechanisms,  Pontifical 
Academy  of  Sciences,  Vatican  City,  pp.  165-178,  1985. 

Desimone,  R. ,  Schein,  S.J.,  Moran,  J.,  and  Ungerleider,  L.   Contour,  color  and 
shape  analysis  beyond  the  striate  cortex.   Vis.  Res.  25:  441-452,  1985. 

Gross,  C.G.,  Desimone,  R. ,  and  Albright,  T.D.,  and  Schwartz,  E.L.   Inferior 
temporal  cortex  as  a  visual  integration  area.   In  F.  Reinoso-Suarez  and  C. 
Ajmone-Marsan  (eds.):  Cortical  Integration,  Raven  Press,  New  York,  pp. 
291-315,  1984. 

Gross,  C.G.,  Desimone,  R. ,  Albright,  T.D.,  and  Schwartz,  E.L.   Inferior 
temporal  cortex  and  pattern  recognition.   In  C.  Chagas,  R.  Gattass,  and  C. 
Gross  (eds.):  Pattern  Recognition  Mechanisms,  Pontifical  Academy  of  Sciences, 
Vatican  City,  pp.  179-199,  1985. 


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Macko,  K.A.  and  Mishkin,  M.   Metabolic  mapping  of  higher-order  visual  areas  in 
the  monkey.   In  L.  Sokoloff-  (ed.):  Brain  Imaging  and  Brain  Function,  Raven 
Press,  New  York,  pp.  73-86,  1985. 

Malamut,  B.,  Saunders,  R.C.,  and  Mishkin,  M.   Monkeys  with  combined 
amygdalo-hippocampal  lesions  succeed  in  object  discrimination  learning  despite 
24-hour  intertrial  intervals.   Behav.  Neurosci.  98:  759-769,  1984. 

Mishkin,  M. ,  Malamut,  B.,  and  Bachevalier,  J.   Memories  and  Habits:  Two  neural 
systems.   In  J.R.  McGaugh,  G.  Lynch,  and  N.M.  Weinberger  Ceds.):  The 
Neurobiology  of  Learning  and  Memory,  Guilford  Press,  New  York,  pp.  65-77,  1984. 

Mishkin,  M.  and  Petri,  H.L.i   Some  implications  for  the  analysis  of  learning 
and  retention.   In  L.  Squire  and  N.  Butters  (eds.):  Neuropsychology  of  Memory, 
Guilford  Press,  New  York,  pp.  287-296,  1984. 

Moran,  J.  and  Desimone,  R.   Selective  attention  gates  visual  processing  in  the 
extrastriate  cortex.   Science.  229:  782-784,  1985. 

Murray,  E.A.  and  Mishkin,  M.   Severe  tactual  as  well  as  visual  memory  deficits 
follow  combined  removal  of  the  amygdala  and  hippocampus  in  monkeys.   J^. 
Neurosci.  4:  2565-2580,  1984. 

Murray,  E.A.  and  Mishkin,  M.   Amydalectomy  impairs  crossmodal  association  in 
monkeys.   Science  228:  604-606,  1985. 

Richmond,  B.J.  and  Goldberg,  M.E.   On  computer  science,  and  the  physiological 
utility  of  models.   Behav.  Brain  Sci.  8:  300-301,  1985. 

Saunders,  R.C.,  Murray,  E.A.,  and  Mishkin,  M.   Further  evidence  that  amygdala 
and  hippocampus  contribute  equally  to  recognition  memory.   Neuropsychologia 
22:  785-796,  1984. 

Tusa,  R.J.  and  Ungerleider,  L.G.   The  inferior  longitudinal  fasciculus:   A 
re-examination  in  man  and  monkey.   Annals  Neurol,  (in  press). 

Ungerleider,  L.G.   The  corticocortical  pathways  for  object  recognition  and 
spatial  perception.   In  C.  Chagas,  R.  Gattass,  and  C.  Gross  Ceds.):  Pattern 
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Annual  Report  of  the  Laboratory  of  Developmental  Psychology 

National  Institute  of  Mental  Health 

October  1,  1984  through  September  30,  1985 

Marian  Radke-Yarrow,  Chief 

Summary 

The  research  of  the  Laboratory  is  focused  on  identifying  and  studying  the 
processes  underlying  children's  affective  and  social  development.   A  develop- 
mental research  perspective  is  crucial  —  development  viewed  as  the  progressive 
and  cumulative  expression  of  the  interaction  of  the  individual's  innate  potentials 
and  the  environment.   A  major  commitment  of  resources  is  to  research  on  children 
at  risk  for  developing  affective  disorders  and  problem  behaviors. 

We  are  investigating  a  wide  range  of  factors  concerned  with  children's 
cognitive,  social  behavioral,  and  emotional  skills  necessary  for  coping  adaptively 
with  the  demands  of  living.   Although  we  have  a  special  interest  in  emotional 
development,  which  has  been  a  little-studied  aspect  of  development,  we  have 
tried  to  deal  with  emotions  not  in  isolation  but  to  study  them  functioning  inter- 
actively with  other  processes.   To  describe  development  adequately  in  any  one 
system,  one  must  draw  upon  understanding  of  development  in  other  systems.   Two 
interests  run  through  the  research:  to  identify  universals  and  to  understand 
individual  differences,  i.e.,  to  describe  norms,  and  to  investigate  factors  in 
individual  adaptive  and  maladaptive  patterns  of  development. 

There  are  two  programmatic  directions  in  the  Laboratory.   One  concerns  the 
study  of  children  of  depressed  parents,  who,  from  the  standpoint  of  genetics 
and  environment,  represent  a  high-risk  population.   Children  from  families  free 
of  psychiatric  disorder  constitute  a  comparison  group  and  permit  us  to  examine 
normative  development.   Both  groups  of  families  are  studied  intensively  over  a 
number  of  years.   A  second  research  program  involves  the  study  of  early  adoles- 
cence, a  risk  period  in  development.   In  this  work,  too,  the  strategy  is  to 
follow  the  children  over  a  period  of  time.   Children  approaching  puberty  are 
followed  into  adolescence;  individual  and  group  patterns  of  psychological 
functioning  and  biological  change  occurring  at  this  time  are  examined. 

The  research  on  children  of  depressed  and  normal  parents  and  the  research 
on  young  adolescents  have  required  extensive  data  collection  on  relatively 
large  samples  and  multiple  measures  made  at  successive  periods  of  time.   For 
both  programs,  methods  of  assessment  have  had  to  be  developed.   Both  programs 
were  initiated  four  and  five  years  ago  and  are  now  at  the  point  of  analysis 
and  early  findings.   Data  collection  has  been  completed  in  the  adolescent  study, 
and  staff  time  is  devoted  entirely  to  analysis.   In  the  study  of  children  of 
depressed  parents,  a  very  substantial  portion  of  data  collection  has  been 
accomplished.   Data  collection  pertains  now  mainly  to  the  second  wave  of  assess- 
ments of  the  families.   Analysis  is  well  under  way. 

Other  studies  in  the  Laboratory  that  have  been  initiated  in  the  past  year 
are  closely  related  to,  or  are  outgrowths  of,  the  projects  just  described. 


85 


These  are  smaller  studies,  generally  using  experimental  or  short-term  designs. 

Most  of  the  research  in  the  Laboratory  is  collaborative.   The  nature  of 
the  problems  dictates  the  need  for  the  combined  expertise  of  investigators 
with  differing  interests  and  from  different  disciplines.   The  specific  projects 
are  elaborated  in  the  following  pages. 

Studies  of  children  of  depressed  parents  and  well  parents. 

Previous  research  in  the  Laboratory  demonstrated  how  early  and  how  severely 
the  developmental  course  can  be  disturbed  and  behavior  become  deviant  in  children 
of  affectively-ill  parents.   The  findings  were  based  on  very  limited  samples  of 
children  and  of  behavior,  but  the  strength  of  the  findings  indicated  the  import- 
ance of  further  research.   In  the  research  that  has  followed,  we  have  obtained 
far  more  extensive  assessment  of  the  children  and  of  the  environments  that 
children  experience  being  reared  by  affectively-ill  parents  and  well  parents. 
Parents  and  children  are  observed  in  an  informal  apartment  setting  over  a 
series  of  days,  in  diverse  circumstances  and  conditions,  which  were  chosen  to 
represent  parenting  functions  and  to  tap  a  range  of  psychological  properties 
of  the  child.   In  addition  to  videotaped  observations  of  interaction,  there 
are  interviews  and  standards  tests.   Psychiatric  assessments  of  children  and 
parents,  family  genetic  history,  and  life  events  history  are  also  obtained. 

In  the  clinical  families,  the  mother  is  diagnosed  as  depressed  (major, 
bipolar,  or  minor),  the  father  is  depressed  or  normal.   In  the  comparison 
families,  both  parents  are  free  of  psychiatric  disorder.   On  entry  into  the 
study,  each  family  has  one  child  between  1-1/2  and  2-1/2  years  of  age  and 
another  child  between  5  and  8  years  of  age.   The  families  are  seen  again  after 
a  2-1/2  year  interval.   The  sample  (N=130)  is  predominantly  middle  class  with 
a  small  subsample  of  the  very  low  economic  class. 

Summaries  of  the  individual  studies  follow.   Findings  are  preliminary  in 
most  instances.   Also,  findings  being  reported  are  mainly  on  the  younger  chil- 
dren; later  analyses  will  include  the  older  siblings.   Emphasis  in  the  pre- 
liminary reports  is  on  group  comparisons  by  diagnosis  of  mother. 

a.   An  important  developmental  task  of  young  children  is  the  formation  of  a 
conception  of  the  self.   It  is  hypothesized  that  early  self-conceptions  and 
evaluations  are  heavily  determined  within  the  family,  and  that  one  important 
source  of  the  child's  information  is  parents'  verbalizations.   Because  self- 
attributions  of  hopelessness,  helplessness,  and  unworthiness  characterize  de- 
pressed adults,  what  depressed  parents  say  to  and  about  their  children  is  of 
particular  interest.   To  investigate  possible  early  origins  of  a  self-conception 
that  fulfills  the  depression  model,  we  are  investigating  the  mother's  comments 
to  and  about  her  child  across  a  sampling  of  situations  (Wylie  &  Yarrow).  Ver- 
balizations are  transcribed  from  videotapes  and  coded.   It  is  evident  that 
children  are  exposed  to  very  high  frequencies  of  attributions  about  themselves. 
Most  attributions  (35%)  are  directed  to  specific  aspects  of  the  child  rather 
than  to  the  total  person.   Comments  regarding  the  child's  competencies  occur 
with  high  frequency  relative  to  other  categories.   Attributions  about  the  child's 
feelings  and  emotions,  lovability,  and  altruism  each  account  for  only  1  to  3% 
of  the  attributions.   Some  aspects  of  the  child's  self  (namely,  carefulness  and 


86 


self-control)  receive  negative  comments  almost  exclusively.   Many  more  attribu- 
tions are  implicit  than  explicit.   Attributions  are  more  often  positive  eval- 
uations than  negative  ones.   However,  when  evaluations  are  strongly  positive  or 
negative,  mothers  tend  to  be  explicit,  and  positives  and  negatives  are  similar 
in  frequency.   Comparison  of  depressed  and  well  mothers  shows  differences. 
Children  of  depressed  mothers  receive  more  negative  attributions  than  do  chil- 
dren of  well  mothers.   Moreover,  many  more  mixed  messages  (positive  comments 
with  negative  comments)  are  given  to  children  of  depressed  mothers,  thus  provid- 
ing an  uncertain  and  possibly  anxiety-provoking  information  about  the  self.  The 
course  of  this  study  will  be  to  examine  further  differences  between  depressed  and 
normal  mothers  and  to  investigate  child  indicators  of  self-conceptions  and 
evaluations  in  relation  to  maternal  verbalizations. 

b.  Another  cognitive  component  of  the  child's  environment  is  the  parent's 
belief  systems  about  their  children.   Beliefs  are  assumed  to  affect  how  parents 
manage  their  children  and  their  expectations  regarding  them.   Well  and  depressed 
mothers  were  asked  to  weigh  the  contribution  of  external  factors  beyond  their 
control,  their  own  behavior  and  personality,  and  behavior  of  the  child's  father 

to  the  child's  behavior  and  outcomes  of  genetics  (Kochanska,  Yarrow,  &  Kuczynski). 
Depressed  mothers  are  more  critical  of  their  children,  particularly  in  affective 
aspects,  than  are  normal  mothers.   Also,  they  perceive  differently  the  determinants 
of  child  behavior.   Mothers  with  major  depression  tend  to  place  responsibility 
for  their  children's  characteristics  on  the  parents;  well  mothers  assign  contri- 
butions more  equally  to  parental  and  biological  factors;  mothers  with  bipolar 
illness  emphasize  biological  and  other  uncontrollable  factors  as  determinants 
of  their  children's  behavior.   Further  data  are  being  obtained  to  determine 
the  origins  of  these  beliefs  and  how  they  relate  to  mothers'  behavior  with 
their  children. 

Other  data  on  parental  beliefs  come  from  another  study  of  physically  abusive 
parents  and  a  matched  nonabusive  sample  (Trickett  &  Susman).   Most  of  the  abusive 
parents  were  diagnosed  depressed.   A  characteristic  of  the  abusive  families  is 
their  isolation  and  nonengageraent  with  the  larger  community,  deriving  in  part 
from  their  beliefs  that  the  outside  world  is  hostile.   Their  beliefs  are  reflected 
in  their  rearing  practices,  namely,  their  discouragement  of  new  and  independent 
experiences  for  their  children. 

c.  In  characterizing  environments  of  children,  the  cognitive  content  and 
style  of  parent-child  exchange  have  not  been  extensively  studied.   In  comparing 
normal  and  depressed  family  environments  it  might  be  expected  that  cognitive 
characteristics  associated  with  depression  would  interfere  with  parents'  abilities 
to  serve  as  mediators  of  their  children's  cognitive  development.   One  study 
(Friedman  &  Gordon)  is  concerned  with  how  and  what  mothers  teach  when  they  are 
asked  to  teach  the  child  a  task  and  also  when  no  teaching  is  assigned.   The 
content  imparted  by  mothers  to  2-  and  3-years-olds  relates  predominantly  to 

facts  and  principles  and  methods  of  knowledge  use,  (e.g.,  reasoning,  planning, 
problem  solving).   Less  information  is  transmitted  regarding  methods  for  knowledge 
acquisition  and  knowledge  representation.   These  latter  findings  are  curious, 
since  theoretically,  knowledge  acquisition  and  representation  are  skills  that 
a  child  of  this  age  must  acquire.   Mothers  give  minimal  encouragement  to  their 
children's  engagement  in  cognitive  activity  such  as  decision  making,  reasoning 
or  correct  labeling  of  objects.   When  an  adequate  sample  has  been  coded,  com- 
parisons of  normal  and  depressed  mothers  will  be  made. 


87 


Another  probe  into  cognitive  aspects  of  development  is  a  study  of  children's 
social  inferential  skills,  i.e.,  their  competence  in  inferring  the  feelings  and 
motives  of  others  (lannotti,  Cummings ,  &  Waxier).   Mother,  child  and  peer  were 
observed  in  the  laboratory  at  2  years  and  at  5  years.   Appraisals  of  inferential 
skills  are  based  on  parents'  and  children's  responses  to  hypothetical  incidents 
about  which  inferences  can  be  made  concerning  feelings  and  motives.   Associations 
between  parental  behaviors  and  child  inferential  skills  were  found.   Children's 
competent  social-cognitive  skills  at  age  2  and  again  at  age  5  were  positively 
related  to  the  mother's  facilitating  the  child's  experiences  with  others,  and 
providing  the  child  with  direct  training  in  attending  to  social  and  psychological 
cues.   These  mothers  directed  their  children's  attention  to  the  feelings  and 
behaviors  of  others  and  encouraged  their  children's  positive  social  interactions. 
Maternal  depression,  however,  was  not  associated  with  differences  in  social  cog- 
nitive skills  of  the  child,  at  least  as  manifested  in  hypothetical  circumstances. 

Cognitive  and  attentional  characteristics  of  the  children  of  the  depressed 
and  well  mothers  are  the  focus  of  two  studies.   In  one,  the  child's  play  behavior 
is  analyzed  (Barrett  &  Friedman).   Play  reflects  children's  capacities  to  use 
their  knowledge,  affect,  and  imagination  in  a  constructive,  coherent  fashion. 
Daughters  of  bipolar  and  normal  mothers  were  compared.   (At  the  time  of  the 
analysis,  there  was  an  insufficient  number  of  sons  in  the  bipolar  group.)   For 
girls  in  both  groups,  play  episodes  were  longer  and  had  greater  sophistication 
when  the  mother  was  present.   Daughters  of  bipolar  mothers  showed  more  complicated 
and  more  symbolic  play.   Whether  this  difference  is  related  to  the  kind  of 
involvement  in  the  play  by  the  mother  or  is  related  to  the  diagnosis  will  be 
investigated. 

In  a  related  analysis,  children's  attentional  properties  are  examined 
(Breznitz  &  Friedman).   The  literature  is  inconsistent  concerning  influences  of 
parental  affective  disorders  on  attentional  characteristics  of  offspring.   Atten- 
tion has  generally  been  investigated  in  the  laboratory  and  on  specific  assigned 
tasks.   It  is  measured  here  in  natural  settings.   Indices  include  children's 
capacities  to  sustain  attention  and  interest  in  objects.   An  attentional  deficit 
in  children  of  mothers  with  depression  is  suggested.   There  is  a  significant 
difference  in  time  spent  on  different  play  objects  by  children  of  the  depressed 
and  normal  groups.   Children  of  mothers  with  a  diagnosis  of  major  depression 
tend  more  to  move  from  object  to  object,  developing  less  sustained  involvements 
than  do  the  children  of  well  mothers. 

d.  The  development  of  self-control  is  a  critical  achievement  in  early  child- 
hood.  Impaired  parental  skills  in  managing  children  have  repeatedly  been 
implicated  in  the  etiology  of  maladaptive  patterns  of  child  behavior  and 
especially  in  problems  of  self-control  in  young  children.  To  this  end,  parental 
discipline  and  control  practices  and  their  outcomes  are  examined  in  families 
with  well  and  depressed  mothers  (Kuczynski,  Kochanska,  &  Yarrow).  In  both  groups, 
overt  noncompliance  accompanied  by  defiance  and  anger  decreased  with  the  age  of 
the  child  (comparing  2  with  3  years),  whereas  indirect  forms  of  noncompliance 
such  as  attempts  to  delay  compliance,  bargain,  or  modify  parental  demands  in- 
creased with  age.   Parental  power-assertive  strategies  such  as  direct  commands 
and  physical  enforcement  were  related  to  overt  defiance.   Comparisons  between 
normal  mothers  and  mothers  with  major  depression  indicate  that  children  of  de- 
pressed mothers  have  more  problems  of  noncompliance  than  children  of  normal 


mothers,  that  depressed  mothers  are  relatively  ineffective  in  controlling  their 
children's  behavior  and  that  this  difficulty  increases  with  the  child's  age. 
Much  more  analytic  work  lies  ahead  before  we  have  an  understanding  of  these 
interactions. 

In  analyses  to  come,  fathers'  contributions  to  child  control  are  also  being 
considered  (Wilson).   Reflecting  as  much  culture  as  biology,  theories  of  develop- 
ment have  dealt  almost  exclusively  with  mothers  as  influences  on  the  behavior 
of  offspring.   The  father  has  been  ignored  or  has  at  best  been  considered  in 
his  absence.   Preliminary  inspection  indicates  that  fathers  with  wives  who  are 
depressed  report  significantly  more  involvement  in  child  care  and  household 
duties  than  do  fathers  with  well  spouses. 

e.  If  parental  affective  illness  spells  cumulatively  stressful  experiences 
for  the  offspring,  one  should  anticipate  the  greatest  carry-over  of  parental  im- 
pairments to  be  in  areas  of  children's  emotions,  social  behavior,  and  relation- 
ships.  A  majority  of  our  studies  focus  on  the  affective  properties  of  the 
child's  experience,  investigated  in  terms  of  specific  maternal  characteristics, 
qualities  of  parent-child  interactions  and  relationships,  and  child  character- 
istics. 

el.   To  characterize  the  emotions  expressed  by  parent  and  child,  each 
minute  of  9  hours  of  observation  was  coded  for  the  specific  emotions  that 
occurred  (Yarrow   &  Kuczynski).   Groups  are  compared  in  terms  of  the  diagnosis 
of  the  mother.   Anger,  sadness,  and  low  negative  mood  were  expressed  least  fre- 
quently by  well  mothers,  and  most  frequently  by  major  depressed  mothers.   Posi- 
tive affect  was  expressed  most  frequently  by  well  mothers  and  least  frequently 
by  bipolar  mothers.   As  groups,  the  2-  to  3-year  olds  closely  match  the  affects 
of  their  mothers,  i.e.,  they  line  up  similarly  on  frequencies  of  each  emotion. 
This  is  not  the  case  for  the  school-age  siblings. 

Epidemiological  studies,  based  mainly  on  interviews,  have  described  depressed 
mothers  as  showing  little  affection.   Our  data  do  not  support  this  generalization. 
Mothers  in  the  three  diagnostic  groups  do  not  differ  in  frequency  of  expressed 
affection.   They  do  differ,  however,  in  a  critical  way.   In  depressed  mothers, 
affection  is  frequently  expressed  along  with  sadness  and  anxiety.   This  carries 
a  complex  and  possibly  stressful  message  to  the  child. 

e2.   Physical  affection  is  analyzed  in  more  detail  (how  it  is  expressed, 
the  functions  it  serves,  and  how  the  recipient  responds  to  it).   The  kind  of 
relationship  between  mother  and  child  is  reflected,  in  part,  in  these  reciprocal 
interactions  (Yarrow,  Kuczynski,  &  Wilson).   Although  major  analyses  are  not 
yet  available,  an  early  exploration  suggests  that  some  seriously  depressed 
mothers  engage  in  very  close  and  prolonged  physical  contact  and  affection  with 
their  young  children. 

e3.   Another  analysis  (Yarrow  &  Kochanska)  is  focused  on  how  mothers  handle 
children's  emotions.   When  a  child  expresses  fear,  sadness,  anger,  or  joy,  how 
does  the  mother  respond,  and  what  does  the  child  learn  with  regard  to  coping 
with  his/her  emotions?   This  study  is  not  yet  involved  in  major  analyses,  but 


89 


some  early  findings  show  differences  in  depressed  and  well  mother-child  dyads. 
Negative  emotional  episodes  are  of  significantly  longer  duration  in  the  depressed 
dyads.   Also,  the  ratio  of  positive  to  negative  emotional  engagements  is 
2  to  1  in  well  mother-child  dyads.   Positive  and  negative  occur  with  equal 
frequences  in  the  depressed  groups. 

When  mother  and  child  are  confronted  with  an  experimentally-introduced 
mild  stress  (an  anthropometric  examination  of  the  child  by  an  unfamiliar 
adult)  (Kuczynski  &  Yarrow),  negative  affect  and  emotionality  tend  to  be 
greater  in  the  depressed  mothers'  handling  of  the  event. 

Communication  between  mother  and  child  has  been  referred  to  earlier 
when  cognitive  issues  were  being  considered.   Characteristics  of  speech  and 
of  verbal  interaction  provide  indicators  also  of  the  parent-child  relationships 
and  the  affective  tone  of  the  relationships.   The  speech  behavior  of  well  and 
depressed  mothers  was  investigated  in  two  settings,  a  relaxed  setting  of 
preparing  and  eating  lunch,  and  a  somewhat  stressful  setting,  (the  examination 
referred  to  above)  (Breznitz  &  Sherman).   Given  the  general  motor  retardation, 
the  reduced  energy  level  and  social  withdrawal  of  depressed  individuals,  it 
was  predicted  that  the  speech  patterns  of  depressed  mothers  would  differ  from 
the  speech  patterns  of  well  mothers.   It  was  found  that  the  total  duration  of 
the  mother's  speech  to  her  child  was  less  for  depressed  women  than  well  women 
in  the  relaxed  situation  of  their  preparing  lunch.   In  the  period  of  stress  in 
which  mother  and  child  are  together  awaiting  a  visit  by  a  doctor,  the  pattern 
of  speech  in  the  dyads  with  depressed  mothers  changes  from  the  pattern  shown 
while  the  mother  was  preparing  lunch  in  a  manner  opposite  to  that  of  the  normal 
mothers.   The  depressed  mothers  change  to  speaking  more  and  to  having  more 
frequent  pauses  in  their  speech,  while  the  normal  mothers  change  to  speaking 
less  with  fewer  pauses.   When  in  a  relaxed  situation,  seated  together  eating 
lunch,  children  of  depressed  mothers  speak  significantly  less  than  do  children 
of  normal  mothers.   However,  in  the  period  while  waiting  for  the  doctor,  these 
differences  disappear.   The  small  stress  created  by  waiting  for  the  doctor  has 
a  dramatic  effect  on  the  children  of  depressed  mothers  causing  the  amount  and 
frequency  of  their  speech  to  appear  similar  to  that  of  the  normals.   The  change 
in  the  pattern  of  speech  observed  in  the  depressed  women  and  their  children  is 
referred  to  clinically  as  one  that  is  indicative  of  high  levels  of  anxiety.   The 
normal  mothers  and  their  children  did  not  show  this  pattern. 

f.  Another  series  of  studies  of  these  children  and  their  siblings  turn  the 
focus  of  analysis  on  child  behavior.   Research  has  demonstrated  increased  likeli- 
hood of  affective  problems  in  the  offspring  of  depressed  parents.   In  most  of 
the  studies,  however,  the  offspring  have  been  adolescents  or  adults.   There  is 
little  information  concerning  children  of  the  ages  studied  here,  and  concerning 
behavioral  characteristics  prior  to  the  onset  of  clear  mood  disorders. 

fl.   Withdrawn,  inhibited  behavior  and  acting-out,  aggressive  behavior 
are  being  investigated.   There  is  evidence  of  the  relative  stability  of  these 
behaviors  over  time;  and  they  are  linked  with  other  forms  of  emotional  and 
behavioral  problems.   Both  attributes  might  be  expected  to  have  an  increased 
likelihood  of  occtirrence  in  offspring  of  depressed  parents  (Kochanska  &  Yarrow). 

Preliminary  findings  on  inhibited  behavior  show  no  differences  in 
toddler-age  children  of  normal  and  depressed  mothers.   However,  the  affective 
interplay  between  mother  and  child  appears  different.   When  children  display 

90 


inhibition  in  encountering  the  unfamiliar,  normal  mothers  use  positive 
affective  expression  to  help  them;  depressed  mothers  respond  with  negative 
affective  expression.   Such  differences  in  socialization  might  be  expected  to 
influence  subsequent  child  patterns. 

(Investigation  of  acting-out  behaviors  is  in  a  planning  stage,  and  not 
reported  this  year.) 

f2.   Extrapolating  from  the  research  evidence  on  offspring  of  older  ages, 
young  children  of  depressed  parents  are  likely  to  manifest  more  affective  and 
behavioral  disturbance  than  offspring  of  normal  parents.   To  investigate  this 
question,  psychiatric  assessments  were  made  of  each  child.   The  pursuit  of  the 
issue  involves  methodological  research  as  well  as  empirical  findings  on  adaptive 
and  maladaptive  behaviors.   Improved  instruments  for  assessing  very  young  chil- 
dren are  much  needed.   Therefore,  one  objective  in  our  research  is  to  develop 
procedures  that  can  be  used  in  clinical  practice  as  well  as  in  research  with 
children  1-1/2  to  5  years  of  age  (Cytryn,  McKnew,  Sherman,  &  Yarrow).   In  one 
procedure,  a  psychiatrist  sees  the  child  in  a  play  interview  consisting  of  three 
segments:   play  with  neutral  toys,  with  family  toys,  and  with  toys  with  aggres- 
sive potential.   Special  attention  is  being  given  to  procedures  for  arriving 
at  assessments  of  the  child's  strengths  and  vulnerabilites :   The  child's  play 
is  rated  on  the  mental  status  scale  of  the  C.A.S.;  each  of  the  play  segments 
is  rated  on  behavioral  scales;  clinical  ratings  are  made  of  emotion  regulation, 
attachment,  coping  and  mastery  skills;  and  finally,  a  clinical  assessment  is 
made  of  specific  areas  of  concern  in  the  child's  behavior  and  overall  psychiatric 
risk.   A  comparison  of  the  clinical  risk  rating  and  assessment  of  risk  calculated 
from  the  behavioral  rating  scales  indicates  that  we  have  developed  a  reasonable 
model  of  the  psychiatrist's  behavior  as  he/she  synthesizes  behavioral  observa- 
tions and  reaches  a  summary  judgment.   The  behavior  summary  predicts  40%  of 
the  variance  of  the  clinical  summary.   Follow-up  research  will  indicate  which 
of  the  procedures  is  more  predictive  of  later  psychopathology. 

In  another  part  of  the  methodological  investigation,  instead  of  the  usual 
clinical  situation  of  child  with  psychiatrist,  the  videotaped  interaction  of 
child  with  mother  is  observed  and  coded  by  the  psychiatrist.   The  behavior 
sample  includes  stresses  that  are  natural  parts  of  the  child's  life  such  as 
waiting  for  lunch  when  hungry,  attempting  to  solve  frustrating  problems,  coping 
with  the  mother's  unavailability  while  she  attempts  to  nap,  meeting  a  stranger 
with  mother  present,  as  well  as  situations  of  pleasure  and  relaxation.   Each 
episode  is  scored  first  for  the  presence  or  absence  of  specific  behaviors. 
Next,  ratings  are  made  on  eight  scales:   level  of  motor  activity,  content  of 
thought,  social  attentiveness,  responsiveness,  initiation,  coping  and  mastery, 
and  emotional  expression  and  regulation.   Although  there  are,  as  one  would 
anticipate,  high  correlations  (_rs  .50  to  .79)  between  the  methods,  they  do  not 
produce  identical  assessments  and  predictions  for  individual  children.   Clinical 
judgments,  for  example,  place  21%  of  the  children  at  moderate  to  high  risk  for 
social  and  emotional  problems;  calculated  behavior  scores,  12%.   The  next 
phase  in  this  study  will  be  to  examine  the  specific  areas  of  differences,  and 
how  well  the  several  assessments  predict  the  status  of  the  child  2-1/2  years 
later. 

For  preliminary  assessment  of  the  psychiatric  status  of  the  2-  to  3-year- 
old  and  the  5-  to  8-year-old  offspring  of  normal,  major  depressive,  and  bipolar 


91 


depressive  mothers,  the  psychiatrists'  clinical  assessments  of  specific  areas  of 
concern  about  the  child's  behaviors  have  been  examined.   Based  on  approximately 
75%  of  the  projected  sample,  findings  are  as  follows:   When  any  area  of  concern 
is  counted  for  the  3-year-olds,  the  offspring  of  normal,  major  depressive, 
and  bipolar  parents  show  no  differences  in  frequency  of  disturbance.   Somewhat 
more  than  a  third  of  the  children  are  rated  in  one  or  more  areas  of  concern. 
When  only  dysthyraia  and  depression  are  considered,  one  child  out  of  50  in  the 
groups  of  major  depression  is  so  rated.   For  the  5-  to  8-year-old  siblings, 
when  any  area  of  concern  is  rated,  45%,  53%,  and  71%  of  the  children  of  normal, 
major  depressive,  and  bipolar  mothers,  respectively,  are  rated  in  one  or  more 
areas.   When  the  same  groups  are  rated  only  for  dysthymia  and  depression,  the 
frequencies  are  21%,  40%,  and  50%  for  normal,  major  depressive,  and  bipolar, 
respectively.   These  findings  begin  to  describe  the  early  developmental  histories 
of  offspring  of  depressed  parents,  and  to  identify  early  signs  of  disturbance 
and  factors  associated  with  such  disturbance.   Further  analyses  will  be  concerned 
with  multiple  assessments  of  the  child.   These  will  be  examined  in  relation  to 
environmental  and  genetic  history  variables.   In  the  study  of  normal  and  de- 
pressed parents  and  their  offspring,  future  work  will  (a)  continue  analysis  of 
diagnostic  group  differences,  (b)  extend  to  analyses  of  individual  differences, 
and  (c)  move  toward  integrating  the  findings  on  diverse  aspects  of  behaviors. 

Studies  of  Adolescence 

Development  in  early  adolescence  is  investigated  in  two  projects.   In  both, 
psychological  and  behavioral  functioning  of  adolescents  are  examined,  but  with 
different  emphases.   In  Dr.  Nottelmann's  study,  the  emphasis  is  on  societal  and 
environmental  factors  in  relation  to  behavior  during  early  adolescence.   Children 
are  assessed  before  and  after  moving  into  secondary  school.   In  the  collaborative 
study  by  Drs.  Susman  and  Nottelmann,  the  interrelations  of  biological  changes 
and  behavior  functioning  are  examined. 

A  major  finding  of  Dr.  Nottelmann's  study  is  that  most  adolescents  have  a 
smooth  school  transition  and  are  able  to  meet  the  demands  for  higher  competence 
in  secondary  school,  but  that  there  are  subgroups  of  adolescents  who  do  experi- 
ence adjustment  problems.   Such  problems  seem  to  be  influenced  by  adolescents 
seeing  themselves  as  being  different  from  their  peers.   For  example,  adolescents 
who  were  out  of  step  with  same-age  peers  in  physical  growth  during  the  year  of 
transition  reported  relatively  low  self-esteem  and  rated  themselves  low  on 
cognitive,  social,  and  physical  competence — in  particular,  girls  who  were 
maturing  early  and  boys  who  were  maturing  late  in  comparison  to  their  peers. 
Other  findings  for  height  differences  indicate  that  adolescent  adjustment 
is  influenced  by  the  larger  environmental  context  as  well  as  by  same-age  peer 
comparisons.   For  instance,  tall  girls  in  the  last  grade  of  elementary  school 
and  short  girls  in  the  first  grade  of  secondary  school  reported  low  self- 
esteem  and  also  rated  themselves  low  on  social  competence. 

Adolescents  identified  as  aggressive  (by  a  projective  test)  also  reported 
lower  self-esteem  and  also  rated  themselves  lower  on  cognitive  competence  than 
did  nonaggressive  adolescents.   Moreover,  they  viewed  their  school  environment 
less  positively  and  nominated  fewer  peers  for  cooperative  activities  than 
nonaggressive  adolescents. 

In  the  collaborative  study  by  Drs.  Susman  and  Nottelmann,  which  examines 
biological  and  psychological  relations,  early  adolescent  adjustment  and 

92 


behavior  are  investigated  in  relation  to  precise  measures  of  pubertal  develop- 
ment.  This  has  not  been  done  in  past  behavioral  studies.   The  biological 
measures  of  the  study  include  hormones  from  two  systems:   the  hypothalamic- 
pituitary-gonadal  (HPG)  axis  and  the  hypothalamic-pituitary-adrenal  (HPA) 
axis.   The  specific  mechanisms  whereby  these  systems  relate  to  behavior  are  not 
yet  known.   What  is  known  is  that  changes  in  levels  of  many  hormones  controlled 
by  these  axes  are  related  to  many  disturbances  in  behavior.   Disturbances  in  the 
HPG  system  hormones  have  been,  and  continue  to  be,  examined  for  their  possible 
role  in  the  etiology  of  depression  and  aggression.   Only  recently  have  disturb- 
ances in  the  HPA  been  linked  to  depression  and  other  mental  health  problems. 
Aspects  of  the  HPG  (gonadotropins  and  gonadol  steroids)  and  HPA  axis  (adrenal 
androgens)  were  examined  because  both  axes  are  responsible  for  the  changes  that 
occur  during  puberty. 

Boys  and  girls  were  enrolled  into  the  study  by  pubertal  status,  as  defined 
by  Tanner's  five  stages  of  development;  assessments  were  made  when  children 
entered  the  study  and  again  six  months  and  twelve  months  later.   Plasma  level 
determinations  include  the  following  hormones:   gonadotropins  (luteinizing 
hormone  and  follicle  stimulating  hormone),  gonadal  steroids  (testosterone  and 
estradiol);  also  assessed  were  testosterone-estradiol  binding  globulin),  and 
adrenal  androgens  (dehydroepiandros terone,  dehydroepiandrosterone  sulphate,  and 
androstenedione).   Psychological  assessments  of  the  adolescent  include  measures 
of  emotions,  self-evaluations,  relationships  in  the  family  and  with  peers,  compe- 
tencies, problem  behaviors,  school  performance,  and  cognitive  abilities.   Adoles- 
cents are  seen  with  their  parents  and  individually. 

As  anticipated,  plasma  levels  of  gonadotropins,  gonadal  steroids,  and 
adrenal  androgens  were  higher  with  increments  in  chronological  age  and  pubertal 
stage.   The  differences  between  Stage  1  and  Stage  5  plasma  testosterone  levels 
in  boys  and  plasma  estradiol  levels  in  girls  indicate  that  the  most  dramatic 
rise  in  hormone  levels  occurs  in  sex  steroids.   For  boys,  testosterone  level 
also  was  the  strongest  correlate  of  indices  of  pubertal  development.   For  girls, 
however,  androstenedione  level  was  the  strongest  correlate  of  pubertal  develop- 
ment, except  for  menarchial  status,  which  was  most  strongly  related  to  estradiol 
level. 

One  focus  of  the  study  is  timing  of  maturation.   Timing  of  maturation,  or 
the  age  at  which  adolescents  progress  through  the  physical  changes  of  puberty,  is 
known  to  affect  emotions,  adjustment,  and  behavior  problems  during  adolescence. 
One  important  question  examined  in  the  study  was  whether  early  adolescent  adjust- 
ment and  behavior  vary  with  hormone  levels,  per  se,  hormone  levels  that  are  low 
or  high  for  age,  pubertal  stage,  or  both  age  and  pubertal  stage. 

Behavior  problems  of  adolescents,  reported  by  their  mothers  were  related  to 
hormone  levels  but  not  to  age  or  stage  of  physical  development.   For  example, 
boys  with  a  profile  of  lower  gonadal  steroids  and  higher  adrenal  androgens  had 
the  higher  scores  for  obsessive-compulsive  (testosterone,  androstenedione)  and 
acting  out  behavior  (estradiol,  androstenedione).   Girls  with  lower  adrenal 
androgen  levels  (dehydroepiandrosterone  sulfate)  had  the  higher  scores  for 
depressive-withdrawal  and  acting  out  behavior  problems. 

Moods  of  boys,  based  on  adolescent  self-ratings  and  maternal  ratings,  also 
were  related  to  hormone  levels,  per  se ,  and  to  hormone  level  for  pubertal 


93 


stage.   Positive  moods  were  related  to  either  lower  gonadal  steroid  or  lower 
adrenal  androgen  levels  or  to  lower  levels  of  these  hormones  at  later 
pubertal  stages. 

Adjustment  problems,  based  on  adolescent  self-ratings,  were  related  to 
hormone  level  for  age  or  hormone  level  adjusted  for  both  age  and  pubertal  stage, 
or  timing  of  maturation.   For  example,  older  boys  with  a  profile  of  low  gonadal 
steroids  (testosterone)  and  high  adrenal  androgen  (androstenedione)  levels  had 
more  problems  with  social  relationships  and  reported  more  confusion  and  sad 
affect.   Older  boys  in  the  early  stages  of  pubertal  development  with  high  adrenal 
androgen  (androstenedione)  levels  had  less  prosocial  attitudes  toward  others. 

In  general,  these  findings  show  that  timing  of  maturation  is  important  for 
the  adjustment  of  adolescents.   Earlier  maturers  may  be  less  prepared  to  cope 
with  hormone-related  emotional  changes,  as  well  as  physical  changes,  than 
later  maturers.   Later  maturers,  though,  may  experience  disadvantages  related 
to  negative  evaluations  by  peers.   Thus,  it  appears  that  hormone  changes  and 
the  timing  of  these  changes  have  implications  for  the  mental  health  status  of 
adolescents.   Longitudinal  analyses  are  planned  to  explore  these  causal  issues. 

Another  focus  of  the  study  derives  from  our  understanding  of  adolescence  as 
a  developmental  period  in  which  there  are  dramatic  increases  in  both  hormones 
and  aggressive  and  rebellious  behavior.   In  animal  and  human  studies,  higher 
levels  of  androgens  relate  to  higher  levels  of  aggression.   A  major  research 
question  then  was  whether  increases  in  androgens  and  other  puberty-related 
hormones  are  related  to  the  rise  in  aggression  during  adolescence  in  humans.   The 
theoretical  approach  that  guided  the  examination  of  this  question  was  that 
hormone  levels  influence  emotions,  which,  in  turn,  are  reflected  in  adolescent 
behavior  like  aggression. 

Findings  for  hormone-aggression  analyses  indicate  that  levels  of  hormones 
Chat  rise  across  puberty  do  relate  to  the  emotions  and  degree  of  aggression 
in  early  adolescent  boys,  but  not  girls.   Boys  who  were  higher  on  self- 
reported  sad  affect  had  a  hormone  profile  of  lower  testosterone  to  estradiol 
ratio,  lower  testosterone-estradiol  binding  globulin,  and  higher  androstenedione. 
Self-reported  anxiety  also  appears  to  play  a  role  in  relations  between  hormones 
and  aggression.   Boys  who  were  higher  on  acting-out  behaviors  were  lower  on 
anxiety.   Boys  who  were  high  on  acting  out-behaviors  had  a  hormone  profile 
of  lower  estradiol,  testosterone-to-estradiol  ratio,  testosterone-estradiol 
binding  globulin,  and  dehydroepiandrosterone  sulphate  as  well  as  higher 
androstenedione.   Further,  boys  who  were  higher  on  rebellious  behavior  (e.g., 
talks  back,  irritable,  irresponsible)  had  a  hormone  profile  of  higher 
luteinizing  hormone  and  dehydroepiandrosterone  and  lower  follicle  stimulating 
hormone. 

The  finding  that  hormone  levels  during  puberty  relate  to  anxiety,  sad  affect, 
and  aggressive  behaviors  indicate  that  perhaps  even  normal  and  subtle  variations 
in  hormone  levels  are  important  factors  in  the  development  of  behavior 
problems  and  psychopathology.   Early  adolescents  may  be  especially  sensitive 
to  the  rises  in  hormone  levels.   These  findings  may  have  implications  for 
the  etiology  of  psychopathology. 


94 


The  hormones  and  aggressive  behaviors  examined  in  these  analyses  were  not 
related  for  girls.   Differences  in  hormone-aggression  relations  for  boys  and 
girls  may  be  affected  by  differences  in  socialization  experiences  of  the  two 
sexes.   However,  there  also  may  be  differences  between  boys  and  girls  in  how 
they  respond  to  stress.   Boys  may  tend  to  react  with  aggression  and  girls  may 
tend  to  react  with  anxiety.   Further  work,  in  the  research  on  adolescents  will 
be  concerned  with  extending  the  analyses  of  hormone-behavior  relations 
and  especially  with  longitudinal  analyses. 

Laboratory  Staff 

The  professional  staff  at  the  Laboratory  includes  a  number  of  disciplines: 
developmental  psychologists,  clinical  psychologists,  child  psychiatrists,  and 
nurses.   About  40%  of  the  junior  professional  and  technical  staff  are  supported 
by  nongovernmental  sources.   Much  of  the  research  is  carried  out  collaboratively 
within  the  Laboratory.   Some  of  the  research  involves  collaborations  with 
other  Laboratories  in  NIH,  and  still  other  studies  are  being  done  jointly  with 
investigators  at  Universities. 

A  number  of  staff  members  have  completed  Staff  Fellow  appointments  and  will 
be  leaving  the  NIMH  this  year  for  University  positions.   Dr.   E.  Mark  Cummings, 
a  Senior  Staff  Fellow,  Dr.  Leon  J.  Kuczynski,  a  Visiting  Foreign  Associate,  and 
Dr.  Karen  Barrett,  a  MacArthur  Foundation  Postdoctoral  Fellow  will  be  leaving  at 
the  end  of  the  summer.   Dr.  Katherine  Suter,  a  Child  Psychiatrist,  has  joined 
the  Laboratory  (July  1985)  as  a  Medical  Staff  Fellow.   Dr.  Grazyna  Kochanska  and 
Dr.  Frances  Bridges-Cline  are  John  D.  and  Katherine  T.  MacArthur  Foundation 
Fellows  and  will  be  guest  researchers  in  the  Laboratory. 

Investigators  in  the  Laboratory  participate  in  many  ways  in  their 
scientific  disciplines  and  in  professional  activities,  serving  on  review 
committees,  editorial  boards,  presenting  at  scientific  meetings  and  conferences 
in  the  United  States  and  Europe,  giving  invited  lectures  and  appearing  in  the 
media  to  present  the  research  of  the  Laboratory  to  the  general  public. 


95 


Annual  Report  of  the 

Laboratory  of  Psychology  and  Psychopathology 

National  Institute  of  Mental  Health 

October  1,  1984  to  September  30,  1985 

Allan  F.  Mirsky,  Ph.D.,  Chief 

This  is  the  fifth  full  year  report  of  the  program  of  the  Laboratory  of 
Psychology  and  Psychopathology  (LPP).  A  number  of  administrative  changes  have 
been  made  which  affect  the  LPP:  the  Clinical  Infant  Research  Unit  (CIRU) 
under  the  direction  of  Dr.  Stanley  I.  Greenspan,  has  been  transferred  to  the 
Bureau  of  Maternal  and  Child  Health;  the  section  on  Clinical  Brain  Imaging 
(SCBI)  has  been  transferred  to  the  Laboratory  of  Cerebral  Metabolism  (LCM). 
The  work  of  that  section  will  be  reported  under  the  heading  of  the  LCM.  Dr. 
Seymour  S.  Kety  has  been  assigned  to  the  LPP  and  his  research  will  be  reported 
under  this  laboratory's  activities. 

We  have  occupied  our  permanent  laboratory  space  in  the  ACRF  since  May  of 
1983;  acquisition  of  the  necessary  major  items  of  research  equipment  is 
complete  as  of  this  year  and  renovation  of  our  laboratory  has  been 
accomplished.  Hundreds  of  patient  sessions  have  been  run  by  the  investigators 
in  the  LPP  during  the  reporting  period,  and  good  progress  is  being  achieved  in 
all  aspects  of  the  research  program. 

A  brief  outline  of  studies  appears  below. 

A.  Clinical  Studies  of  Attention  and  Brain  Function 

1.  Human  Clinical  Studies  of  Attention  Disorder 

2.  Attention  Disorders  as  Assessed  by  Event-related  Brain  Potentials 

3.  Neuropsychological  Evaluation  of  Psychiatric  and  Neurological  Patients 

4.  Topographic  Analysis  of  Brain  Activity 

B.  Animal  models  of  attention  disorders 

1.  Models  in  the  Monkey  of  Generalized  Seizures  of  the  Absence  Type 

2.  Brain  Lesion  and  State  Change  Effects  on  Visual  Attention 

3.  Attention-related  Neurons  in  the  Brain  of  the  Rhesus  Monkey 

C.  Psychobiology  of  Cognitive  Processes 

D.  Autonomic  Nervous  System  Activity  in  Attention  and  Psychopathology 

1.  Psychophysiological  Responsivity  and  Behavior  in  Schizophrenia 

2.  Psychophysiological  Concomitants  of  Minimal  Brain  Dysfunction  in 
Children 

3.  Personality  Factors  and  Psychophysiological  Responses  to  Changing 
Stimulus  Input 


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E.  Interaction  of  Nature  and  Nurture  In  Schizophrenia 

1.  Studies  of  Heredity  and  Environment  in  Schizophrenia 

2.  Studies  on  Etiological   Factors  in  Schizophrenia 

F.  Cognitive  and  Perceptual   Changes  in  Affective  Illness 

A.     Clinical   Studies  of  Attention  and  Brain  Function 

1.  Human  Clinical   Studies  of  Attention  Disorder 

Many  of  the  members  of  the  LPP  are  involved  in  a  joint  project  in  which  a 
wide  variety  of  attentional,  cognitive,  autonomic,  and 

electroencephalographically-derived  tests  are  applied  to  the  same  populations 
of  experimental   and  control    subjects.     These  will   include  epileptic  persons, 
schizophrenic  subjects,  brain-lesioned  subjects,  dementing  subjects,  and 
controls.     The  aim  is  to  develop  a  profile  of  functioning  for  the  several 
groups  that  will   highlight  the  similarities  and  differences  among  them  and 
will   lead  to  a  better  neuropsychological  characterization  of  their 
impairment. 

The  major  elements  within  this  program  include:     the  work  of  the  Cognitive 
Psychophysiology  Unit  (Dr.   Connie  C.  Duncan-Johnson,  head);  the  Imaging 
Studies  of  Dr.   Richard  Coppola;  the  development  and  implementation  of  the 
Neuropsychological   Test  Battery  by  Drs.  Goldberg  (consultant)  and  Dr. 
Duncan-Johnson.     Ms.  Squillace  participated  in  the  development  of  the  battery 
before  her  departure  in  1984.     Her  role  as  neuropsychological   examiner  has  now 
been  assumed  by  Dr.   Emile  Brouwers.     Dr.   Duncan-Johnson's  Unit  is  applying 
techniques  of  cognitive  psychophysiology  to  elicit  and  evaluate  the  several 
event  related  potential   components   (auditory  and  visual)  which  have  been  shown 
to  relate  to  attention,  uncertainty  and  surprise   (P300,   N140,  etc.).     Similar 
experimental   paradigms   (e.g.,  the  "odd-ball"  method)  will   be  applied  to 
various  groups  of  subjects  with  attentional   disorders  so  as  to  be  able  to 
compare  and  contrast  ERP  component  amplitudes,  latencies  and  distributions. 
It  is  anticipated  that  these  studies  will   help  to  illuminate   (in  conjunction 
with  other  methods)  the  nature  of  the  similarities  and  differences  among 
various  attentional   disorders. 

2.  Attention  Disorders  as  Assessed  by  Event-Related  Brain  Potentials 

Major  progress  has  been  made  this  year  in  testing  various  groups  of 
disordered  patients  with  ERP  techniques.     Significant  new  findings,  in 
particular,  appear  to  be  emerging  in  characterizing  and  contrasting  the 
cognitive/attentive  disturbances  in  patients  with  various  types  of  eating 
disorders,  adult  dyslexia,  and  schizophrenia.     More  details  are  found  in  the 
project  description  of  ZOl   MH  00509-03  LPP. 

3.  Neuropsychological   Evaluation  of  Psychiatric  and  Neurological   Patients 
The  Neuropsychological    Test  Battery  was  developed  by  various  LPP  staff. 


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including  Dr.  Connie  Duncan-Johnson,  Ms.  Kathleen  Squillace  and  outside 
consultant  Dr.  Elkhonon  Goldberg  of  Einstein  Medical  School.  After 
considerable  discussion,  review  of  the  literature  and  consultation  with  IRP 
staff,  a  battery  of  neuropsychological  tests  was  devised  which  provides  a 
reasonably  complete  assessment  of  the  various  executive,  mnemonic,  linguistic 
and  attentive  capacities  of  the  human  brain.  The  test  battery  has  been 
administered  in  whole  or  in  part  to  about  150  persons  so  far,  and  the 
information  it  provides  will  form  the  neurobehavioral  data  base  which  will  be 
used  to  interrelate,  evaluate  and  integrate  the  various  electrographic, 
biochemical  and  other  physiological  measures  applied  to  various  groups  of 
patients  with  psychiatric  and  neurological  disorders.  Additional  details  can 
be  found  in  the  project  description  of  ZOl  MH  00508-03  LPP. 

4.  Topographic  Analysis  of  Brain  Activity 

Dr.  Coppola's  imaging  laboratory  became  operational  in  the  fall  of  1982. 
Continuing  from  the  work  begun  with  Dr.  Buchsbaum  a  32  lead  continuous  EEG 
mapping  system  was  implemented.  The  laboratory  is  now  regularly  recording  32 
lead  studies  from  a  variety  of  patient  groups.  It  appears  that  in  some 
situations  the  additional  imaging  resolution  gained  from  32  leads  vs  16  on  one 
hemisphere  can  be  quite  useful.  In  other  cases  the  32  lead  capability  is  used 
to  record  from  both  hemispheres  simultaneously,  allowing  bilateral 
comparisons.  Aside  from  evaluation  of  this  new  technique,  studies  are  in 
progress  examining  patients  with  depression  (under  IV  procaine  HCL  challenge); 
examining  cases  with  presumed  Alzheimer's  disease;  developing  EEG  imaging  in 
patients  with  epilepsy;  evaluating  the  effects  of  various  psychotropic  drugs 
on  the  EEG;  comparing  groups  with  differing  major  foci  of  alpha  EEG  activity. 

B.  Animal  Models  of  Attention  Disorders 

1.  Models  in  the  Monkey  of  Generalized  Seizures  of  the  Absence  Type 

Over  the  course  of  the  last  15  years,  staff  now  in  the  LPP  have  been 
interested  in  the  development  of  techniques  for  simulating  the  generalized 
seizure  patterns,  as  well  as  the  behavioral  accompaniments,  of  absence 
epilepsy.  We  have  explored  the  use  of  electrical,  chemical,  and  metabolic 
methods  of  inducing  discharges.  In  our  laboratories,  we  have  used  in  past 
studies:  electrical  stimulation  of  subcortical  and  cortical  structures, 
administration  of  chlorambucil  and  pentylenetetrazol,  subcortical  implantation 
of  aluminum  hydroxide  cream,  and  application  of  conjugated  estrogen 
symmetrically  to  forebrain  cortical  surfaces.  Most  recently,  we  have  done 
some  experiments  with  compounds  which  block  metabolism  of  GABA  (i.e., 
gamma-vinyl  GABA)  and  produce  the  paradoxical  effect  of  inducing  symmetrical 
and  synchronous  SW-like  activity  in  the  EEG  of  the  monkey. 

Such  models  of  generalized  seizure  activity  permit  the  testing  of  various 
hypotheses  and  provide  an  experimental  tool  for  study  of  single  unit  activity 
(as  an  example)  under  conditions  of  seizure. 

We  expect  to  continue  with  some  of  these  studies  during  the  next  year 
under  the  direction  of  Dr.  Michael  Myslobodsky. 


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1.     Attention-Related  Neurons  in  the  Brain  of  the  Rhesus  Monkey 

Our  method  of  identifying  this  cell  type  in  the  Rhesus  monkey  brain  has 
been  based  upon  an  experimental  test  paradigm  in  which  animals  are  trained  to 
perform  in  a  go,  no-go  visual  discrimination  task.  Attention-related  cells 
recorded  from  microelectrodes,  are  those  which  fire  in  both  go  and  no-go 
trials.  That  is,  they  fire  on  trials  when  the  animal  has  merely  to  attend  to 
a  stimulus  display  and  performs  no  obvious  motor  response  except  holding  a  paw 
on  a  response  key  (no-go  trials);  they  fire  as  well  on  trials  on  which  a  brisk 
lift  and  hit  response  to  obtain  a  fruit  juice  reward  is  necessary.  The 
cellular  response  may  be  identical  on  both  trial  types  and  may  in  addition 
anticipate  the  onset  of  the  cue  stimulus  by  several  hundred  milliseconds. 
This  type  of  cell  we  have  labeled  as  type  II  and,  as  noted  above,  is  found 
distributed  throughout  certain  regions  of  the  brain  stem  and  prefrontal 
cortex.  Further  studies  will  be  aimed  at  providing  a  systematic  functional 
analysis  of  this  cell  type,  searching  for  other  loci  in  the  brain  where  they 
may  be  found,  characterizing  the  relation  among  the  several  loci,  and 
beginning  pharmacological  studies  of  these  cells.  As  is  documented  in  project 
report  201  MH  00506-05  LPP,  a  gradient  of  type  II  through  possibly 
transitional  cells  through  type  I  cells  has  been  described  in  certain  regions 
of  the  monkey  frontal  lobe.  This  work  suggests  that  there  may  exist  in  the 
premotor  area  of  the  monkey  a  transitional  cell  type  between  the  "attention" 
cell  and  the  motor  execution  cell.  This  cell  type  may  serve  some  bridging  or 
connecting  function  between  the  attentional  and  motor  execution  systems. 

During  the  past  year,  this  experimental  (go/no-go)  paradigm  has  been 
extended  successfully  to  the  preoccipital  and  inferior  parietal  cortices  of 
the  monkey.  Type  II  cells  are  found  in  these  brain  areas  and  seem  to  serve 
functions  similar  to  those  in  other  association  cortices.  More  details  can  be 
found  in  the  above-referenced  project  report. 

3.  Brain  Lesion  and  State  Change  Effects  on  Visual  Attention 

There  is  collaboration  with  LN  and  LCM  in  some  parts  of  this  work.  One  of 
the  goals  has  been  to  specify  the  role  of  non-visual  regions  of  the  primate 
cerebrum  in  visual  attention,  and  this  work  has  used  several  techniques 
including  lesions,  stimulation,  and  electrographic  recording  of  brain 
potentials  under  various  lesion  and  non-lesion  conditions.  Other  studies  have 
been  concerned  with  the  recording  and  analysis  of  visually-evoked  potentials 
under  a  variety  of  perceptual  conditions,  and  with  drug  or  sleep  state  effects 
on  the  mechanisms  of  visual  attention. 

One  of  the  behavioral  measures  used  in  this  study  is  the  Generalized 
Attention  Test  (GAT)  which  is  being  developed  at  the  NIMH  by  LPP  staff.  It 
represents  an  extension  and  modification  of  the  Continuous  Performance  Test 
(CPT)  of  attention  which  was  originally  refined  and  perfected  at  the  NIMH 
during  the  period  1954-1961.  The  CPT  has  been  used  extensively  by  various 
groups  of  investigators,  particularly  in  the  study  of  epilepsy,  schizophrenia, 
and  metabolic  illnesses.  The  GAT  extends  the  concept  of  the  CPT  to  involve  a 
variety  of  cognitive  dimensions  (such  as  intra  and  inter-dimensional  shifts), 
as  well  as  parametric  control  of  other  perceptual  variables.  The  GAT  is 


100 


designed  for  use  with  both  humans  and  monkeys  so  that  direct  comparisons  can 
be  made  of  the  results  in  each  species.  Preliminary  results  on  monkeys 
indicates  that  they  can  be  successfully  trained  to  perform  on  this  task  and 
some  of  the  data  from  the  first  monkey  subjects  are  extremely  promising. 

C.  Psychobiology  of  Cognitive  Processes 

The  aim  of  this  research  has  been  to  relate  psychological  and  biological 
determinants  of  various  components  of  cognition  that  are  involved  in  the 
acquisition,  processing,  retention,  consolidation,  and  retrieval  of 
information  or  experience. 

Experiments  have  been  designed  to  characterize  and  contrast  cognitive 
failures  in  patients  with  disturbances  of  mood,  children  with  various  forms  of 
impairment  in  learning,  and  populations  of  neuropsychiatric  patients  including 
those  with  Huntington's  disease,  Korsakoff's  psychosis,  and  Alzheimer's 
disease.  How  are  specific  forms  of  central  nervous  system  changes  related  to 
discrete  alterations  in  cognitive  processes?  Components  of  information 
processing  in  unimpaired  individuals  are  also  studied.  A  third  type  of  study 
of  both  unimpaired  individuals  and  patient  groups  uses  various  types  of  drug 
manipulation  to  alter  specific  aspects  of  cognitive  processes.  How  do  drug 
treatments  that  alter  the  activity  of  discrete  aggregates  of  neurons  relate  to 
qualitatively  different  changes  in  cognitive  processes? 

a.  Studies  of  depression 

Depressed  patients  are  impaired  in  some  but  by  no  means  all  aspects  of 
learning,  memory,  and  retrieval.  In  part,  this  is  a  function  of  the  intensity 
of  their  depression;  moreover,  the  retrieval  of  experience  is  in  part  mood 
state-dependent.  However,  some  aspects  of  information  processing  are 
particularly  likely  to  be  interrupted.  Using  models  that  distinguish  between 
automatic  versus  effortful  (or  passive  versus  active)  processing  in  cognition, 
we  have  noted  that  depressed  patients  may  be  profoundly  impaired  when 
cognitive  processes  require  active  or  effortful  as  opposed  to  passive  or 
automatic  kinds  of  operations.  For  the  latter,  the  depressed  patient  may  be 
indistinguishable  from  a  normal  control.  The  extent  to  which  depressed 
patients  are  provided  with  organization  of  structure  (as  opposed  to  random 
uses)  in  information-processing  tasks  is  the  extent  to  which  they  are 
relatively  indistinguishable  from  normal  controls  in  terms  of  learning  and 
memory . 

Recent  studies  in  patients  with  Parkinson's  Disease  (PD)  further  highlight 
the  distinction  between  impairments  in  automatic  and  effortful  cognitive 
operations.  Early  stage  PD  patients  experience  memory-learning  failures  on 
tasks  that  require  effort  but  not  those  that  can  be  accomplished 
automatically.  The  memory-learning  pathology  is  very  much  like  that  evident 
in  depressed  patients  and  unlike  that  seen  in  progressive  dementia  patients  of 
an  Alzheimer's  type.  We  have  also  been  able  to  show  that  mood  changes  along 
with  changes  in  personality  configurations  (as  seen  in  multiple  personality 
syndrome)  affect  changes  in  how  experiences  are   encoded  and  organized  and  also 
how  they  are  retrieved  from  memory.  Those  cognitive  changes  are  similar  to 
drug  related  state-dependent  learning  and  state-dependent  retrieval  effects. 


101 


b.  studies  of  dementia 

Patients  with  progressive  idiopathic  dementia  (most  probably  of  the 
Alzheimer's  type)  have  classically  been  diagnosed  on  the  basis  of  the 
cognitive  state  that  they  present  clinically.  These  patients  demonstrate 
profound  impairments  in  learning  and  memory  as  well  as  other  kinds  of 
cognitive  operations.  We  have  now  described  more  fully  the  characteristics  of 
the  cognitive  dysfunction  in  these  patients  and  defined  some  of  the  mechanisms 
that  determine  their  learning/memory  failure.  In  a  study  of  a  large  series  of 
early  stage  Alzheimer's  patients,  we  have  demonstrated  that  information  is 
lost  from  memory  relatively  rapidly,  that  immediate  memory  is  relatively 
unimpaired,  and  that  any  type  of  learning/memory  operation  that  requires  the 
establishment  of  permanent  trace  events  is  dramatically  disrupted.  These 
impairments  are   due  in  large  part  to  processing  or  acquisition  deficits  which 
then  result  in  weak  trace  formation  and,  therefore,  failures  to  remember.  We 
have  shown  that  the  extent  to  which  Alzheimer's  patients  have  access  to 
structures  in  semantic  memory  is  the  extent  to  which  they  are  relatively 
unimpaired  on  many  tasks  of  learning  and  memory.  These  results  can  have 
important  implications  both  diagnostically,  in  distinguishing  this  group  of 
patients  from  other  groups,  as  well  as  for  potential  treatment  strategies. 

Cholinergic  drug  treatment  of  progressive  dementia  patients  has  proved  to 
produce  weak  enhancement  of  some  components  of  information  processing.  It 
woulG  appear  that  those  patients  who  are  least  cognitively  impaired  are  most 
likely  to  benefit  from  such  treatment.  In  contrast,  vasopressin-treated 
progressive  dementia  patients  have  shown  temporary  marked  changes  in  learning 
and  memory,  particularly  in  those  subjects  who  can  access  previous  knowledge 
better,  following  such  drug  treatment. 

The  mechanisms  and  determinants  of  the  cognitive  impairments  in  depression 
and  dementia  have  allowed  us  to  devise  strategies  to  distinguish  between  these 
two  groups  clinically.  Three  dimensions  that  are  particularly  useful  in 
distinguishing  the  cognitive  impairment  in  depression  from  that  of  the 
progressive  idiopathic  dementia  patient  are:  (1)  that  of  automatic  vs. 
effortful  processing;  (2)  the  degree  to  which  effort  is  extended  in 
accomplishing  tasks;  and  (3)  the  extent  to  which  the  information  being 
processed  is  either  random  or  highly  structured. 

c.  Learning  disabilities 

We  have  examined  cognitive  changes  in  children  with  various  forms  of 
learning  disability.     Two  groups  of  these  children  have  been  studied:     in  one, 
hyperactivity  is  part  of  the  syndrome;  in  the  second,   there  is  no  evidence  of 
hyperactivity  or  generalized  retardation.     All   of  these  children  demonstrated 
aramatic  impairments  in  learning  and  memory  that  resemble  the  kinds  of 
cognitive  losses  seen  in  some  groups  of  adults.     The  resemblance  is  closest  to 
depressed  patients,  but  it  also  mimics  the  kinds  of  cognitive  changes  that  are 
produced  by  drugs  which  disrupt  cholinergic  and  noradrenergic  activity.     Thus, 
both  hyperactive  and  learning-disabled  children  demonstrate  impairments  in 
effortful   processing  of  information,  whereas  automatic  processing  is  left 
relatively  intact. 


102 


d.  Pharmacological  studies  of  cognition 

Recent  studies  have  shown  that  the  dopamine  system  is  involved  in  those 
types  of  cognitive  operations  that  require  effort  but  not  for  cognitive 
processes  that  can  be  accomplished  automatically.  When  elderly  unimpaired 
subjects  were  treated  with  L-Dopa/carbidopa  their  recall  of  information  was 
enhanced  (effortful  processes)  but  their  memory  for  how  often  an  event  took 
place  (automatic  processing)  was  unaffected. 

We  have  also  begun  to  explore  the  role  of  the  serotonin  system  in 
cognition.  We  have  been  able  to  show  that  zimelidine,  a  relatively  specific 
5HT  reuptake  blocker,  can  substantially  reverse  the  cognitive  impairing 
effects  of  acute  doses  of  ethanol  in  man.  Ethanol  disrupts  the  memory  for 
weakly  processed  information  and  zimelidine  reverses  this  acute  amnesic-like 
effect.  It  would  appear  that  the  serotonin  system  may  serve  to  amplify  weak 
memory  traces. 

In  both  patient  groups  and  unimpaired  subjects,  we  have  found  that 
cholinergic  antagonists  and  agonists  produce  cognitive  effects  that  are 
qualitatively  different  from  those  of  drugs  that  have  their  major  action  on 
catecholamine  activity  (either  as  agonists  or  antagonists).  A  series  of 
studies  has  been  completed  demonstrating  the  role  of  neuropeptides,  such  as 
synthetic  vasopressin-like  substances,  in  enhancing  aspects  of  learning  and 
memory.  The  enhancement  occurs  not  only  in  cognitively  impaired  depressed 
patients  but  in  unimpaired  subjects  and  in  patients  with  progressive 
idiopathic  dementia  as  well.  The  general  pattern  of  these  findings  implies 
that  different  neurotransmitter  systems  and/or  neurochemical  mediators  may  be 
involvea  in  the  regulation  of  various  aspects  of  the  acquisition,  retention, 
and  retrieval  of  information. 

We  have  now  completed  a  series  of  cholinergic  trials  in  Alzheimer's 
patients  and  have  demonstrated  that:  (a)  cholinergic  antagonists,  such  as 
scopolamine,  mimic  many  of  the  characteristics  of  progressive  idiopathic 
dementia;  and  (b)  combinations  of  cholinergic  agents  produce  small  but 
reliable  enhancements  of  some  aspects  of  learning  and  memory  in  such 
patients.  The  amount  of  enhancement  in  learning  and  memory  is  limited  by  the 
degree  of  cognitive  intactness  of  the  patients.  While  alcohol  has  been  viewed 
traditionally  as  one  type  of  pharmacological  manipulation  that  reliably 
produces  learning  and  memory  impairments  in  man,  recent  work  from  our 
laboratory  (in  collaboration  with  NIAAA)  has  demonstrated  that  post-processing 
manipulation,  including  treatment  with  alcohol,  can  produce  some  enhancement 
in  learning  and  memory.  This  paradoxical  improvement,  as  well  as  that  evident 
in  the  effect  of  vasopressin  on  reversing  retrograde  amnesia  following 
administration  of  ECT,  implies  that  it  is  important  to  continue  to  explore  the 
biological  events  that  succeed  information  processing. 

D.  Autonomic  Nervous  System  Activity  in  Attention  and  Psychopathology 

The  central  focus  of  this  research  is  the  role  of  attentional  processes 
and  autonomic  nervous  system  (ANS)  functioning  in  psychopathology,  especially 
schizophrenia.  Studies  are  directed  toward  several  basic  issues:  (1)  the 


103 


nature  of  the  attention  and  ANS  dysfunction,  (2)  the  diagnostic  specificity  of 
the  dysfunction,  (3)  state  vs.  trait  issues,  (4)  the  neurobiological  basis  of 
attention  and  ANS  functioning. 

In  a  completed,  but  as  yet  unpublished  study,  we  have  replicated  earlier 
findings  that  a  pattern  of  high  baseline  ANS  activity,  slow  adaptation  and 
habituation,  and  a  sluggish  ANS  response  to  stress  were  associated  with  a 
schizophrenic  diagnosis.  We  have  examined  ANS  activity  in  different  subgroups 
of  patients.  Only  unremarkable  differences  were  found  between  patient  groups 
who  improved,  worsened,  or  showed  no  effects  from  IV  dextroamphetamine 
compared  to  a  placebo  infusion.  However,  significantly  reduced  ANS 
responsivity  to  significant  stimuli  and  situations  were  found  in 
schizophrenics  with  cortical  atrophy  compared  to  patients  with  normal  CT 
scans.  Since  the  ANS  results  for  the  patient  groups  differed  from  control 
values  in  the  same  direction,  the  data  suggest  a  quantitative  rather  than  a 
qualitative  difference  between  groups  contrary  to  speculations  that  have  been 
advanced  elsewhere.  In  a  current  study  on  schizophrenia,  a  major  aim  is  to 
compare  several  RT  methods  of  assessing  attention  deficits  in  order  to  define 
more  precisely  the  nature  of  attention  impairments  in  schizophrenia  and  to 
investigate  the  role  of  ANS  activity  in  attention  impairments. 

Diagnostic  specificity  of  the  measures  is  being  investigated  by  testing, 
on  part  of  the  schizophrenia  protocol,  patients  with  a  major  depressive 
illness,  obsessive-compulsive  neuroses,  and  panic-anxiety  disorder  and  adults 
with  a  history  of  infantile  autism.  Comparisons  of  obsessive-compulsive 
adults  with  controls  indicate  higher  baseline  ANS  arousal  in  the  patients  but 
no  differences  in  habituation.  In  a  group  of  adolescent  obsessive- 
compulsives,  only  the  boys  showed  this  pattern  of  results  compared  to  matched 
controls.  Obsessive  girls  had  low  arousal  and  ANS  responsivity.  Hopefully, 
correlations  with  other  biological  markers  will  shed  light  on  this  puzzling 
result. 

Our  prior  work  suggested  that  many  ANS  variables  represent  stable  traits 
that  may  be  partly  genetically  determined.  We  are  continuing  to  study  their 
relationships  to  such  personality  variables  as  sensation  seeking, 
extraversion,  and  psychoticism  in  normal  subjects  and  are  collaborating  with 
investigators  who  are   obtaining  biological  and  neuropsychological  measures  on 
the  same  subjects.  State  influences  are  being  studied  in  patients  diagnosed 
as  multiple  personality.  Nine  patients  and  five  controls  have  been  tested  in 
four  or  five  sessions  in  a  design  which  enables  comparison  of  within 
personality  with  between  personality  variations  for  each  subject.  Results 
show  that  eight  of  nine  patients  had  significant  day-to-day  consistency  in 
between-personal ity  variations  in  reaction  time  and  in  more  of  the  ANS 
variables  than  would  be  expected  by  chance.  Controls  produced  reliable 
consistency  only  in  hypnotic  states.  In  patients,  there  was  a  partial 
carryover  of  habituation  from  one  alternate  personality  to  the  next  almost  as 
large  as  that  seen  in  the  controls.  This  suggests  an  influence  of  the 
experience  of  one  personality  on  the  ANS  reactions  of  others.  The 
relationship  of  this  to  conscious  awareness  among  alternate  personalities  will 
be  investigated  on  a  case-to-case  basis.  In  another  study  related  to  state 
influences  on  ANS,  normals  are   tested  when  they  are  either  supine  or 


104 


standing.  Standing  doubles  plasma  norepinephrine  and  our  results  show  it 
elevates  electrodermal  and  heart  rate  base  levels  and  increases  ANS  orienting 
responses  to  tones  in  frequency  and  amplitude.  Effects  on  two-flash  threshold 
differed  for  subjects  differing  in  "psychoticism".  Standing  seems  to  increase 
ANS  activity,  without  confounded  changes  in  attention  or  distraction,  and  it 
could  have  wider  application  in  studying  the  effects  of  "arousal"  on 
behavior.  Our  results  also  show  inverse  correlation  between  ANS  activity  and 
psychoticism,  suggesting  that  psychoticism  may  be  more  akin  to  sociopathy  than 
to  clinical  psychosis. 

Neurobiological  bases  of  attention  and  ANS  activity  are  studied  by 
investigation  of  the  effects  of  drugs  with  fairly  specific  actions.  Analyses 
will  begin  shortly  on  the  comparative  effects  of  several  drugs  on 
schizophrenia  including  pimozide,  a  dopamine  receptor  blocker,  and  propanolol, 
a  beta  receptor  blocker.  The  effects  in  schizophrenics  of  prazosin,  a 
specific  alpha  noradrenergic  antagonist,  were  an  increase  in  ANS  arousal  but 
no  clinical  changes.  The  ANS  effects  are  likely  due  to  an  increase  in  plasma 
Nt.  The  results  confirm  our  previous  conclusion  of  an  independence  of 
variations  in  ANS  activity  and  in  psychosis.  A  recent  study  compared  the 
effects  of  an  MAO  type  A  inhibitor  (clorgyline)  with  clomipramine,  a  tricyclic 
antidepressant,  in  obsessive-compulsive  patients.  Although  only  clomipramine 
was  clinically  effective,  both  drugs  reduced  electrodermal  base  levels, 
however,  only  clomipramine  reduced  ANS  responding  to  simple  tones  and  task 
related  stimuli.  This  result  is  of  interest  because  ANS  hyperresponsivity  has 
been  implicated  as  an  etiological  factor  in  theories  of  obsessive-compulsive 
disorder.  Moreover,  the  patients  with  the  greatest  clinical  improvement 
showed  the  largest  drug  effects  on  several  of  these  variables. 

The  ANS  effects  of  a  chronic  (2-week)  period  of  high  dietary  caffeine 
consumption  vs.  placebo  in  normal  children  was  investigated  with  Dr.  Rapoport 
using  a  design  that  also  permits  testing  the  effects  of  habitual  caffeine 
use.  Results  replicated  previous  studies  from  this  project  using  both  acute 
and  chronic  caffeine  dosage  in  finding  increased  ANS  arousal  indices  and 
retarded  habituation  in  subjects  taking  caffeine.  Also  confirmatory  were 
findings  that  low  users  had  higher  arousal  indices  on  placebo  compared  to 
habitual  users.  This  suggests  that  the  characteristic  level  of  ANS  activity 
of  a  child  may,  in  part,  determine  the  propensity  for  caffeine  use.  Since 
caffeine  affects  adenosine  and  benzodiazapine  receptors,  the  data  suggest  that 
these  systems  are  involved  in  the  control  of  ANS  activity. 

E.  Interaction  of  Nature  and  Nurture  in  Schizophrenia 

1.  Studies  of  Heredity  and  Environment  in  Schizophrenia 

The  project  is  composed  of  the  following  studies: 

a.  An  intensive  multi disciplinary  study  of  a  family  with  monozygous 
quadruplets  (daughters)  concordant  as  to  schizophrenia  but  discordant  as 
to  severity  and  outcome. 


105 


The  first  study  of  this  family  was  completed  and  published  in  book  form  in 
1963.  We  have  continued  our  contacts  with  this  family  to  follow  the  clinical 
course  of  these  women  and  to  see  how  the  course  is  related  to  earlier  and 
current  life  experiences.  A  second  intensive  multi disciplinary  study  of  these 
women  was  completed  in  June  of  1981. 

This  study  includes  a  number  of  the  same  variables  that  were  examined  in 
the  quadruplets  in  the  late  50's  (e.g.,  CPT,  reaction  time,  autonomic  nervous 
system  arousal  and  habituation)  and  adds  a  considerable  number  of  new  methods 
and  techniques  for  assessing  behavioral  and  biological  factors.  Among  the  new 
methods  are  the  CAT  scan,  the  PET  scan,  far-field  brainstem  auditory  evoked 
potentials,  cerebral  mapping  of  various  EEG  components  and  measures  of  many 
biogenic  compounds  from  blood,  urine,  and  CSF  whose  existence  was  unknown  or 
for  which  no  link  to  psychopathology  had  been  established  twenty  years  ago. 
The  focus  of  this  effort  remains  the  same  as  it  v/as  in  1963:  to  help 
illuminate  the  differences  in  the  severity  of  illness  among  these  women.  We 
have  added,  where  possible,  studies  of  the  mother  of  the  quadruplets,  of  the 
husband  of  the  one  who  has  been  married,  and  of  the  two  sons  who  have  issued 
from  that  marriage.  This  additional  information  may  also  shed  some  light  on 
possible  genetic  factors  in  schizophrenia.  These  studies  (a  series  of  three 
papers)  were  published  in  1984  in  Psychiatry  Research. 

b.  Studies  of  adoptees  with  schizophrenic  parents  and  their  biological 
and  adoptive  families. 

This  represents  a  completion  of  work  done  with  a  cohort  of  adoptees  and 
controls  that  were  obtained  in  Denmark  in  the  1960's.  Although  a  considerable 
portion  of  this  work  has  been  published,  much  of  the  psychological  information 
was  not  analyzed.  A  reanalysis  of  the  original  case  material  has  appeared  in 
the  American  Journal  of  Psychiatry.  Another  paper  comparing  disordered  and 
non-disordered  subjects  in  terms  of  reported  stresses  during  development  is 
under  editorial  review  at  this  time. 

c.  A  study  of  children  of  schizophrenic  and  control  parents  reared  in 
town  or  kibbutz  in  Israel. 

This  study,  begun  in  1965,  has  involved  a  multi disciplinary 
(psychological,  psychiatric,  neurological)  examination  of  a  cohort  of  50 
children  with  schizophrenic  parents  (index  cases)  together  with  50  matched 
controls.  Half  of  each  group  was  raised  on  a  kibbutz,  and  half  was  raised 
with  their  nuclear  families  in  cities  and  towns  in  Israel.  The  children  were 
seen  three  times:  once  in  1966-67,  when  they  were  about  10-11  years  of  age, 
once  in  1973  when  they  were  about  17,  and  most  recently  in  1981  when  they  were 
in  their  mid-twenties.  This  last  study  found  significant  amounts  of 
psychopathology  in  the  index  cases.  Aside  from  several  reports  of  the 
neurologic  findings  (suggesting  more  "soft"  signs  in  the  index  cases)  none  of 
the  work  has  been  published  before  this  year.  Major  progress  has  been  made  in 
the  last  two  years:  (1)  Major  portions  of  the  data  have  been  analyzed;  (2)  A 
series  of  manuscripts  have  been  prepared  describing  the  1966,  1973,  and  1981 
studies.  These  form  the  basis  of  a  comprehensive  report  that  was  published  in 
a  single  issue  of  the  Schizophrenia  Bulletin  in  1985.  The  results  of  the  1981 


106 


study  are  startling,  since  they  suggest  that  being  raised  in  a  kibbutz 
environment  is  more  likely  to  lead  to  major  psychopathology,  given  a 
schizophrenic  diathesis,  than  is  growing  up  in  the  nuclear  family  within  a 
city  environment.  This  follow-up  report  is  included  in  the  comprehensive 
report  in  the  Schizophrenia  Bulletin.  Some  of  the  results  were  presented  at 
the  1983  meeting  of  the  American  Psychiatric  Association  and  at  a  number  of 
recent  lectures  and  seminars.  Plans  are  now  being  implemented  to  conduct 
another  major  follow-up  of  the  Israeli  cohort,  including  brain  CT  scans, 
measures  of  psychiatric  status,  and  behavior  on  cognitive  and  attention  tests. 

.  The  objectives  of  all  of  these  projects  are  to  understand  how  hereditary 
and  environmental  factors  interact  to  make  for  schizophrenic  outcomes  of 
varying  types  and  degrees. 

2.  Studies  on  Etiological  Factors  in  Schizophrenia. 

Studies  of  the  occurrence  of  mental  illness  in  families  have  been  useful 
in  identifying  familial  forms  of  the  illnesses  and  in  the  development  of 
hypotheses  regarding  the  form  and  strength  of  genetic  and  environmental 
factors  in  etiology.  Where  these  major  variables  are  separated  by  the  process 
of  adoption,  specific  etiologic  hypotheses  can  be  tested  separately  and  in 
combination.  A  total  national  sample  of  14,500  adult  adoptees  in  Denmark, 
including  76  who  have  developed  schizophrenia,  provide  the  basis  of  one  phase 
of  this  research;  the  other  phase  is  represented  by  schizophrenic  patients  and 
their  families  residing  in  Roscommon  County,  Ireland,  where  the  prevalence  of 
schizophrenia  appears  to  be  three  times  higher  than  its  prevalence  in  England 
and  other  Western  countries. 

F.  Cognitive  and  Perceptual  Changes  in  Affective  Illness 

This  project  has  involved  a  variety  of  studies,  of  which  two  are  still 
being  completed.  All  are  at  the  stage  of  final  data  collection  and  are  also 
at  or  near  the  completion  of  data  analysis.  Progress  over  the  past  year  will 
be  summarized  below,  while  more  detailed  descriptions  appear  in  the  individual 
project  reports.  Since  these  projects  were  under  the  direction  of  Dr.  Edward 
Silberman,  who  left  the  LPP,  no  additional  data  are  being  collected.  It  is 
expected  that  all  the  projects  will  be  concluded  shortly.  Dr.  Silberman 
remains  a  Guest  Researcher  in  the  laboratory. 

a.  Psychomotor  and  psychosensory  symptoms  in  affective  illness 

Data  have  been  collected  on  a  total  of  111  patients  with  affective 
illness,  partial  complex  epilepsy,  and  hypertension  (controls).  Preliminary 
analyses  indicate  that  affective  and  epileptic  patients  are  indistinguishable 
in  frequency  of  certain  kinds  of  visual,  auditory,  visceral,  and  cognitive 
illusions  and  hallucination,  while  paroxysmal  motor  phenomena  are  present  only 
in  the  epileptic  group.  The  relationship  of  such  symptomatology  to  underlying 
personality  factors  and  life  course  of  illness  in  the  affective  group  is  now 
being  analyzed. 


107 


b.  Lateral ized  hemispheric  function  in  depression 

Depressed  female  subjects  have  been  found  to  show  right  hemisphere 
advantage  in  a  verbal  processing  task  which  normally  displays  left  hemisphere 
advantage.  Since  such  a  task  is  an  obligatory  left  hemisphere  task  in 
normals,  these  results  suggest  shifts  of  hemispheric  function  as  a  concomitant 
of  the  depressed  state.  Further  studies  involving  male  depressed  subjects  and 
depressed-recovered  patients  are  now  being  planned.  A  manuscript  has  been 
submitted  for  publication. 


108 


ANNUAL  REPORT  OF  THE  LABORATORY  OF  SOCIO-ENVIRONMENTAL  STUDIES 

NATIONAL  INSTITUTE  OF  MENTAL  HEALTH 

October  1,  1984  through  September  30,  1985 

Melvin  L.  Kohn,  Ph.D.,  Chief 


This  is  ray  twenty-fifth  and  last  annual  report  as  Chief  of  the 
Laboratory  of  Socio-environmental  Studies  of  the  National  Institute  of 
Mental  Health.   On  September  first,  I  shall  join  the  faculty  of  the 
Department  of  Sociology  at  The  Johns  Hopkins  University.   1  leave  NIMH  and 
NIH  with  gratitude  for  the  administrative,  financial,  and  even  emotional 
support  I  have  received  for  nearly  all  of  the  thirty-three  years  I  have 
worked  in  these  remarkable  institutions.   I  leave,  also,  with  pride  in  the 
accomplishments  of  the  Laboratory,  which  has  long  been  at  the  forefront  of 
basic  research  in  social  structure  and  personality.   I  regret,  though,  that 
one  major  item  on  my  agenda  has  yet  to  be  achieved.   I  had  hoped  that  NIH 
would  use  our  Laboratory  as  a  prototype  for  social  science  Laboratories  in 
other  Institutes.   This  has  not  yet  happened.   I  remain  confident,  however, 
that  one  day  NIH  will  recognize  the  need  to  establish  social  science 
Laboratories  in  several  of  its  Institutes.   At  that  time,  I  hope  that  NIH 
will  call  upon  me,  as  a  knowledgeable  alumnus,  for  advice  and  help. 

During  the  current  year,  the  Laboratory's  research  efforts  have  been 
addressed  to:   analyses  of  the  relationship  between  educational  experience 
and  children's  personality  development,  the  intergenerational  transmission 
of  values,  and  intra-family  dynamics  and  children's  personality 
development;  continued  cross-national  comparative  analyses  —  for  the  U.S. 
and  Poland  and  for  the  U.S.  and  Japan  —  of  the  relationships  between  job 
conditions  and  psychological  functioning;  and  differentiation  of  the 
principal  sectors  of  the  U.S.  economy.   This  annual  report  summarizes  all 
of  these  activities. 

EDUCATIONAL  EXPERIENCE  AND  CHILDREN'S  PSYCHOLOGICAL  DEVELOPMENT 

The  purpose  of  Karen  Miller,  Melvin  Kohn,  and  Carmi  Schooler's 
analysis  has  been  to  examine  the  processes  by  which  students'  educational 
experiences,  particularly  the  degree  of  self-direction  they  exercise  in 
their  educational  endeavors,  affect  their  psychological  functioning.   Data 
for  this  analysis  were  collected  in  a  1974  follow-up  to  the  Laboratory's 
original  1964  cross-sectional  survey  of  employed  men.   In  the  follow-up 
study,  a  subsample  of  the  men  were  re-interviewed  and  the  wife  and  one 
pre-selected  child  of  each  father  in  the  subsample  were  also  interviewed. 
The  interview  schedule  for  these  "children"  —  by  then  aged  13  to  25  — 
contains  an  intensive  battery  of  questions  about  the  current  educational 


109 


experiences  of  all  those  respondents  still  in  school.   These  questions, 
designed  to  parallel  those  earlier  found  to  be  useful  for  analyzing  adults' 
occupational  experience,  focus  on  such  dimensions  of  the  educational 
experience  as  its  substantive  complexity  and  how  closely  it  is  supervised. 
The  underlying  hypothesis  is  that,  just  as  occupational  self-direction 
affects  the  psychological  functioning  of  employed  adults,  so  does 
educational  self-direction  affect  the  psychological  functioning  of 
students.   Insofar  as  it  is  meaningful  to  do  so,  the  investigators  have 
tried  to  conceptualize  and  measure  educational  self-direction  as  being 
parallel  to  occupational  self-direction. 

Past  annual  reports  have  discussed  the  relationships  of  educational 
self-direction  with  three  major  facets  of  personality  —  ideational 
flexibility,  self-directedness  of  orientation,  and  distress.   The  major 
accomplishment  this  year  has  been  the  development  of  a  comprehensive  model 
of  educational  self-direction  and  all  three  facets  of  personality.   The 
model  shows  an  intricate  system,  in  which  educational  self-direction 
directly  or  indirectly  affects  all  three  facets  of  personality;  distress 
and  ideational  flexibility  affect  educational  self-direction;  distress 
affects  both  self-directedness  of  orientation  and,  indirectly,  ideational 
flexibility;  and  self-directedness  of  orientation  and  ideational 
flexibility  directly  or  indirectly  affect  each  other.   These  findings  have 
important  theoretical  and  practical  implications  for  our  understanding  of 
the  psychological  impact  of  education,  for  the  understanding  of  work  in  the 
labor  force,  and  for  the  understanding  of  the  link  between  the  two. 

First,  the  findings  illuminate  some  of  the  major  processes  by  which 
education  affects  cognitive  functioning.   The  model  certainly  confirms  that 
educational  self-direction  affects  students'  cognitive  functioning.   The 
model  also  suggests,  though,  that  a  large  part  of  this  effect  comes  about 
through  educational  self-direction  affecting  self-directedness  of 
orientation  and  distress.   Thus,  non-cognitive  aspects  of  personality  play 
a  considerable  role  in  how  schooling  contributes  to  students'  intellectual 
development.   Educational  self-direction  leads  to  more  effective 
intellectual  performance  as  much  because  it  leads  to  a  self-directed 
orientation  and  because  it  mitigates  distress  as  because  of  its  role  in 
cognitive  training,  per  se. 

A  second  major  conclusion  is  that  the  causal  relationship  between 
personality  and  the  exercise  of  self-direction  in  work  is  remarkably 
similar  for  students  and  employed  adults.   The  similarity  of  findings 
suggests  some  fundamental  links  between  work,  regardless  of  setting,  and 
the  personality  of  the  worker.   For  schooling,  as  for  paid  employment,  the 
opportunity  to  exercise  self-direction  in  one's  own  work  has  powerful 
psychological  effects.   This  finding  constitutes  a  striking  affirmation 
that  an  interpretative  model  designed  to  explain  the  social  psychology  of 
work  in  paid  employment  is  applicable  to  the  social  psychology  of  work  in 
school. 


110 


Finally,  the  research  corroborates  essential  elements  of  the 
interpretation  that  schooling  reproduces  social  class  by  differentially 
training  students  to  be  independent  and  self-directed  in  their 
orientations.   The  investigators  find,  as  expected,  that  students  from 
families  of  higher  socio-economic  status  are  more  likely  to  be 
educationally  self-directed.   They  also  find  that  as  students  make  their 
way  through  the  educational  system,  they  are  progressively  trained  to  have 
more  and  more  self-directed  orientations.   Indeed,  the  research  goes 
further:   it  identifies  a  crucial  element  of  that  training.   Students  in 
higher  grade  levels  experience  more  educational  self-direction;  this 
increased  exercise  of  initiative,  thought,  and  independent  judgment  in 
schoolwork  changes  the  self-directedness  of  students'  orientations,  their 
sense  of  distress  or  well-being,  and  the  effectiveness  of  their  cognitive 
functioning.   Because  educational  self-direction  has  these  powerful 
effects,  it  proves  to  be  a  central  mechanism  through  which  the  educational 
system  molds  the  personalities  of  students  in  ways  consonant  with  their 
likely  positions  in  the  hierarchical  division  of  labor.   Educational 
self-direction  is  a  critical  link  between  schooling  and  adult  occupational 
role. 


THE  INTERGENERATIONAL  TRANSMISSION  OF  VALUES 

Melvin  Kohn,  Kazimierz  Slomczynski,  and  Carrie  Schoenbach's 
cross-national  analysis  of  the  intergenerational  transmission  of  values 
addresses  two  principal  issues:   To  what  extent  does  the  social 
stratification  position  of  the  parental  family  affect  the  values  of  its 
adolescent  and  young-adult  offspring?   What  are  the  processes  by  which  that 
position  affects  offsprings'  values?   Both  questions  are  classic  issues  for 
the  sociology  and  social  psychology  of  socialization. 

Although  there  is  considerable  evidence  that  social  stratification 
has  substantial  effects  on  adults'  —  hence,  parents'  —  values  and 
orientations,  there  is  much  less  evidence  that  the  family's  stratification 
position  has  appreciable  effects  on  the  values  and  orientations  of  its 
adolescent  and  young-adult  offspring.   Nor,  for  that  matter,  is  there  much 
evidence  that  parents '  values  have  an  appreciable  effect  on  their 
children's  values.   Quite  the  contrary:   prior  research  has  indicated 
suprisingly  weak  relationships  between  parents'  and  children's  values.   And 
yet,  as  Clausen  put  it,  "[a]  basic  tenet  of  socialization  theory  —  almost 
all  socialization  theory  —  is  that  the  child's  core  value  orientations  are 
learned  in  the  family...." 

The  inquiry  is  premised  on  the  belief  that  it  is  not  socialization 
theory  but  past  empirical  evidence  that  is  deficient.   The  investigators 
therefore  hypothesized  that  the  stratification  position  of  the  parental 
family  appreciably  affects  the  values  of  adolescent  and  young-adult 
offspring  and,  furthermore,  that  the  effect  is  primarily  through  parents' 
values. 


Ill 


Tliey  further  hypothesized  that  the  relationship  between  social 
stratification  and  children's  values,  as  mediated  through  parents'  values, 
is  not  peculiar  to  any  particular  economic  or  political  system,  but  is 
built  into  the  structure  of  industrial  society.   The  inquiry  was  therefore 
cross-national,  based  on  nore-or-less  parallel  analyses  of  one  capitalist 
society  (the  United  States)  and  one  socialist  society  (Poland).   Obviously, 
even  identical  findings  for  these  two  countries  would  not  ensure  that  the 
findings  could  be  generalized  to  other  capitalist  and  socialist  societies. 
Similar  findings  would,  however,  demonstrate  that  the  relationship  between 
stratification  and  children's  values  is  not  unique  to  the  economic  and 
political  system  of  either  the  U.S.  or  Poland.   The  intent  of  the  inquiry 
was  to  show  that  the  relationship  between  social  stratification  and 
children's  values  transcends  the  differences  between  capitalist  and 
socialist  societies  because  the  same  mechanisms  underlie  this  relationship 
in  both  systems. 

The  further  intent  of  this  inquiry  was  to  explicate  the  mechanisms 
by  wliich  family  stratification  position  affects  children's  values.   Here 
the  investigators  built  on  their  earlier  analyses,  which  point  to  the 
pivotal  role  of  occupational  experience,  particularly  the  opportunity  to 
exercise  self-direction  in  one's  work.   They  extended  those  analyses  in 
several  ways:   by  simultaneously  examining  the  relationship  between 
occupational  self-direction  and  the  values  of  husband,  wife,  and  child;  by 
assessing  the  influence  of  each  family  member's  values  on  the  values  of  the 
others;  and,  basic  to  all  the  rest,  by  treating  the  family  as  a  subsystem 
of  the  larger  social  system. 

The  analyses  are  based  on  representative  samples,  in  the  United 
States  and  Poland,  of  parents  and  their  adolescent  or  young-adult 
offspring.   In  both  countries,  the  samples  consist  of  triads:   father, 
mother,  and  one  selected  child. 

The  U.S.  data  are  partially  longitudinal.   The  baseline  data  consist 
of  interviews  conducted  in  1964  with  3101  men,  representative  of  all  men 
employed  in  civilian  occupations  in  the  continental  United  States.   Every 
man  who  was  the  father  of  at  least  one  child  living  at  home  and  aged  3-15 
years  was  asked  about  his  values  vis-a-vis  one  of  those  children,  randomly 
selected.   Ten  years  later,  in  1974,  a  representative  subsample  of 
one-fourth  of  the  men  in  the  baseline  survey  (687  in  all)  were 
re-interviewed.   Also  interviewed  were  the  wives  of  men  then  married  and 
the  "child"  about  whom  the  values  questions  had  been  asked  ten  years  before 
—  these  "children"  being  13-25  years  old  when  interviewed.   There  are  352 
families  in  the  U.S.  sample. 

The  Polish  data  are  cross-sectional.   Interviews  were  conducted  in 
1978  with  1557  men,  representative  of  all  men  employed  in  civilian 
occupations  in  the  urban  areas  of  Poland.   As  in  the  U.S.  survey,  each 
father  who  had  at  least  one  child  aged  3-15  living  at  home  was  asked  about 
his  values  vis-a-vis  a  randomly  selected  one  of  those  children.   In 


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1979-1980,  interviews  were  conducted  with  those  children  who  were  then 
13-17  years  old  (N=177)  and  with  their  mothers.   This  age-range  was 
intended  to  maximize  the  overlap  with  the  age-range  of  the  U.S.  "children". 
Although  the  data  from  mothers  and  children  were  secured  one-and-a-half  to 
two  years  later  than  those  from  fathers,  the  investigators  are  treating  the 
Polish  data  as  if  tVie  interviews  with  father,  mother,  and  child  were 
contemporaneous . 

The  analyses  provide  evidence,  for  both  the  U.S.  and  Poland,  not 
only  of  a  close  relationship  between  parents'  and  children's  values,  but  of 
an  actual  influence  of  parents'  values  on  children's  values.   Most  past 
studies  have  underestimated  the  magnitudes  of  the  correlations  between 
parents'  and  children's  values,  because  they  have  not  taken  measurement 
error  into  account  and,  more  fundamentally,  because  they  have  not  dealt 
with  so  important  a  dimension  of  values  as  valuation  of  self-direction. 
And,  by  and  large,  past  studies  stopped  with  correlations.   The  crux  of  the 
matter,  however,  is  not  the  magnitudes  of  the  correlations  but  the  part 
played  by  parental  values  in  the  actual  process  of  intergenerational 
value-transmission.   The  present  study  demonstrates  that  parental  values 
have  a  considerable  impact  on  offspring's  values,  even  with  many  other 
pertinent  aspects  of  social  structure  taken  into  account.   A  fundamental 
but  contested  tenet  of  socialization  theory  —  almost  all  socialization 
theory  —  has  hereby  been  confirmed. 

The  investigators  have  further  found  that,  in  both  the  U.S.  and 
Poland,  the  stratification  position  of  the  parental  family  has  a 
considerable  impact  on  the  values  of  its  adolescent  and  young-adult 
offspring.   This  effect  results  primarily  from  the  stratification  position 
of  the  family  affecting  parents'  values  and  parents'  values,  in  turn, 
affecting  offspring's  values.   Moreover,  these  analyses  show  that  all  the 
links  in  the  causal  chain  are  strong:   Social  stratification  affects 
parental  occupational  self-direction;  occupational  self-direction  affects 
parental  values;  parental  values  affect  children's  values.   Whatever  doubts 
past  studies  nay  have  raised  about  this  causal  chain  can  now  be  resolved. 

In  both  countries,  parents'  occupational  self-direction 
substantially  affects  (and  is  affected  by)  their  values.   Moreover, 
adolescents'  and  young  adults'  educational  self-direction  affects  their 
values,  just  as  parental  occupational  self-direction  affects  parental 
values.   These  findings  lend  support  to  the  further  argument  that  people's 
own  experiences  become  more  and  more  important  for  their  values,  with  their 
own  social-structural  positions  and  attendant  experience  eventually 
overshadowing  the  influences  of  their  parental  families. 

The  findings  also  provide  an  intergenerational  extension  of  Kohn  and 
Schooler's  interpretive  model  of  social  stratification,  job  conditions,  and 
adult  personality.   In  that  model,  social  stratification  affects 
occupational  self-direction,  which  is  reciprocally  related  to  adult 
personality,  which  in  turn  affects  job  conditions  and  one's  place  in  the 
stratification  order.   We  can  now  add  that  social  stratification  affects 


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not  only  adults'  own  values  but  also  their  children's  values  and  their 
children's  opportunities  for  educational  self-direction.   Parental 
stratification  position  thus  affects  children's  values  not  only  through  the 
transnission  of  values  from  parents  to  children,  but  also  through  its 
effect  on  children's  opportunities  for  educational  self-direction. 

Finally,  the  investigators  have  found  one  substantial  difference 
between  the  U.S.  and  Poland:   the  relative  roles  of  fathers  and  mothers  in 
the  intergenerational  transmission  of  values.   In  the  U.S.,  fathers  play  at 
least  as  large  a  role  as  do  mothers;  in  Poland,  mothers  play  the 
predominant  role.   This  finding  does  not  seem  to  be  an  artifact  of  study 
design.   Nor  does  this  cross-national  difference  seem  to  result  from 
differing  economic  or  political  systems.   Rather,  it  appears  to  be  a 
cultural  difference:   Polish  fathers  play  a  more  traditional  role  in  the 
division  of  labor  within  the  family  and  in  the  socialization  of  children. 
This  pattern,  originally  characteristic  of  the  peasantry  and  diffused  to 
all  segments  of  the  society  through  rural-to-urban  migration,  is  also 
supported  by  the  influential  Catholic  church.   More  important  than  this  one 
difference,  however,  is  the  basic  similarity  of  findings  for  the  U.S.  and 
Poland:   Family  stratification  position  greatly  affects  adolescents' 
values;  half  or  more  of  this  effect  is  through  parents'  values;  and  there 
is  a  strong  reciprocal  relationship  between  occupational  self-direction  and 
values.   The  processes  are  built  into  the  structure  of  industrial 
societies,  both  capitalist  and  socialist. 

INTRA-FAtllLY  DYNAMICS  AND  CHILDREN'S  PERSONALITY  DEVELOPMENT 

Carmi  Schooler  has  continued  working  on  a  complementary  analysis  of 
the  ways  that  reported  child-rearing  practices  and  family  relationships 
affect  children's  psychological  development.   He  has  been  using 
confirmatory  factor  analysis  to  develop  measures  of  the  many  aspects  of 
parent-child  relationships  covered  by  the  interviews,  using  the  same  data 
as  in  the  foregoing  analyses.   His  measures  include  parental  strictness, 
warmth,  and  degree  of  dominance  over  the  child,  as  well  as  the  degree  to 
-hich  children  feel  free  to  talk  things  over  with  each  parent  and  the 
likelihood  that  they  will  turn  to  either  parent  when  troubled.   New 
measures  developed  this  year  include  indices  of  children's  perceptions  of 
their  closeness  to  each  of  their  parents,  as  well  of  the  communication 
processes  that  take  place  within  the  family. 

During  this  year.  Schooler  has  examined  the  links  between  family 
behavior  patterns  and  children's  psychological  functioning.   These  analyses 
not  only  take  into  account  family  social  background,  but  also  statistically 
control  the  parents'  own  psychological  functioning.   The  findings  indicate 
that  parents  who  give  their  children  the  opportunity  to  be  self-directed 
have  intellectually  flexible,  self-directed  children.   Thus,  the  children 
of  parents  who  tend  not  to  control,  dominate,  or  lay  down  the  law  to  their 
children  are  more  likely  to  have  high  levels  of  intellectual  flexibility 
and  to  be  self-directed  in  both  their  values  and  their  orientations. 


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Parents  who  praise  their  children  are  also  more  likely  to  have  children  who 
are  intellectually  flexible  and  have  self-directed  values. 

A  certain  amount  of  parental  emotional  distance  from  children  seems, 
however,  to  be  conducive  to  children's  intellectual  flexibility  and 
self-directedness .   Parental  warmth  is  related  to  lower  levels  of 
children's  intellectual  flexibility,  as  is  a  close  relationship  with  the 
mother,  as  perceived  by  the  child.   A  close  relationship  with  the  father  is 
related  to  a  conformist  orientation,  and  a  close  relationship  with  either 
parent  is  related  to  conformist  values.   On  the  other  hand,  close 
parent-child  relationships  are  positively  related  to  children's  sense  of 
well-being.   In  addition,  children  of  parents  who  are  warm  and  praising  and 
to  whom  the  children  speak  freely  suffer  less  distress.   The  importance  of 
the  links  between  close  parent-child  relationships,  communication,  and  the 
child's  sense  of  well-being  is  also  attested  to  by  the  strongest  finding  of 
the  analysis:   a  powerful  relationship  between  children's  sense  of 
well-being  and  the  family's  openness  and  interest  in  communication. 

Although  the  pattern  of  relationships  that  has  emerged  from  these 
analyses  seems  coherent  and  meaningful,  the  nature  of  the  underlying  causal 
connections  is  uncertain  (e.g.,  parents'  praise  can  be  either  the  cause  or 
the  result  of  children's  superior  intellectual  flexibility).   Because  of 
this  ambiguity.  Schooler  has  devoted  considerable  effort  this  year  to 
trying  to  devise  linear  structural-equation  analyses  that  will  permit  the 
assessment  of  possibly  reciprocal  relationships  (e.g.,  between  children's 
and  parents'  psychological  functioning;  between  parents'  behavior  and 
children's  psychological  functioning).   This  analysis  has  proved  to  be 
exceptionally  difficult;  it  may  even  be  impossible  to  definitively 
establish  causal  directionality.   However,  even  if  the  causal  nature  of  the 
relationships  between  family  behavior  patterns  and  children's  psychological 
functioning  remains  ambiguous,  establishing  that  parental  childrearing 
practices  are  related  to  children's  psychological  functioning  independently 
of  parents'  psychological  functioning  and  of  social  background  represents 
an  increase  in  our  knowledge  of  the  relationships  between  family  life  and 
children's  psychological  functioning. 


THE  POLISH  REPLICATION 

The  main  purpose  of  the  Polish  replication  has  been  to  see  whether 
the  interrelationship  of  social  stratification,  job  conditions,  and 
psychological  functioning  is  similar  in  socialist  and  capitalist  societies. 
Three  principal  co-investigators,  Kazimierz  Slomczynski,  Krystyna  Janicka, 
and  Jadwiga  Koralewicz-Zebik,  carried  out  in  1978  in  Poland  a  precise 
replication  of  the  survey  originally  conducted  by  Kohn  and  Schooler  in  1964 
in  the  United  States.   After  the  data  had  been  collected,  coded,  and  edited 
in  Poland,  Slomczynski  brought  them  to  NIH,  where  he,  Kohn,  and  at  times 
other  members  of  the  Laboratory  have  been  analyzing  them.   Previous  annual 
reports  have  reviewed  the  development  of  methods  designed  to  assure 
cross-national  comparability  of  indices  and  the  analysis  of  two  of  the 


115 


central  questions  of  the  Polish  replication:   Do  people's  positions  in  the 
system  of  social  stratification  bear  the  same  relationships  to  their  values 
and  orientations  in  socialist  Poland  as  in  the  capitalist  U.S.?   if  so,  do 
these  relationships  result  from  the  greater  opportunities  for  occupational 
self-direction  enjoyed  by  men  of  higher  social-stratification  position?   As 
discussed  in  detail  in  earlier  reports,  the  answers  to  both  questions  are 
positive  with  respect  to  values  and  social  orientations,  but  not  with 
respect  to  self-conception. 

Further  comparative  analysis  of  the  Polish  and  U.S.  data  has  focused 
on  social  stratification  and  the  intergenerational  transmission  of  values 
(discussed  above).   Current  work  focuses  on  extending  these  analyses  to 
other  facets  of  children's  personality  development  —  cognitive 
development,  self-conceptions,  and  social  orientations.   The  investigators 
have  also  been  filling  in  gaps  in  their  analyses  and  reassembling  their 
findings  for  publication  as  a  book.   This  book  is  to  be  published  in  Polish 
by  the  Polish  Scientific  Publishers,  on  behalf  of  the  Polish  Academy  of 
Sciences,  who  provided  the  Polish  data  and  have  supported  the  research 
throughout.   With  appropriate  modifications,  the  book  may  also  be  published 
in  English  in  the  United  States. 


THE  JAPANESE  REPLICATION 

Another  major  replication  of  the  Kohn-Schooler  occupations  study  has 
been  conducted  in  Japan  by  Atsushi  Naoi  of  Osaka  University  and  Ken'ichi 
Toninaga  of  Tokyo  University,  in  collaboration  with  Carmi  Schooler. 
Data-collection  took  place  during  the  summer  and  fall  of  1979.   At  that 
time,  a  probability  sample  of  more  than  800  employed  men  was  interviewed, 
using  a  questionnaire  that  asked  about  job  conditions  and  aspects  of 
psychological  functioning  in  ways  comparable  to  those  of  the  1964  U.S. 
study.   Data-analysis  began  in  October,  1980,  when  Naoi  came  to  the 
Laboratory  as  a  Visiting  Scientist  to  work  collaboratively  with  Schooler. 
As  reported  in  previous  Annual  Reports,  Naoi  and  Schooler  developed 
confirmatory  factor-analytic  measurement  models  of  occupational 
self-direction,  intellectual  flexibility,  and  several  facets  of 
self-conception  and  social  orientation.   These  models  proved  to  be 
generally  similar  to  those  that  had  previously  been  developed  for  the  U.S. 
sample.   Causal  analyses  of  these  data  generally  confirmed  the 
generalizeability  to  Japan  of  the  U.S.  findings. 

This  year,  Carmi  Schooler's  work  on  the  replication  and  extension  to 
Japan  of  our  U.S.  research  involved  both  the  continued  analysis  of 
previously  collected  data  and  negotiation  and  planning  for  the  acquisition 
of  new  data. 

Early  in  the  year,  Schooler  developed  a  model  of  the  reciprocal 
effects  of  job  conditions  and  parental  valuation  of  self-direction.   The 
model  demonstrates  that  parents'  own  experience  of  occupational 
self-direction  leads  them  to  value  self-direction  for  their  children;  by 


116 


contrast,  ownership,  high  hierarchical  position,  and  employment  in  a 
bureaucracy  lead  parents  to  value  conformity  to  external  authority.   The 
effect  of  occupational  self-direction  is  entirely  consonant  with  our  U.S. 
findings;  the  effects  of  organizational  position  are  distinctive  to  Japan. 

The  same  analytic  model  was  used  to  examine  the  relationships 
between  Japanese  men's  occupational  conditions  and  what  Schooler  terms 
their  psychological  integration  into  their  social  position  —  their  level 
of  alienation;  their  identification  with  their  social  stratification 
position  and  with  their  social  class  position;  and  the  importance  they 
place  on  their  jobs,  their  firms,  and  their  families.   Occupational 
self-direction  reduces  alienation  and  promotes  identification  with  higher 
social  classes  and  strata.   In  addition,  although  ownership,  high 
hierarchical  position,  and  employment  in  a  bureacracy  do  not  affect 
alienation,  they  all  lead  men  to  identify  themselves  with  higher  social 
strata  and  higher  social  classes,  suggesting,  contrary  to  the  beliefs  of 
many  Japanese  area  specialists,  that  socio-economic  position  does  affect 
the  ways  Japanese  men  identify  themselves  and  their  interests. 

Another  set  of  analyses  added  an  index  (earlier  developed  by 
Tominaga  and  Schooler)  of  the  relative  traditionalism  of  Japanese 
industries  to  models  of  the  interrelationship  of  job  conditions  and 
psychological  functioning.   Although  the  inclusion  of  this  index  of 
traditionalism  does  not  substantially  change  any  of  the  previous  findings, 
employment  in  a  relatively  traditional  industry  does  have  psychological 
repercussions.   Employment  in  such  an  industry  leads  to  conformist  values, 
less  personally  responsible  standards  of  morality,  and  greater 
authoritarian-conservatism.   It  also  results  in  less  alienation,  greater 
identification  with  higher  social  strata,  greater  self-confidence,  and  less 
self-deprecation.   This  pattern,  in  which  participation  in  a  traditional 
social  setting  leads  to  a  sense  of  social  integration,  to  acceptance  and 
identification  with  authority,  and  to  comfort  with  oneself  is  congruent 
with  the  interpretations  of  a  school  of  social  theorists  that  traces  back 
to  Durkheim. 

Given  the  richness  of  the  comparative  findings,  it  seems  highly 
desirable  that  the  parallel  analyses  of  U.S.  and  Japanese  data  be  extended. 
To  that  end.  Schooler  is  working  intensively  on  an  attempt  to  replicate  in 
Japan,  albeit  with  limited  resources,  the  full  set  of  studies  on  the 
interrelationship  of  occupational  conditions,  family  conditions,  and 
psychological  functioning  that  we  have  carried  out  in  the  United  States. 
Plans  are  currently  under  way  for  our  Japanese  collaborators  to 
re-interview  the  original  sample  of  employed  men,  to  obtain  longitudinal 
data  parallel  to  those  we  have  for  U.S.  men.   The  wives  of  the  original 
respondents  have  already  been  interviewed  by  the  Japanese  investigators. 
Schooler  is  presently  conducting  negotiations  to  obtain  these  data  for 
comparative  analyses  of  the  psychological  effects  of  housework  and  paid 
employment  for  women  in  Japan  and  the  United  States  —  countries  in  which 
the  social  position  of  women  is  quite  different.   Finally,  plans  have  been 
initiated  for  a  jointly-sponsored  effort  to  parallel  in  Japan  our  research 


117 


on  the  intergenerational  transmission  of  values  and  modes  of  psychological 
functioning.   This  is  to  be  done  by  interviewing  the  child  who  was  the 
focus  of  the  childrearing  questions  asked  in  the  men's  interview.   The 
replication  of  this  aspect  of  the  U.S.  study  is  seen  as  particularly 
important  because  there  is  evidence  suggesting  marked  differences  between 
generations  in  Japan;  a  great  deal  might  be  learned  by  a  comparative 
examination  of  the  nature  of  psychological  continuity  in  Japanese  and  U.S. 
families. 


SECTORS  OF  THE  U.S.  ECONOMY 

This  year  Ronald  Schoenberg  and  Carmi  Schooler  have  continued  their 
investigation  of  the  hypothesis,  current  in  the  fields  of  sociology  and 
economics,  that  there  are  two  principal  sectors  in  the  United  States 
economy,  a  primary  sector  and  a  secondary  sector.   Proponents  of  this 
position  maintain  that  the  sector  in  which  people  work  affects  their 
economic  compensation  and  occupational  conditions.   If  so,  it  seems  likely 
that  the  sector  in  which  people  work  would  also  affect  their  psychological 
functioning. 

To  explore  the  consequences  of  economic  sector  for  the  psychological 
functioning  of  workers,  Schoenberg  and  Schooler  have  developed  an  approach 
to  its  definition  that  differs  somewhat  from  previous  approaches,  one  that 
reflects  more  completely  the  idea  that  the  sectors  may  differ  in  their 
underlying  structure.   Their  approach  does  not  assume  in  advance  how  many 
sectors  there  might  be. 

Variance  in  underlying  structure  implies  that  the  distributions  of 
industry  measures  in  each  sector  will  have  different  moments,  that  is,  the 
means,  variances,  and  covariances  will  be  different  in  each  sector.   By 
applying  a  statistical  method  called  mixture  analysis  to  the  industry 
measures,  it  is  possible  to  estimate  the  means,  variances,  and  covariances 
for  each  sector  without  assuming  in  advance  which  industry  belongs  in  which 
sector.   Thus,  sector  membership  of  each  industry  is  an  additional 
parameter  that  is  estimated  in  the  model.   Provided  there  are  enough 
industries  in  a  given  sector,  it  should  be  possible  to  use  the  estimated 
sector  variance-covariance  matrix  to  estimate  parameters  of  a  structural 
model  describing  the  interrelationships  of  the  industry  variables  and  their 
relationship  with  workers'  psychological  functioning.   If  this  is  done  for 
each  sector,  it  should  then  be  possible  to  determine  the  precise  effect  of 
sector  on  workers'  psychological  functioning. 

To  carry  out  this  analysis,  Schoenberg  and  Schooler  secured  two 
data-sets  that  purport  to  contain  the  necessary  industry  data.   Their 
analyses  quickly  revealed  that  one  of  these  data-sets  was  riddled  with 
inaccuracies.   The  mixture  analysis  of  the  second  data-set  has  now  been 
completed.   Schoenberg  and  Schooler  have  found  not  two,  but  at  least  six, 
sectors  in  the  U.S.  industrial  economy.   Seven-,  eight-,  and  nine-sector 
models  fit  the  data  significantly  better  than  does  a  six-sector  model,  but 


118 


this  appears  to  capitalize  on  chance,  since  the  three  additional  sectors  do 
not  add  substantively  to  the  model. 

whatever  the  number  of  sectors,  two  sectors  consistently  emerge  from 
what  had  previously  been  considered  to  be  the  primary  sector.   One  of  them 
contains  industries  that  tend  to  have  a  high  proportion  of  government  sales 
(ordnance,  aircraft  and  ships,  communication,  industrial  chemicals)  and  the 
other  contains  other  important  large-scale  industries  (steel,  machinery, 
computers,  household  appliances,  photographic  equipment,  textiles, 
plastics,  footwear).   These  two  sectors  rank  first  and  second, 
respectively,  in  size,  profit,  and  concentration.   The  second  sector  ranks 
first  in  growth,  autonomy,  assets,  employee  benefits,  and  unionization,  and 
outranks  the  first  in  productivity  and  employee  salaries.   Other  sectors 
seem  to  center  on  raw  material  processing  and  refining;  retail  sales; 
wholesale  sales;  printing,  commercial  research  and  general  contracting.   In 
evaluating  the  apparent  raeaningfulness  and  cohesiveness  of  these  sectors, 
it  should  be  borne  in  mind  that  the  sectoral  distinctions  are  not  based  on 
similarity  of  product  but  on  the  similarity  of  such  industry 
characteristics  as  average  number  of  workers,  amount  of  assets  per  company, 
and  degree  of  concentration. 

Schoenberg  and  Schooler  are  continuing  to  refine  these  analyses  of  the 
structure  of  the  economy  and  shall  soon  begin  their  investigation  of  ways 
that  sectorial  locus  may  affect  individual  psychological  funcntioning. 


METHODOLOGICAL  INQUIRY 

During  the  course  of  the  year,  Ronald  Schoenberg  made  significant 
progress  in  two  other  areas  of  methodology.   An  important  problem  in  the 
analysis  of  survey  data  is  that  the  measures  are  frequently  categorical 
rather  than  continuous.   Schoenberg  this  year  developed  a  statistical 
procedure  for  the  analysis  of  categorical  variables  in  the  context  of  the 
measurement  models  presently  used  in  this  Laboratory.   This  work  will  have 
important  implications  for  the  analysis  of  social  science  data  generally. 

In  another  area,  a  method  for  mixture  analysis  was  developed  for 
exploratory  data  analysis.   Its  application  to  the  analysis  of  data  on 
industries  to  determine  whether  there  are  effects  of  type  of  industry  on 
the  psychological  functioning  of  workers  is  described  above.   In  mixture 
analysis,  the  underlying  model  is  assumed  to  differ  by  subgroups  of  the 
data  but  it  is  not  known  which  individual  case  belongs  to  which  group.   The 
number  of  subgroups  is  also  not  known.   A  computer  program  was  written  to 
implement  the  estimation  of  the  membership  parameters,  that  is,  parameters 
that  describe  the  subgroup  to  which  each  case  belongs,  as  well  as  the 
computation  of  a  statistic  to  help  determine  how  many  subgroups  there  are. 

In  the  coming  year  these  methods  for  categorical  and  mixture  analysis 
will  be  extended  to  applications  in  medical  diagnosis,  in  particular  the 
diagnosis  of  schizophrenia. 


119 


Annual  Report  of  Laboratory  of  Cell  Biology 

National  Institute  of  Mental  Health 

Michael  J .Brownstein ,  M.D.,  Ph.D.,  Chief 

October  1,  1984  -  September  30,  1985 


INTRODUCTION 

The  members  of  the  Laboratory  of  Cell  Biology  listed  in  last 
year's  annual  report  have  been  Joined  by  a  number  of  new 
people:   D.  Marshak,  W.S.  Young,  M.  Brann  (all  P.R.A.T.  fellows), 
A.  Rokaeus,  D.  Kligman,  T.  Zoeller,  K.  Roller,  J.  Waschek  and  J. 
Wallingford.   In  addition,  a  new  senior  investigator,  Audrey 
Stone,  has  transferred  into  the  Laboratory.   Dr.  Palkovits 
continues  to  function  as  half-time  chief  of  the  anatomy  group  and 
Dr.  David  Brown  has  been  awarded  a  Fogarty  Scholarship  and  is 
working  in  the  LCB  for  a  year. 

Several  people  plan  to  come  to  the  LCB  by  autumn  of  this 
year  including  S.J.  Lolait,  N.  Buckley,  R.  Burch,  L.  Mahan ,  and 
D.  Rausch.   Each  of  these  will  bring  new  skills  and  perspectives 
to  the  lab  and  we  look  forward  to  their  arrival. 


Our  work  has  broadened  its  base  in 
Marshak  has  introduced  protein  microseq 
synthesis  into  the  lab,  Dr.  Brownstein 
oligonucleotides  for  members  of  the  LCB 
other  NIH  laboratories,  Drs  .  Young  and 
technique  for  in  situ  hybridization  cyt 
has  perfected  methods  for  colocalizing 
cell  at  the  light-  and  electron  microsc 
Drs.  Barbet,  Rougon  ,  and  Reisine  have  d 
technique  for  delivering  drugs  into  eel 
allowed  us  to  embark  on  new  projects  th 
undertaken  a  year  ago.   These  new  lines 
in  the  descriptions  of  our  specific  pro 


the  last  year.   Dr. 
uencing  and  peptide 
synthesizes 

and  for  workers  in  three 
Siegel  have  developed  a 
ochemistry,  and  Dr.  Mezey 
two  antigens  in  the  same 
opic  level.   In  addition, 
evised  a  lysosorae-based 
Is.   These  methods  have 
at  we  could  not  have 

of  research  are  reflected 
jects . 


BIOLOGICALLY  ACTIVE  PEPTIDES  IN  THE  BRAIN 


Our  goal  is  to  understand  the  development  and 
the  nervous  system.   In  particular,  we  are  attempti 
characterize,  and  study  molecules  with  important  ro 
brain  and  periphery.   Examples  include  peptide  neur 
receptors,  factors  that  regulate  neuronal  developme 
proteins  involved  in  the  uptake,  storage  and  biosyn 
neurotransmitters.   The  first  step  in  this  process 
developing  assays  for  molecules  of  interest  and  usi 
to  detect  these  molecules  in  the  course  of  their  pu 
In  this  way  we  have  succeeded  in  isolating  a  gonado 
releasing  factor  from  lamprey,  a  neurite  extension 
neuronal  cell  adhesion  molecule.   Antibodies  agains 
factors  can  be  prepared  and  used  for  anatomical,  bi 


function  of 
ng  to  isolate , 
les  in  the 
o transmitters  , 
nt ,  and 
thesis  of 
involves 
ng  the  assays 
rif ication . 
tropin 

factor ,  and  a 
t  the  purified 
ochemical , 


121 


cell  biological  and  molecular  biological 
proteins  can  be  subjected  to  chemical  ana 
amino  acid  sequence  of  a  peptide  or  prote 
the  molecular  biologist  for  isolating  DNA 
particular  messenger  RNA  species.  In  the 
exploited  such  sequence  information  to  de 
including  those  for  chromagran in ,  galanin 
and  substance  P.  The  cDNA's  can  be  seque 
of  the  proteins  that  they  encode  inferred 
cDNA's  can  be  employed  to  isolate  their  c 
as  probes  for  studies  of  messenger  RNA  pr 
particularly  exciting  method  based  on  the 
cDNA  probes  has  been  perfected:  in  situ 
cytochemistry.  This  technique  allows  one 
principle,  to  quantitate)  mRNA  levels  in 
on  brain  sections.  It  should  be  very  use 
central  nervous  system  development  and  fu 


stud  ies ;  or 
lyses.  Eve 
in  can  be  v 

complement 

last  year 
tect  a  numb 
e  ,  the  raf 
need  and  th 
Furtherm 
orrespond  in 
eduction  . 

use  of  sma 
hybridizati 

to  visuali 
ind  ividual 
ful  for  stu 
nc  tion  . 


the 
n  th 
ery 
ary 
we  h 
er  o 
gene 
e  st 
ore  , 
g  ge 
A 

11  s 
on 

ze  ( 
nerv 
dies 


purified 
e  partial 
useful  to 
to  a 
ave 
f  cDNA's 

product , 
rue  ture 

the 
nes  and 

ynthetic 

and ,  in 
e  cells 
of 


Pep  tide/protein  isolation  and  characterization 

Drs.  Marshak,  Fraser,  Sherwood,  and  Sower  have  purified  the 
gonadotropin  releasing  hormone  from  lamprey,  determined  its  amino 
acid  sequence,  and  synthesized  the  peptide.   Their  work  may 
contribute  to  a  better  understanding  of  lamprey  reproduction  and 
allow  a  method  for  eradicating  this  fish  predator  from  the  Great 
Lakes  to  be  developed. 

Drs.  Kligman  and  Marshak  have  isolated  a  neurite  extension 
factor  from  the  bovine  telencephalon  and  have  nearly  completed 
their  characterization  of  it.   They  hope  now  to  study  the  role  of 
this  factor  in  normal  development. 

Dr.  Rougon  has  purified  the  rat  and  bovine  neuronal  cell 
adhesion  molecules  (N-CAM);  she  and  Dr.  Marshak  have  sequenced 
the  N-termini  of  these  proteins,  synthesized  a  peptide 
corresponding  to  the  N-terminus  of  the  bovine  molecule,  and 
raised  an  antibody  against  this  peptide  which  recognizes  the 
native  N-CAM  molecule. 

Cloning  and  sequencing  of  cDNA's  and  genomic  DNA's 

Dr.  Bonner  has  continued  a  collaboration  with  Dr.  Rapp  of 
NCI  to  characterize  the  raf  oncogene.   The  gene  encodes  a  serine- 
threonine  specific  protein  kinase  which  is  expressed  in  a  variety 
of  tissues  including  brain.   They  have  deduced  the  complete  amino 
acid  sequence  of  the  human  protein  from  a  nearly  complete  cDNA 
and  have  expressed  the  protein  in  E.  coli.   They  have  proposed 
that  the  protein  is  a  protease-ac tivated  kinase  which  mediates 
growth  factor  signals  and  are  currently  testing  this 
hypothesis.   They  have  also  used  the  cDNA  to  identify  all  but  the 
first  exon  in  the  cloned  human  gene  and  have  identified  other 


122 


cDNA's  which  encode  a  related  protein  with  a  different  tissue 
distribution . 

Dr.  Bonner  has  cloned  the  complete  rat  substance  P  gene  and 
most  of  the  human  gene  and  has  mapped  it  to  human  chromosome  7. 
Drs .  Af folter ,  Bonner  and  Lazzarini  have  cloned  a  nearly  complete 
human  substance  P  cDNA.   Sequence  comparisons  of  the  rat,  human 
and  bovine  genes  indicate:   (a)  that  the  amino  acid  sequence  of 
the  entire  precursor  protein  is  highly  conserved,  suggesting  a 
function  for  the  portions  not  encoding  substances  P  and  K,  and 
(b)  that  there  is  a  very  extensive  region  (200  nucleotides)  of 
conserved  (80%)  sequence  in  the  promoter  region  which  suggests  an 
unusual  complexity  to  the  regulation  of  the  gene.   Dr.  Bonner  has 
recently  cloned  a  second  human  tachykinin  gene  which  encodes 
neuromedin  K  (neurokinin  6)  and  is  in  the  process  of 
characterizing  it. 


Chromogranin  A  is  a  member  of  a  famil 
glycoproteins.  This  72000  dalton  protein 
protein  of  the  adrenal  medulla  and  is  also 
parathyroid,  pituitary,  brain,  enteric  ner 
pancreas,  thyroid,  thymus,  retina  and  natu 
immune  system.  Wilson  et  al.,  have  shown 
chromogranin  is  contained  in  chromophobe  c 
pituitary,  making  chromogranin  the  first  i 
product  of  these  cells.  In  order  to  under 
of  this  ubiquitous  protein  might  be,  and  t 
marker  of  the  diffuse  neuroendocrine  syste 
have  cloned  and  are  characterizing  the  bov 
chromogranin  A.  To  begin  to  understand  th 
chromogranin  A  might  play  in  endocrine  eel 
transfected  Ms.  lacangelo's  chromogranin  A 
fibroblasts  and  isolated  several  cell  line 
the  chromogranin  processed  gene  and  stably 
messenger  RNA.  It  is  hoped  that  a  careful 
phenotype  of  these  cells  will  afford  some 
function  of  chromogranin  A  in  the  endocrin 
lacangelo  are  also  studying  the  regulation 
biosynthesis  in  cultured  chromaffin  cells, 
elevated  potassium  and  veratridine,  which 
enkephalin  mRNA  in  these  cells  (see  below) 
the  expression  of  the  chromogranin  message 
two  major  secreted  proteins  of  the  adrenal 
coordinate  regulation  in  these  cells. 


y  of  acidic,  secre 
is  the  main  secret 

present  in 
vous  system,  endoc 
ral  killer  cells  o 
in  fact  that 
ells  of  the  anteri 
dentified  secretor 
stand  what  the  fun 
o  characterize  it 
m  Eiden  and  lacang 
ine  cDNA  and  gene 
e  function  that 
Is,  Chang-Mei  Hsu 

cDNA  clone  into  r 
s  that  have  integr 

express  chromogra 

examination  of  th 
clues  as  to  the 
e  system.   Hsu  and 

of  chromogranin  A 
Interestingly , 
strongly  induce 
,  do  not  seem  to  a 
,  indicating  that 

medulla  are  not  u 


ted 
ory 

r  ine 
f  the 

or 

y 

ction 
as  a 
elo 
for 

has 

at 

ated 

nin 

e 


ffect 

the 

nder 


Dr.  Rokaeus  has  cloned  and  characterized  cDNA  for  porcine 
galanine.   Dr.  Roller  has  made  good  progress  toward  cloning  the 
cDNA  for  peptide  VQY. 

Drs.  Brownstein,  Siegel,  and  Okayama  are  in  the  process  of 
developing  a  novel  bacterial  expression  cloning  system  that 
should  allow  fast  and  simple  selection  of  positive  clones  with 


123 


antibod  ies  . 

Drs.  Siegel,  Brownstein,  and  Okayama  have  constructed 
several  very  large  cDNA  libraries  from  neural  and  endocrine 
tissues  and  have  begun  their  attempts  at  expression  cloning 
receptors  in  mammalian  cell  lines. 

Use  of  cDNA's  and  cRNA's  for  in  situ  hybridization  cytochemistry 

Drs.  Siegel  and  Young  have  succeeded  in  developing  a  fast 
and  reliable  technique  for  visualizing  specific  mRNA's  in  neurons 
on  brain  sections  and  cells  in  tissue  culture.   It  should  prove 
possible  to  convert  this  from  a  qualitative  to  a  quantitative 
method.   Drs.  Siegel  and  Young  have  used  the  method  to  visualize 
proopiomelanocortin-,  enkephalin-,  vasopressin-,  oxytocin-,  CRF-, 
and  CCK-mRNA's  to  date.   Drs.  Young,  Mezey,  and  Siegel  have  been 
able  to  combine  the  immunocy tochemical  and  in  situ  hybridization 
methods,  allowing  them  to  see  both  peptide  hormones  and  mRNA's  in 
the  same  cell.   Dr.  Zoeller  has  begun  to  examine  the  distribution 
of  cells  that  manufacture  GnRH  mRNA. 

Light  and  EM  immunocy tochemistry 

Dr.  Mezey  has  continued  to  study  the  coexistence  of  peptides 
in  the  hypothalamic  paraventricular  nucleus  (PVN).   She  has 
detected  VIP  positive  neurons  in  the  PVN  after  adrenalectomy  and 
during  lactation.   In  addition,  she  has  succeeded  in 
demonstrating  the  coexistence  of  CCK  and  vasopressin  in  cells 
that  contain  corticotropin  releasing  factor  (CRF).   The  former 
two  peptides,  given  together,  stimulate  ACTH  release  from  primary 
cultures  of  pituitary  as  well  as  CRF  does.   Using  electron 
microscopic  immunocy tochemistry  Dr.  Mezey  has  visualized  CCK  and 
vasopressin  in  the  same  secretory  granules  in  the  median 
eminence . 

Dr.  Mezey  has  detected  the  glycopeptide  portion  of  the 
vasopressin  precursor  in  the  vasopressin  deficient  Brattleboro 
rat.   Furthermore,  she  has  found  that  adrenalectomy  results  in  an 
increase  in  urine  osmolarity  in  these  rats,  suggesting  that  they 
may  have  a  limited  capacity  for  vasopressin  biosynthesis. 

Drs.  Mezey  and  Seeburg  have  mapped  the  distribution  of  the 
recently  discovered  GnRH  associated  peptide  (a  prolactin  release 
inhibiting  protein  itself)  in  the  brain  of  the  Rhesus  monkey,  and 
have  shown  that  it  coexists  with  GnRH. 

Drs.  Forman  (Dept.  of  Anatomy,  USUHS),  Mezey,  and  Pruss  have 
demonstrated  that  both  Neuropeptide  Y  and  enkephalin  are  stored 
in  the  same  secretory  granules:   Drs.  Pruss  and  Forman  have 
developed  methods  for  using  video  intensified  immunofluorescence 
microscopy  to  identify  colocalized  neuropeptides  in  purified 
chromaffin  granules,  and  Dr.  Mezey  used  double-label 


124 


immunoelectron  microscopy  as  a  second  method  for  demonstrating 
peptide  colocalization  within  cells  of  the  bovine  adrenal 
medulla.   The  identification  of  these  two  peptides  in  the  same 
secretory  granule  is  of  interest  since  they  are  not  synthesized 
on  a  common  precursor,  and  appear  to  be  independently  regulated 
in  chromaffin  cells. 

Dr.  Pruss  has  also  collaborated  with  Dr.  Rokaeus  in 
localizing  the  peptide  galanin  in  adrenal  medulla  and  in 
chromaffin  cell  culture  where  it  coexists  with  enkephalin. 

Studies  of  receptors 

Dr.  Antoni  has  examined  the  vasopressin  receptors  in  several 
tissues  in  detail.   He  has  shown  that  the  receptor  in  the 
anterior  pituitary  is  pharmacologically  unique. 

Dr.  Brann  has  attempted  to  purify  and  characterize  the 
dopamine  receptor  and  has  had  some  success  in  solubilizing  this 
receptor  and  in  developing  useful  affinity  columns.   He  and  Dr. 
Jelsema  have  explored  the  interactions  between  the  retinal  DA 
receptor  and  GTP-binding  membrane  proteins. 

Studies  of  peptide  and  protein  production,  secretion,  and 
function 


intr 

cell 

lipo 

has 

T20, 

the 

indu 

and 

Affo 

the 

the 


Drs.  Rougon,  Reisine,  and  Barbet  have  developed  a  method  for 
oducing  a  heat-stable  protein  kinase  inhibitor  into  cultured 
s.   They  have  used  antibody  targeting  of  protein  A-coupled 
somes  loaded  with  the  protein  kinase  inhibitor.   Dr.  Rougon 
found  that  the  ACTH-secreting  mouse  pituitary  cell  line,  At- 

expresses  the  embryonic  form  of  N-CAM.   Using  anti-N-CAM  and 
loaded  liposomes,  they  have  been  able  to  prevent  the  cAMP 
ced  secretion  of  ACTH  from  these  cells,  while  phorbol  ester 
potassium  stimulated  release  are  unaffected.   Along  with  Dr. 
Iter,  Drs.  Rougon,  Barbet,  and  Reisine  have  shown  that  when 
targeted  liposomes  deliver  the  protein  kinase  inhibitor  to 
cells,  cAMP  stimulated  ACTH  mRNA  synthesis  is  also  blocked. 


Bee 
subpopul 
have  scr 
phorbol 
cell  lin 
three  hu 
human  ce 
ob  tained 
Waschek , 
and  pept 
response 
strong  s 
treatmen 


ause  prima 
ations  of 
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ester,  or 
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11  lines  a 
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Eiden  and 
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Iso  make 
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Pruss  a 
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phorbol 
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VIP  mRNA 


affin  cell  cultures  contain  multiple 
in  cells,  Drs.  Pruss,  Waschek  and  Eiden 
neuroblastoma  cell  lines  for  cAMP, 
zation  inducible  VIP.   The  mouse  N18 
ma  glioma  hybrid,  NG108,  and  two  out  of 
cell  lines  have  inducible  VIP.   Both 

cytochrome  bcgi*   Dr.  Eiden  has 
the  human  VIP  prohormone.   Drs. 
re  studying  the  regulation  of  VIP  mRNA 
the  human  neuroblastoma  cell  lines  in 
ester,  and  depolarization.   Despite 
AMP  elevation  and  phorbol  ester 

and  peptide  levels,  cAMP  alone  has 


125 


little  or  no  effect  on  either  VIP  mRNA  or  VIP  peptide 
synthesis.   Since  these  cell  lines  also  make  cytochrome  h^-r.,    Dr 
Pruss  will  determine  whether  a  secretory  vesicle  fraction  exists 
in  the  human  cell  lines,  and  determine  whether  secretory  vesicle 
synthesis  is  regulated  by  factors  which  induce  VIP  synthesis  in 
these  cell  lines. 


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Dr.  Waschek  has  be 
cyclic  AMP  and  calcium 
vasoactive  intestinal  p 
cells  in  primary  cultur 
human  neuroblastoma  eel 
Dave  of  the  NIAAA,  it  h 
stimulates  the  release 
from  the  adrenal  medull 
cyclase  in  chromaffin  c 
AMP  causes  an  elevation 
levels.   Dr.  Affolter  d 
enkephalin  mRNA  caused 
preceded  by  an  increase 
preproenkephalin  mRNA, 
enkephalin  peptide  prod 
by  a  direct  activation 
Affolter,  and  Giraud  ha 
of  both  enkephalin  rele 
dependent  on  calcium  in 
dependence  of  secretory 
calcium  influx  "stimulu 
nomenclature  of  Douglas 
dependent  release  of  ca 
in  response  to  nicotini 
demonstrated  that  cell 
also  strongly  stimulate 
chromaffin  cells.   Elev 


en  examining  the  roles  of  intracellular 
in  regulation  of  the  expression  of 
olypeptide  and  enkephalin  in  chromaffin 
e  and,  in  collaboration  with  Dr.  Pruss,  in 
1  in  culture.   In  collaboration  with  Dr. 
as  been  shown  that  nicotine,  which 
of  enkephalin  peptides  and  catecholamines 
a,  also  directly  stimulates  adenylate 
ell  membranes,  and  that  elevated  cyclic 

of  enkephalin  mRNA  and  enkephalin  peptide 
emonstrated  that  the  increase  in 
by  both  nicotine  and  elevated  cAMP  was 

in  the  nuclear  RNA  precursor  for 
suggesting  that  transsynaptic  induction  of 
uction  in  the  adrenal  medulla  is  mediated 
of  the  enkephalin  gene.   Drs.  Eiden, 
ve  demonstrated  that  nicotinic  activation 
ase  and  elevation  of  enkephalin  mRNA  is 
flux,  and  have  termed  this  mutual 

hormone  release  and  biosynthesis  on 
s-secre tion-synthesis  coupling"  after  the 

and  Rubin  who  first  described  calcium- 
techolaraines  from  adrenomedullary  tissue 
c  stimulation.   Dr.  Siegel  has 
depolarization  with  elevated  potassium 
d  enkephalin  biosynthesis  in  cultured 
ated  potassium  also  causes  an  increase  in 


126 


enkephalin  mRNA  which  is  comple 
influx.  Dr.  Waschek  has  since 
which  facilitates  calcium  entry 
enkephalin  biosynthesis  in  chro 
causes  a  calcium-independent,  b 
enkephalins  and  a  calcium-depen 
of  enkephalin  mRNA  under  the  sa 
tentatively  concludes,  and  is  i 
while  secretion  and  biosynthesi 
coupled  by  a  dependence  on  calc 
activated  by  cation  influx  thro 
channe Is ,  one  activated  by  bari 
D600  and  the  other  activated  by 
to  D600. 


tely  dependent 
examined  the  e 
into  chromaff 
maffin  cells  a 
ut  D600-sensit 
dent,  D600-ins 
me  conditions 
n  the  process 
s  of  secreted 
ium  influx,  th 
ugh  two  differ 


upon  calcium 
ffects  of  barium, 
in  cells ,  on 
nd  shown  that  barium 
ive  release  of 
ensitive  elevation 
of  stimulation.   He 
of  proving,  that 
neuropeptides  are 
ese  processes  are 
ent  sets  of 


um  and  sensiti 
calcium  or  ba 


ve  to  blockade  by 
rium  and  insensitive 


Drs.  Hook  and  Pruss  have  studied  the  subcellular 
distribution  of  neuropeptide  processing  enzymes  as  well  as  the 
regulation  of  these  enzymes'  levels  and  specific  activities  in 
chromaffin  cells  which  have  been  stimulated  to  synthesize  or 
store  more  neuropeptides. 


Reserpine  treatment  of  chromaffin  cells 
carboxypeptidase  activity  and  (Met) enkephali 
elevated.  The  carboxypeptidase  exhibited  a 
its  substrate  (K  ).  Further,  the  number  of 
(measured  by  RIA;  remained  constant.  This  s 
active  enzyme  molecules  may  have  been  conver 
molecules  displaying  a  higher  affinity  for  s 
populations  of  carboxypeptidase  enzyme  at  hi 
activation  have  been  found  in  different  subc 
In  contrast  to  reserpine,  forskolin  had  no  e 
carboxypeptidase  activity  although  (Met)enke 
also  increased.  Other  studies  by  Dr.  Eiden 
reserpine  enhances  processing  of  existing  en 
forskolin  induces  synthesis  of  new  enkephali 
elevated  enkephalin  mRNA.  Processing  enzyme 
peptide  hormone  precursors  may  represent  two 
control  in  the  regulation  of  peptide  hormone 


increases 
n  levels  are  also 
higher  affinity  for 
enzyme  molecules 
uggests  that  less 
ted  to  more  active 
ubstrate .   Indeed  , 
gh  and  low  states  of 
ellular  fractions, 
ffect  on 

phalin  levels  were 
have  shown  that  while 
kephalin  precursors, 
n  precursor  through 
s  and/or  mRNA  for 

different  points  of 

production . 


Dr.  Pruss  has  raised  a  monoclonal 
bi-g^.   She  and  Ms.  Shepard  have  been  ma 
this  cytochrome  in  bovine  and  primate  n 
tissue.   The  distribution  is  being  comp 
chromaffin  granule  components:  neuropep 
hydroxylase  (DBH),  chromogranin  A,  and 
processing  enzyme  (using  antibodies  to 
prepared  in  collaboration  with  Dr.  Hook 
cytochrome  b^g-,  in  peptidergic  cells  of 
nervous  system  and  in  all  three  lobes  o 
provided  evidence  that  this  cytochrome 
processing  enzymes  as  well  as  the  catec 
enzyme ,  DBH . 


antibody  to  cytochrome 
pping  the  distribution  of 
eural  and  endocrine 
ared  with  that  of  other 
tides,  dopamine   (?- 
to  a  carboxypeptidase 
the  purified  enzyme 
)  .   The  identification  of 

the  retina,  enteric 
f  the  pituitary  have 
may  be  required  by  peptide 
holamine  biosynthetic 


127 


Physiological  studies  of  the  role  of  VIP  in  the  developing 
nervous  system  are  being  carried  out  by  Drs.  Eiden  and  Siegel  in 
collaboration  with  Dr.  Brenneman  of  the  NICHHD.   These 
investigators  have  shown,  using  mouse  spinal  cord  cells  in 
primary  culture,  that  the  ontogeny  of  VIP  expression  in  the 
spinal  cord  is  paralleled  by  the  tetrodotoxin-sensitive  release 
of  VIP  into  the  culture  medium  of  these  cells,  and  the  ability  of 
exogenous  VIP  to  reverse  the  developmentally-spec if ic  neurotoxic 
effects  of  tetrodotoxin  applied  to  these  cells.   Since  one  action 
of  tetrodotoxin  is  to  inhibit  the  release  of  endogenous  VIP  into 
the  culture  medium,  these  results  suggest  that  VIP  may  be  an 
endogenous  neuronal  growth/survival  factor  in  spinal  cord.   Dr. 
Brenneman  has  shown  that  glial  cells  cultured  from  developing 
spinal  cord  elaborate  and  release  into  the  medium  (but  only  in 
the  presence  of  VIP)  a  factor  which  does  indeed  enhance  the 
survival  of  spinal  cord  neurons. 

Drs.  Moskal  and  Pruss  have  examined  the  effect  of 
gangliosides  on  neuropeptide  expression  in  primary  chromaffin 
cell  cultures.   Since  gangliosides  may  be  involved  in  a  number  of 
cell  surface  recognition  phenomena,  Drs.  Pruss  and  Moskal  will 
determine  if  exogenously  added  gangliosides  can  mimic  the  cell 
density  effects  on  neuropeptide  expression.   In  collaboration 
with  Dr.  Eiden,  they  have  already  shown  that  bovine  brain 
gangliosides  can  specifically  elevate  enkephalin  peptide  levels 
as  well  as  enkephalin  mRNA.   They  will  determine  whether  the 
ganglioside  mediating  this  effect  is  specific  to  brain  and  try  to 
identify  the  specific  ganglioside  involved. 

Drs.  Moskal,  Schaffner,  and  Rougon  have  found  that  a 
specific,  blood  group-related  glycolipid  galactosyltransf erase 
activity  is  stimultated  by  dibutyr 1-cAMP  in  the  synapse-competent 
cell  line  NG108-15.   When  NG108-15  cells  are  cocultured  with 
fetal  rat  muscle,  under  conditions  that  induce  synapse  formation, 
this  enzyme  activity  is  also  stimulated.   Studies  with  a 
monoclonal  antibody  directed  against  the  neuronal  cell  surface 
adhesion  molecule,  N-CAM,  have  shown  that  dibutyrl-cAMP  inhibits 
the  expression  of  N-CAM  whereas  coculture--as  above--stimulates 
its  expression.   Muscle  conditioned  medium  and  muscle  exudate 
have  been  shown  to  stimulate  galac tosyl transferase  activity, 
whereas  this  enzyme  is  inhibited  as  cultures  of  NG108-15  cells 
approach  confluency.   These  studies  further  support  the  idea  that 
specific  enzymes  involved  in  glycosylation  play  a  key  role  in 
synapse  formation. 

Dr.  Moskal  has  identified  several  monoclonal  antibodies  that 
bind  to  cell  surface  proteins  found  highly  restricted  to  the 
hippocampal  formation  in  the  adult  rat.   One  antibody,  G6E3,  has 
been  used  successfully  in  concert  with  a  fluorescence  activated 
cell  sorter  to  isolate  and  maintain  in  culture  hippocampal 
neurons  (in  collaboration  with  A.  Schaffner).   This  antibody  also 
cross  reacts  with  murine  neurons  and  may  be  useful  in 


128 


neurological  mutant  studies. 


A  second  antib 
specific  for  hippoc 
protein  that  is  dev 
weight  during  early 
and  Sarvey ,  Dept.  o 
antibody  can  block 
potentiation  (LTP) 
able  to  show  that, 
antibody  is  an  effe 
potentiated  neurons 
body  layer  no  LTP  b 


ody  ,  B6E1 1  ,  has 
ampal  neurons, 
elopmentally  re 
postnatal  deve 
f  Pharmacology , 
the  formation  a 
in  hippocampal 
in  the  pyramida 
ctive  blocker  o 
When  the  ant 
lockade  was  obs 


also  been  found  to  be  highly 
It  recognizes  cell  surface 
gulated  and  changes  molecular 
lopment.   Drs.  Moskal,  Stanton 

USUHS,  have  found  that  this 
nd  maintenance  of  long  term 
slices.   They  have  also  been 
1  layer  of  the  hippocampus,  the 
nly  at  dendritic  sites  of 
ibody  is  ejected  into  the  cell 
erved . 


In  collaboration  with  Dr.  Conner  (Bowling  Green  State 
University),  Dr.  Moskal  has  found  that  affinity-purified  B6E11 
injected  intrahippocampally  into  neonatal  rats  was  able  to 
significantly  elevate  activated  (REM)  sleep. 

Dr.  Stone  has  utilized  low  uv  CD  spectroscopy  to  determine  a 
number  of  disaccharide  sequences  of  a  multifunctional 
OG tadecasaccharide  of  the  heparin  (H)-heparan  sulfate  (HS)  class 
of  proteoglycans.   This  enabled  a  proposed  structure  containing 
two  similar  functional  oligosaccharide  (OligoS)  units  and  the 
speculation  of  the  general  significance  of  such  units.   Drs. 
Stone  and  DeVoe  have  shown  by  their  method  of  experimental  and 
theoretical  extrinsic  CD  spectroscopy  that  these  OligoS  may  also 
have  special  binding  properties  for  cationic  aromatic  ligands. 

Drs.  Stone  and  Dohadwala  have  found  that  GAG  and  OligoS 
render  the  plasma  membranes  of  up  to  50  percent  of  dissociated 
adrenal  chromaffin  cells  permeable  to  erythrosin  B  (a  'small- 
molecule'  probe),  apparently  nonspec if ically  due  to  their 
polyanionic  character. 

Drs.  Stone  and  Martenson  have  determined  the  conformations 
of  various  sequential  peptides  of  myelin  basic  protein  (MBP)  in 
aqueous  trif luoroe thanol.   The  finding  of  a  sizeable  degree  of 
3-structure  that  is  both  stable  in  one  portion  and  undergoes 
B-structure^-a-helix  transitions  in  other  portions  of  the  protein 
is  significant  in  understanding  the  manner  of  dimerization  and 
aggregation  of  MBP  during  compaction  of  the  myelin  sheath  around 
CNS  neurons. 

NEUROPHARMACOLOGY  OF  CIRCADIAN  RHYTHMS 

Dr.  Zatz  and  his  coworkers  have  developed  a  system  for  the 
study  (and  assay)  of  [^H ] -melatonin  secretion  by  dispersed  chick 
pineal  cells  in  static  primary  culture  (for  several  weeks). 
These  cells  maintain  a  circadian  rhythm  of  melatonin  secretion 
for  several  days  and  a  driven  rhythm  for  several  weeks.   They  can 
be  driven  by  light-dark  cycles,  alternating  high  and  low 


129 


potassium  concentrations,  or  alternating  presence  and  absence  of 
norepinephrine.   Norepinephrine,  low  potassium,  and  light  have 
similar  effects . 

The  cultured  chick  pineal  cells  are  photosensitive  and  the 
action  spectrum  suggests  mediation  by  a  rhodopsin-like  molecule, 
which  contains  retinaldehyde  .   The  cells  take  up  [^Hj-retinol  and 
synthesize  [ -^H  ] -retinaldehyde  and  [^Hl-retinyl  palmitate. 
Preliminary  data  suggest  that  retinaldehyde  is  also  secreted  into 
the  medium.   Vitamin  A  metabolism  is  a  critical  component  of 
visual  function  and  is  primarily  regulated  by  light  and  circadian 
rhythms.   A  photo- sens itive  cultured  cell  system  (with  a 
biochemically  measurable  output)  has  unique  advantages  for  the 
investigation  of  the  biochemical  mechanisms  regulating  vitamin  A 
f unc  tions . 

BIOCHEMISTRY  OF  MEMBRANES 


D 
unexp  e 
inc  rea 
anter i 
lith  iu 
pretre 
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(IP's) 
phosph 
reduce 
Elevat 
the  pr 
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lithiu 
lithiu 
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growth 
lithiu 
therap 
ACTH  s 


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Zatz  and  Reis 
ly  stimulates 
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(DAG's).   DAG 

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t  therapeauti 
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A  number  of  membrane  proteins,  including  receptors,  have 
long  chain  fatty  acids  (usually  palmitate)  bound  in  ester 
linkage.   Drs .  Zatz  and  O'Brien  showed  previously  that  bovine 
retinas  incorporate  [ -'H] -pa  Imi  tate  into  rhodopsin,  the  receptor 
for  light.   They  are  extending  these  observations  in  purified 
preparations.   Evidence  suggests  that  the  enzyme  involved  is  an 
acyltransf erase  which  uses  palmityl  coenzyme  A  as  a  donor  and 
that,  like  its  substrate,  it  is  an  intrinsic  membrane 
glycoprotein.   After  solubilization  and  column  chromatography,  its 
specific  activity  is  increased  about  50-fold.   Acylation  of 


130 


membrane  proteins  provides  a  mechanism  for  posttranslational 
modification  of  receptors,  ion  channels,  etc.  which  could  alter 
their  function  and  regulate  their  interactions  with  drugs, 
hormones,  or  neurotransmitters. 

ON  THE  MECHANISM  OF  GLUCOCORTICOID  ACTION 


Glucocorticoids  exert  a  variety  of  actions  on  various 
tissues  and  organs.   These  hormones  act  in  concert  with  other 
hormones  in  a  permissive  way.   In  addition,  they  are  widely  used 
as  therapeutic  agents  for  inflammatory  and  immunological 
diseases.   This  is  based  upon  their  anti-inflammatory  and 
immunosuppressive  actions.   Dr.  Hirata  and  his  colleagues 
recently  isolated  lipocortin,  a  phospholipase  inhibitory  protein, 
from  media  conditioned  with  glucocorticoid-treated  U937  cells  (a 
human  monocyte  cell  line)  and  glucocorticoid-treated  human 
peripheral  leukocytes.   Highly  purified  preparations  of 
lipocortin  can  inhibit  phospholipase  A-  in  vitro  and  inhibit 
chemotaxis  of  leukocytes  in  situ,  suggesting  that  anti- 
inflammatory activity  of  glucocorticoids  is  mediated  through  this 
protein.   Since  these  preparations  also  alter  the  glycolysis  in 
rat  hepatocytes  mediated  through  a-and  B-adrenergic  receptors, 
lipocortin  has  also  been  suggested  to  be  involved  in  the  signal 
transduction  of  these  receptors.   These  observations  imply  that 
lipomodulin  might  mimic  some  of  the  permissive  effects  of 
glucocorticoids. 


Mari 

spec 

and 

frag 

pur  i 

trea 

puri 

Ap  2. 

"lip 

II) 

tryi 

N-ac 


In  CO 
e  of  F 
ies  of 
15 ,000 
ments 
fy  the 
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n  vitr 


ocorti 
is  a  m 
ng  to 
etylgl 


llaboration  with  Dr.  Flower  of  England  and  Dr.  Russo- 
rance ,  Dr.  Hirata  has  recently  identified  several 

lipocortin;  the  molecular  weights  are  40,000,  30,000 
,  respectively.   The  smaller  species  are  thought  to  be 
of  the  40,000  peptide.   Dr.  Hirata  has  attempted  to 
se  peptides  from  human  sources  such  as  glucocorticoid- 
37  cells  and  peripheral  blood  leukocytes.   Highly 
reparations  of  the  40,000  peptide  inhibit  phospholipase 
o  and  leukocyte  chemotaxis  in  situ.   Sequencing  of  the 
n""  revealed  that  human  high  density  lipoprotein  (HL  A- 
ajor  component  of  this  preparation.   Thus  Dr.  Hirata  is 
purify  lipocortin  further,  using  phospholipase  A2-  and 
ucosa mine-affinity  chromatography . 


Highly  purified  preparations  of  human  lipocortin  can 
induce  S-adrenergic  receptors  in  lung  type  II  cells  and  the 
adrenergic  phenotype  in  NH15  CA2  neuroblastoma-glioma  hybrid 
cells  as  glucocorticoids  do.   HDL-AII,  a  major  contaminant  in  the 
preparations,  has  no  effect,  but  several  monoclonal  antibodies 
raised  against  lipocorotin  can  block  the  effects  of 
glucocorticoids.  These  observations  suggest  that  the  induction  of 
the  synthesis  of  some  proteins  by  glucocorticoids  is  a  secondary 
effect  mediated  through  the  synthesis  of  lipocortin.^ 
Furthermore,  glycolysis  in  isolated  hepatocytes  is  time- 
dependently  converted  from  an  a-adrenergic  receptor  mediated 


131 


function  to  a  f^adrenergic  receptor  mediated  function.   This 
conversion  is  blocked  by  glucocorticoids  as  well  as  by  lipocortin 
and  accelerated  by  anti-lipocort in  antibodies.   These  findings 
suggest  that  lipocortin  is  somehow  involved  in  the  permissive 
effect  of  glucocorticoids  at  physiological  concentrations. 

REGULATION  OF  HORMONE  SECRETION  BY  MOUSE  PITUITARY  TUMOR  CELLS 

Drs.  Reisine,  Luini,  Axelrod,  and  their  colleagues  have 
investigated  the  cellular  mechanisms  involved  in  regulating 
adrenocorticotropin  (ACTH)  release  in  a  tumor  cell  line  of  the 
mouse  anterior  pituitary  ( AtT-20/D 16- 1 6 ) .   CRF,  the  natural 
stimulant  of  ACTH  release,  stimulates  secretion  through  a  cAMP- 
dependent  mechanism.   This  was  demonstrated  by  incorporating 
cAMP-dependent  protein  kinase  inhibitor  into  AtT-20  cells  using  a 
liposome  technique.   This  manipulation  abolished  CRF  stimulated 
ACTH  release.  In  contrast,  K"*"  and  phorbol  ester  evoked  hormone 
secretion  was  not  affected  by  this  treatment  indicating  that 
there  are  multiple  intracellular  pathways  involved  in  regulating 
the  release  of  ACTH.   CRF  and  8-bromo-cyclic  AMP  also  increase 
the  levels  of  proopiomelanocortin  (POMC)  m-RNA  in  AtT-20  cells. 
This  effect  is  blocked  by  the  protein  kinase  inhibitor  indicating 
that  CRF  stimulates  both  ACTH  release  and  synthesis  through  an 
activation  of  cAMP-dependent  protein  kinase. 

Somatostatin  (SRIF)  is  a  potent  inhibitor  of  ACTH  release 
from  AtT-20  cells.   This  peptide  can  block  adenylate  cyclase 
activity  so  as  to  prevent  CRF  and  forskolin  from  evoking  hormone 
secretion.   SRIF  also  lowers  intracellular  calcium  levels.   This 
effect  is  blocked  by  pertussis  toxin  suggesting  that  either  it  is 
mediated  by  a  guanine  nucleotide  inhibitory  protein  (N-)  or 
pertussis  toxin  desensitizes  the  SRIF  receptor.   Receptor  binding 
studies  show,  in  fact,  that  pertussis  toxin  does  desensitize  the 
SRIF  receptor.   SRIF  also  inhibits  8-bromo-cAMP  and  K"^  evoked 
ACTH  release  but  does  not  affect  the  ability  of  these 
secretagogues  to  increase  calcium  mobilization.   These  data 
indicate  that  SRIF  acts  through  multiple  mechanisms  to  regulate 
ACTH  secretion. 

Mechanisms  of  receptor  mediated  hormone  secretion  were 
studied  in  mouse  anterior  pituitary  cells  (AtT-20)  and  in  thyroid 
cells  (FRTL  5).   In  AtT-20  cell  adrenocorticotropin  (ACTH)  is 
secreted  in  response  to  CRF  and  noradrenaline.   The  primary 
effect  of  these  hormones  is  the  stimulation  of  cAMP  synthesis. 
Cyclic  AMP  induces  the  activation  of  voltage-dependent  calcium 
channels  and  increases  the  levels  of  cytosolic  free  calcium,  an 
event  which  is  crucial  for  ACTH  secretion.   Somatostatin,  a 
hypothalamic  peptide,  inhibits  the  stimulation  of  ACTH  secretion 
evoked  by  secretagogues.   Somatostatin  has  at  least  two  different 
mechanisms  of  action.   One  is  the  suppression  of  the 
secretagogue-s timula ted  cAMP  synthesis  and  the  other  is  the 
inhibition  of  voltage-dependent  calcium  channels.   A  third 


132 


mechanism  can  also  be  postulated.   It  is  involved  in  the 
suppression  of  ACTH  release  evoked  by  calcium  ionophores,  agents 
whose  activity  is  independent  of  cAMP  information  and  calcium 
channel  activation.   Multiple  mechanisms  of  action  might  allow 
somatostatin  to  inhibit  ACTH  release  induced  by  different  stimuli 
in  different  states  of  the  cell. 


In  FRTL5  cells  norepinephrine  stimulates  the  fluxes  of 
iodide  and  the  iodination  of  thyroglobulin  via  an  ct  receptor. 
The  mechanism  involves  an  increase  in  cytosolic  calcium  which,  in 
contrast  to  the  action  of  norepinephrine  in  AtT-20  cells,  is  cAMP 
and  calcium  channel  independent.   The  source  of  the  elevated 
calcium  is  at  least  in  part  intracellular.   Norepinephrine  has  a 
larger  effect  on  calcium  in  cells  treated  with  thyrotropin  (TSH), 
suggesting  that  TSH  might  increase  the  number  or  the  efficiency 
of  ct   receptors  . 


appe 

FRTL 

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cell 

lipo 

acid 

find 

invo 

in  F 

the 

curr 


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5  eel 
rates 
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xygen 

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ings 
Ived 
RTL5 
forma 
ently 


ddition  to 

o  be  involv 

Is  .   Blocke 

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of  ACTH  fr 

similar  bl 

ase  and  epo 

leukotrien 

suggest  tha 

in  receptor 

cells  stimu 

tion  of  fiv 

being  inve 


cAMP  and 
ed  in  hor 
rs  of  pho 
c  acid  fr 
om  AtT-20 
ockade  is 
xygenase 
es  and  ep 
t  a  metab 
mediated 
lates  the 
e  metabol 
stigated  . 


calcium,  ara 

raone  secreti 

spholipase  A 

om  membrane 

cells  and  i 

produced  by 

enzymes ,  wh  i 

oxides ,  resp 

olite  of  ara 

hormone  sec 

liberation 

ites,  the  fu 


chidonic 
on  in  bo 
P ,  the  e 
lipids  , 
odide  fl 
inhib  it 
ch  conve 
ec tively 
chidonic 
retion • 
of  arach 
notion  o 


aci 
th  A 
nzym 
inhi 
uxes 
ors 
rt  a 
.  T 
aci 
Nor 
idon 
f  wh 


d  also 
tT-20  and 
e  which 
bit  both 

from  FRTL5 
of  the 
rachidonic 
hese 

d  could  be 
epinephrine 
ic  acid  and 
ich  is 


133 


Annual  Report  of  the  Laboratory  of  Cerebral  Metabolism 
National  Institute  of  Mental  Health 

Louis  Sokoloff,  M.D. ,  Chief 

October  1,  1984  through  September  30,  1985 

The  Laboratory  of  Cerebral  Metabolism  has  undergone  a  couple  of  key 
changes  within  the  last  several  months.   Its  Section  on  Myelin  Chemistry  has 
been  abolished  and  the  Section  on  Clinical  Brain  Imaging,  which  is  responsible 
for  the  PET  research  of  the  Intramural  Research  Program  of  the  NIMH,  was 
transferred  into  the  Laboratory.   The  consequences  of  these  major 
organizational  changes  are  potentially  profound,  but  the  impact  of  the  changes 
has  not  yet  been  integrated  into  the  operations  of  the  Laboratory.   This 
integration  is  a  major  goal  of  the  Laboratory  in  the  next  fiscal  year.   For 
the  purposes  of  the  present  report,  each  of  the  three  Sections  have  prepared 
their  individual  reports  on  the  basis  of  how  they  have  operated  throughout 
most  of  the  past  fiscal  year. 

Section  on  Developmental  Neurochemistry 
Louis  Sokoloff,  M.D. ,  Chief 

The  major  efforts  of  this  Section  during  the  past  year  have  been  directed 
toward  responses  to  published  attacks  on  the  validity  of  the  deoxyglucose 
method  which  was  developed  in  this  Section,  has  been  repeatedly  validated  in 
this  Laboratory  and  in  many  other  laboratories  throughout  the  world  during  the 
past  eight  years,  and  has  formed  the  main  basis  for  the  use  of 
positron-emission  tomography  for  studying  the  human  brain  in  nervous  and 
mental  diseases.   There  have  been  previous  such  attacks  which  had  little 
influence  on  impact,  but  in  the  last  2-3  years  a  triumvirate  of  3  groups,  not 
independent  of  one  another,  who  have  been  promoting  an  alternative  method, 
have  mounted  a  concerted  effort  to  discredit  the  deoxyglucose  method  to  make 
room  for  theirs.   Their  efforts  were  also  without  important  effect  until  they 
elicited  the  aid  of  a  journalist  to  write  a  highly  biased  article  in  the  News 
and  Views  section  of  Science  that  questioned  the  validity  of  the  deoxyglucose 
method  on  the  basis  of  the  criticisms  of  these  antagonists.   The  consequences 
of  the  journalist's  article  were  far  more  profound  and  widespread  than  those 
of  the  published  scientific  articles,  and  the  Laboratory  felt  it  necessary  to 
respond  by  scientific  experimentation  and  publication  in  scientific  journals. 
In  a  series  of  beautifully  designed  experiments  a  group  of  investigators  led 
by  T.  Nelson  and  including  G.  Lucignani  and  G.  Dienel  confirmed  the  validity 
of  the  deoxyglucose  method  and  demonstrated  that  each  of  the  criticisms  was 
invalid,  often  because  of  incredibly  bad  science  and,  in  at  least  one  case, 
probably  even  worse.   Their  results  have  already  been  published  in  part  in  our 
report  in  Science,  and  the  remainder  has  been  submitted  for  publication  in 
another  journal.   It  is  hoped  that  these  studies  will  put  an  end  to  this 
artificial  controversy  once  and  for  all. 

Previously  initiated  studies  on  neurophysiological  and  neuropsychological 
applications  of  the  deoxyglucose  method  have  been  continued  and  in  some  cases 


135 


completed.  M.  Kadekaro  and  L.  Sokoloff  have  demonstrated  in  the  dorsal  root 
ganglion  and  spinal  cord  that  electrically  stimulated  afferent  input  to  this 
sensory  pathway  activates  glucose  utilization  in  the  axonal  terminals  of  the 
pathway  and  not  the  cell  bodies.  These  results  suggest  that  metabolic  mapping 
of  neural  pathways  reflects  mainly  metabolic  changes  in  the  neuropil  and  not 
in  cell  bodies,  indicating  that  synaptic  activity  underlies  the  evoked 
metabolic  response.   These  studies  are  currently  in  press. 

L.  Porrino  and  R.  Esposito  have  extended  their  studies  on  the 
identification  of  the  neural  substrates  of  the  behavioral  phenomenon  of 
rewarding  self-stimulation.   The  self-stimulation  phenomenon  is  widely 
believed  to  be  a  model  for  motivational  behavior.   They  have  studied  rats 
carrying  out  self-stimulation  to  the  ventral  tegmental  area  and  to  the  zona 
compacta  of  the  substantia  nigra  and  found  patterns  of  distribution  of  changes 
in  local  cerebral  glucose  utilization  distinctly  different  from  those  seen 
during  comparable  experimenter-administered  stimulation  to  the  same  areas. 
Self-stimulation  to  the  two  areas  produced  changes  in  different  regions 
corresponding  to  the  terminal  zones  in  the  projection  areas  from  the  two 
stimulated  areas,  but  superimposed  on  these  changes  was  a  common  pattern  of 
bilateral  changes,  particularly  in  the  limbic  system,  which  may  represent  the 
neural  substrates  of  the  self-stimulation  behavior.   Portions  of  this  work 
have  been  published,  and  more  is  in  the  process  of  preparation  for 
publication. 

L.  Porrino  has  been  continuing  her  studies  in  collaboration  with  S.  Burns 
and  I.  Kopin  of  the  NINCDS  on  the, local  changes  in  cerebral  glucose 
utilization,  measured  with  the  [   C]deoxyglucose  method,  that  occur  in  the 
MPTP-induced  model  of  Parkinsonism  in  the  monkey.   She  has  found  selective 
decreases  in  glucose  utilization  in  the  pars  compacta  of  the  substantia  nigra, 
subthalamic  nucleus,  and  external  segment  of  the  globus  pallidus.   All  effects 
were  confined  to  the  nigrostriatal  and  basal  ganglia  dopaminergic  pathways;  no 
effects  were  seen  in  other  dopaminergic  systems,  such  as  the  mesolimbic  and 
mesocortical  systems.   Chronic  L-DOPA  treatment  reversed  the  signs  and 
symptoms  of  Parkinsonism  and  increased  glucose  utilization  throughout  the 
motor  and  premotor  cortex,  motor  nuclei  of  the  thalamus,  and  especially  in  the 
subthalamic  nucleus  and  globus  pallidus.   Surprisingly,  equivalent  DOPA 
administration  to  normal  animals  showed  no  effects  on  local  cerebral  glucose 
utilization.   These  indicate  a  role  of  the  globus  pallidus--subthalamic 
nucleus  circuit  in  the  organization  and  performance  of  normal  movement  and 
suggest  that  the  therapeutic  effects  of  L-DOPA  in  Parkinsonism  are  mediated  by 
pharmacological  mechanisms  quite  different  from  those  previously  proposed. 
Manuscripts  on  this  work  are  currently  being  prepared  for  publication. 

C.  Smith  and  N.  Eng  are  continuing  the  work  on  the  refinement  of  the 
newly  developed  technique  for  the  measurement  of  local  cerebral  protein 
synthesis.   At  the  present  time  the  method  measures  at  worst  the  minimal 
possible  rates  of  protein  synthesis  and  possibly  the  exact  rates.   All  that  is 
needed  is  to  define  the  influence,  if  any,  of  recycled  amino  acid  derived  from 
the  slow  breakdown  of  endogenous  protein.   The  experiments  to  determine  this 
have  been  fully  designed  and  await  implementation  as  soon  as  a  technical 
analytic  procedure  for  the  measurement  of  picomole  quantities  of  amino  acids 


136 


is  perfected.   In  the  meantime  the  method,  even  though  still  not  fully 
refined,  has  proved  to  be  useful  to  identify  local  regions  in  the  brain  with 
impaired  protein  synthesis  in  aging,  to  determine  the  time  courses  of  the 
increases  in  protein  synthesis  that  accompany  regeneration  in  the  central 
nervous  system,  and  to  identify  plastic  changes  in  the  binocular  visual  system 
of  the  newborn  monkey.   Portions  of  this  work  were  published  during  the  last 
year,  but  work  in  the  CNS  plasticity  in  the  newborn  primate  is  being  continued 
at  a  rate  limited  by  the  availability  of  newborn  monkeys. 

E.  Kaufman  and  T.  Nelson  are  in  the  process  of  completing  their  studies 
on  the  metabolic  origin  and  degradation  of  'y-hydroxybutyrate  in  the  CNS  and 
extraneural  tissues  and  on  the  physiological  role  of  endogenous 
Y-hydroxybutyrate  in  the  brain.   Their  work  thus  far  has  been  published  in 
part,  and  manuscripts  are  being  prepared  to  report  their  more  recent  findings. 

Section  on  Myelin  Chemistry 
Marian  W.  Kies,  Ph.D.,  Chief 

The  current  report  includes  four  projects,  slated  for  termination,  which 
were  previously  included  under  the  Section  on  Myelin  Chemistry.   Members  of 
the  Section  have  made  many  important  contributions  in  the  field  of 
neurochemistry  as  well  as  in  the  study  of  autoimmune  damage  to  the  central 
nervous  system  (CNS).   Dr.  Kies  was  the  first  to  isolate  and  identify  myelin 
basic  protein  (BP)  as  the  major  (perhaps  the  only)  encephalitogenic 
constituent  of  the  CNS.   Dr.  Martenson  established  the  existence  of  two 
similar  BPs  in  the  myelin  of  certain  species  of  rodents,  and  showed  that  the 
smaller  of  the  two  proteins  was  identical  to  the  larger  except  for  a  deletion 
of  40  residues  in  the  C-terminal  region  of  the  polypeptide.   He  also 
demonstrated  the  existence  of  charge  heterogeneity  in  the  BP  molecule  and 
provided  data  to  show  that  this  heterogeneity  was  partly  caused  by 
phosphorylation  in  vivo.   Dr.  Kies,  in  collaboration  with  Dr.  Alvord,  found 
that  experimental  allergic  encephalomyelitis  (EAE)  could  be  suppressed  or 
prevented  by  injections  of  BP  without  mycobacteria.   (BP  injected  in  an 
emulsion  containing  mycobacteria  is  the  antigen  which  induces  EAE.)  Dr.  Driscoll 
discovered  the  phenomenon  of  enhanced  transfer  (100  to  1000-fold  increase  in 
the  ability  of  BP-sensitized  cells  to  transfer  EAE  after  culture  with  specific 
antigen  (BP) .   This  observation  has  revolutionized  studies  on  pathogenesis  of 
EAE  because  of  the  in  vitro  studies  which  are  now  possible.   In  the  present 
report  are  two  other  very  important  discoveries  of  Dr.  Driscoll' s — induction 
of  chronic  demyelinating  EAE  in  adult  guinea  pigs.   This  technique  is 
important  because  it  provides  a  much  better  experimental  model  for  study  of 
demyelination  comparable  to  the  lesions  found  in  MS.   The  second  important 
discovery  is  his  demonstration  that  enhanced  transfer  requires  more  than  the 
simple  induction  of  a  proliferative  response  in  the  BP-sensitized  cells--other 
active  factors,  perhaps  even  other  subsets  of  cells,  are  required  for  the  full 
expression  of  the  lesion. 

These  and  other  important  contributions  of  the  Section  have  been  possible 
because  of  the  careful  critical  work  of  these  and  other  investigators,  as  well 
as  the  dedicated  skillful  support  staff  which  Dr.  Kies  has  recruited  and 


137 


trained.   It  is  unfortunate  that  the  demands  and  resources  of  the  IRP  have 
necessitated  the  breakup  of  this  productive  group  of  scientists. 

During  the  past  year  Dr.  Martenson's  main  interests  have  been  directed 
toward  an  elucidation  of  the  three-dimensional  structure  of  BP  under  a  variety 
of  solution  conditions  in  an  attempt  to  understand  the  function  of  the  protein 
in  the  myelin  sheath.   He  and  Mrs.  Pedersen  have  investigated  the  primary 
structure  of  various  species  of  BP  utilizing  specific  proteinases  to  prepare 
known  fragments  of  BP  for  use  in  biological  and  physicochemical  studies. 

Dr.  Monferran,  a  guest  worker,  has  been  studying  the  effects  of 
crosslinking  reagents  on  BP  to  determine  the  manner  in  which  the  polypeptide 
chain  folds  in  three  dimensions.   These  data  will  also  provide  information  on 
the  mechanism  of  BP  dimerization.   It  has  been  proposed  that  compaction  of 
myelin  occurs  as  a  result  of  the  dimerization  of  BP  across  the  cytoplasmic 
surfaces  of  the  myelin  lamellae. 

With  Dr.  Audrey  Stone  Dr.  Martenson  has  studied  the  "conformational 
adaptability"  of  various  fragments  of  BP  in  water-trif luroethanol  (TFE) 
solutions,  to  determine  the  types  and  locations  of  secondary  structures  which 
BP  could  assume  under  conditions  which  favor  intramolecular  hydrogen  bonding. 

In  a  collaborative  study  with  Dr.  Alvord,  a  monoclonal  antibody  to  GPBP 
has  been  used  to  interpret  the  specific  folding  of  the  polypeptide  chain  of 
BP.   The  specificity  of  binding  of  this  antibody  to  fragments  could  not  be 
interpreted  in  terms  of  its  reactivity  with  a  single  sequence  in  the  molecule; 
by  utilizing  several  known  fragments  they  were  able  to  propose  a  stable 
conformation  of  BP  which  accounted  for  the  specificity  of  the  monoclonal 
antibody. 

Studies  with  Dr.  George  Mendz  on  the  antigenic  site  in  fragment  1-14  have 
shown  that  interactions  occur  between  Ala-1  and  Gly-11  and  between  His-10  and 
Tyr-14,  indicating  a  good  deal  of  folding  within  this  relatively  small 
peptide. 

With  Mrs.  Deibler  Dr.  Kies  has  investigated  the  multiple  peptide  forms  of 
human  myelin  basic  protein,  which  until  recently  were  considered  by  these 
investigators  to  be  proteolytic  fragments  of  the  18.5  K  protein.   It  is  now 
apparent  that  several  17  K  forms  are  present  in  the  BP  preparation  and  they 
probably  arise  from  m-RNA  processing  rather  than  from  autolysis  of  the  18.5  K 
form  in  situ.   Proof  of  the  latter  has  been  possible  with  some  of  the 
methodology  developed  by  Mrs.  Deibler  in  other  studies  on  BP--tryptic  mapping 
on  HPLC,  use  of  the  amino  acid  analyzer  not  only  for  amino  acid  analysis  but 
for  C-terminal  analysis,  FPLC  fractionation  of  proteins  of  similar  charge  and 
size,  and  electrophoresis  on  slab  gells  of  various  fractions  isolated  in  the 
above  fractionation  procedures. 

In  collaboration  with  Dr.  Stone,  Dr.  Kies  and  Mrs.  Deibler  have 
investigated  the  influence  of  phosphorylation  on  the  conformation  of  BP.   CD 
studies  show  that  the  percent  of  beta-structure  increases  with  increased 
phosphate  content.   This  suggests  that  phosphorylation  of  BP  may  plan  a  role 
in  the  orientation  of  BP  in  myelin. 

138 


Dr.  Driscoll  has  been  investigating  the  step-by-step  mechanisms  by  which 
antigen-specific  cells  infiltrate  the  CNS  and  cause  inflammatory  lesions  as 
well  as  demyelination.   These  studies  are  technically  difficult  and  very 
time-consuming.   He  has  demonstrated  that  transfer  of  EAE  and,  by  analogy, 
induction  of  EAE  requires  not  only  BP-sensitized  cells  but  the  production  and 
interaction  of  several  soluble  factors,  including  Il-l,  Il"2,  and  gamma-lFN. 
These  results  have  important  bearing  not  only  on  the  pathogenesis  of  EAE  but 
also  on  our  understanding  of  the  pathogenesis  of  multiple  sclerosis. 

In  addition  to  the  cellular  mechanisms  described  above,  Dr.  Driscoll 
recently  developed  an  ingenious  method  for  inducing  demyelination  in  adult 
guinea  pigs  by  a  combination  of  cell  transfer  and  sensitization  of  recipients 
with  non-encephalitogenic  whole  CNS.   With  this  technique  Dr.  Driscoll  and  Dr. 
Kira  demonstrated  that  myelin  from  chicken  brain  (which  is  itself 
non-encephalitogenic)  induces  demyelination  when  used  in  combination  with 
cell-transfer  and  simultaneous  sensitization  with  guinea  pig  BP.   Sera  from 
these  guinea  pigs  are  capable  of  inhibiting  myelination  in  vitro.   This  is  the 
first  time  the  in  vitro  and  in  vivo  demyelinating  factors  have  been  shown  to 
be  directly  related  (collaborative  experiments  with  Dr.  Sell  and  Bourdette). 

Section  on  Clinical  Brain  Imaging 
Robert  M.  Cohen,  M.D. ,  Ph.D.,  Chief 

The  major  effort  of  this  section  continues  to  be  the  refining  of  existing 
methodologies  for  the  study  of  cortical  functioning  based  on  positron  emission 
tomography  (PET)  and  to  a  lesser  degree  electrical  brain  mapping  procedures 
particularly  as  they  complement  PET  information.   We  continue  to  contribute  to 
the  development  and  comparison  of  three  methods  of  data  abstraction,  the 
"peel",  the  "ring"  and  the  region  of  interest  (ROI).   We  have  been  examining 
the  benefits  of  standardizing  the  abstracted  PET  data  to  emphasize  that 
information  that  directly  pertains  to  the  activity  of  localized  regions  of  the 
brain  as  opposed  to  the  overall  glucose  metabolic  rate  (GMR)  of  the  brain.   To 
accomplish  this  we  have  explored  multivariate  analytical  approaches  other  than 
that  which  has  been  previously  used  (repeated  measures  analysis  of  variance) 
which  we  feel  is  not  suitable  to  the  important  biomedical  questions  that  PET 
should  be  addressing. 

We  have  pursued  as  thorough  an  analysis  as  possible  of  the  FDG  PET 
representations  of  two  behavioral  paradigms,  repeated  somatosensory  stimuli 
and  an  auditory  continuous  performance  test.   They  complement  each  other  in 
that  in  the  former  the  "normal"  response  is  to  habituate  to  a  "benign"  sensory 
stimulus,  whereas  the  latter  task  requires  selective  attention.   Differences 
are  observable  and  suggest  that  PET  is  a  modality  where  specific  functionally 
distinct  regions  of  the  frontal  cortex  can  be  examined.   These  paradigms  are 
currently  in  use  to  study  normal  habituation  and  selective  attention  and  to 
evaluate  whether  these  fundamental  processes  are  altered  in  mental  illness. 
To  date,  schizophrenic  patients  do  not  show  the  diminution  in  electrical 
response  to  repetitive  somatosensory  stimuli  observed  in  normals.   These 
differences  appear  to  be  reflected  in  a  different  PET  pattern  of  GMR. 


139 


Annual  Report  of  the 

Laboratory  of  General  and  Comparative  Biochemistry 

National  Institute  of  Mental  Health 

October  1,  1984  to  September  30,  1985 

Giulio  L.  Cantoni,  M.D.,  Chief 


After  the  stormy  and  administratively  difficult  fiscal  year  1984,  fiscal 
year  1985  has  been  relatively  uneventful  from  an  administrative  point  of  view. 
Researchwise  the  year  has  been  very  productive  in  spite  of  considerable  loss 
of  time  in  the  early  part  of  the  year  due  to  relocation  in  the  limited  space 
assigned  to  the  Laboratory  of  General  and  Comparative  Biochemistry. 

The  research  program  of  Dr.  Cantoni' s  group  revolved  around  three  projects 
that  are  closely  interrelated  and  all  deal  with  the  regulation  of  the  biological 
utilization  of  S-adenosylmethionine  (AdoMet)  and  the  significance  of  methylation 
reactions  for  various  physiological  processes.   Our  approach  is  two  pronged: 

1)  in  vitro  we  pursue  our  long  lasting  interest  in  the  basic  enzymology  of 
AdoMet  and  Adenosylhomocysteine  (AdoHcy). 

2)  in  vivo  we  analyze  the  biological  role  of  AdoMet  in  2  different  systems: 
macrophage  chemotaxis  and  muscle  differentiation. 

The  different  members  of  the  research  group  (R.  Aksamit,  P.  Backlund,  C.  Unson, 
A.  Merta  and  G.L.  Cantoni)  work  as  a  team  and  participate  in  one  way  or  another 
in  all  the  different  projects  that  are  closely  interrelated  conceptually  as  well 
as  technologically.   The  different  expertise  of  various  members  of  the  team  and 
their  different  contribution  to  any  given  project  is  recognized  in  various  ways: 
authorship,  presentation  at  meetings,  etc. 

A  more  detailed  discussion  of  the  various  projects  and  a  projection  of  the  future 
direction  follows,  the  individual  projects  may  also  be  consulted  for  further 
details. 

1)  For  several  years  we  have  been  studying  the  hypothesis  that  the  ratio  of 
AdoMet/AdoHcy  is  the  principal  determinant  for  the  utilization  of  AdoMet 
by  various  biological  systems.   The  key  enzyme  that  regulates  AdoMet/ AdoHcy 
ratio  is  AdoHcyase,  an  enzyme  first  discovered  and  crystallized  in  this 
laboratory.   We  have  now  obtained  a  potent  antibody  against  the  rat  liver 
enzyme  and  are  using  this  tool: 

a)  to  study  the  distribution,  level  and  turnover  of  the  enzyme  in  different 
tissues  and  under  different  physiological  conditions; 

b)  to  search  for  the  mRNA  coding  for  AdoHcyase.   This  is  one  of  the  two 
possible  approaches  to  isolation  and  characterization  of  the  gene  coding 
for  the  enzyme; 

(R.  Aksamit  has  taken  the  primary  responsibility  for  the  immunological 
characterization  of  the  antibody  and  its  interaction  with  the  antigen, 
while  P.  Backlund  will  be  involved  in  the  search  for  mRNA  and  its 
translation) . 


141 


c)  peptide  fragments  derived  from  the  rat  liver  enzyme  are  being  sequenced 
in  order  to  enable  us  to  contract  out  for  the  corresponding  deoxyoligo- 
nucleotides,  that  can  serve  as  probes  for  the  cDNA  coding  for  the  enzyme. 
This  is  an  alternative  approach  to  the  identification  of  the  cDNA  gene. 

(C.  Unson  is  primarily  responsible  for  this  phase  of  the  work,  but  we 
anticipate  that  R.  Aksamit  will  utilize  some  of  these  peptides  to 
generate  antibodies  that  will  contribute  to  our  understanding  of  the 
conformation  of  the  enzyme  in  solution.   P.  Backlund  is  also  involved 
in  the  characterization  of  the  various  peptides  derived  from  AdoHcyase, 
and  eventually  we  all  will  be  involved  in  the  characterization  of  the 
AdoHcyase  gene) 

d)  AdoHcyase  is  composed  of  4  subunits;  each  subunit  binds  tightly  one 
mole  of  NAD  an  essential  cofactor  for  the  enzymatic  catalysis.   With  G. 
de  la  Haba  we  have  found  conditions  that  in  vitro  lead  to  a  reversible 
removal  of  the  NAD  and  to  inactivation  of  the  enzyme.   This  is  an 
important  finding  especially  if  it  should  be  possible  to  establish  that 
this  reversible  inactivation  operates  in  vivo. 

2)   The  role  of  AdoMet  in  various  biological  processes  in  eukaryotes  can  be 

studied  either  by  use  of  specific  inhibitors  or  by  selection  of  behavioral 
mutants.   We  are  using  successfully  both  approaches. 

a)  We  were  the  first  to  introduce  specific  inhibitors  of  AdoHcyase,  (the 
patent  of  one  of  these,  DZA-arysteromycin,  has  recently  been  granted  as 
an  exclusive  royalty-bearing  license  to  SRI).   We  will  continue  to 
examine  other  compounds,  especially  those  like  deazaadenosine  (DZA) 
that  can  give  rise  intracellularly  to  congeners  of  AdoHcy  endowed  with 
different  inhibitory  specificity.   It  may  be  recalled  that  DZA  specifi- 
cally inhibits  macrophage  chemotaxis  and  stimulates  the  differentiation 
of  myoblasts  into  rayofibers. 

b)  Macrophage  chemotaxis  may  be  viewed  as  model  for  behavioral  responses 
to  sensory  stimuli,  or,  more  generally,  as  a  model  for  cell  to  cell 
communication. 

In  our  earlier  work  on  the  inhibition  of  chemotaxis  by  DZA  and  other 
methylation  inhibitors  we  have  used  a  macrophage  cell  line  RAW  264  that 
respond  to  a  chemically  undefined  raacromolecule.   Mammalian  neutrophils 
respond  to  a  synthetic  tripeptide  fMLP  but  do  not  grow  in  tissue  culture. 
In  order  to  bridge  these  difficulties  R.  Aksamit,  by  fusion  of  RAW  264 
with  human  leukocytes,  has  been  able  to  construct  a  new  hybrid  cell  line 
that  responds  to  fMLP  and  can  be  maintained  in  tissue  culture. 

Macrophage  chemotaxis  is  a  process  that  involves  an  as  yet  unknown  but 
probably  large  number  of  gene  products.  The  steps  involved  in  chemo- 
taxis can  be  divided  into  three  groups:  recognition  and  binding  of  the 
attractant,  transduction  of  the  signal  to  the  motor  and  motor  response 
(In  E^.  coll  at  least  seven  genes  or  gene  families  have  been  identified 
as  required  for  chemotaxis). 

With  the  new  hybrid  cell-line  that  B.  Aksamit  has  isolated  we  have  been 
able  to  determine  that  DZA  does  not  inhibit  binding  of  the  attractant  to 


142 


its  receptor,  and  therefor  that  the  step  inhibited  by  DZA  is  related  to 
signal  transduction  or  motor  response. 

c)  Using  the  hybrid  cell-line,  P.  Backlund  has  been  investigating  the  role 
of  a  family  of  multi-subunit  nucleotide  binding  proteins  in  the  transduc- 
tion of  the  chemotactic  signal.   Two  bacterial  toxins.  Pertussis  and 
Cholera,  after  entry  into  the  cells  catalyze  the  ADP  ribosylation  of  Ni 
and  Ns  subunits  of  the  nucleotide  binding  proteins,  and  thereby  interfere 
with  the  transduction  of  a  variety  of  hormonal  or  neuroef fector  stimuli. 
P.  Backlund  and  B.  Aksamit  have  found  that  both  of  these  toxins  inhibit 
chemotaxis  and  we  have  obtained  evidence  that  the  inhibition  is  related 
to  the  ADP-ribosylation  of  Ni  and  Ns  subunits,  respectively.   These 
observations  are  particularly  exciting  because  they  suggest  a  new  role 

in  signal  transduction  for  one  of  the  subunits  of  N  proteins,  the  3  or 
3y  subunit,  that  is  common  to  both  Ni  and  Ns  complexes. 

R.  Aksamit  has  recently  isolated  fourteen  non-chemotactic  mutants  of 
RAW264  and  in  the  next  several  months  we  will  attempt  to  determine  in 
the  next  few  months  if  any  is  defective  in  the  N  proteins. 

We  should  also  be  able  to  determine  how  the  inhibitory  effect  of  DZA  is 
related  to  the  N  proteins  and  thus  begin  to  identify  the  various  steps 
involved  in  the  chemotactic  response  in  eukaryotes. 

d)  The  capacity  of  a  gene  to  be  transcribed  appears  to  be  inversely  related 
to  the  degree  of  DNA  methylation. 

It  has  been  found  that  modification  of  the  pattern  of  DNA  methylation 
alters  phenotypic  expression.   The  differentiation  of  myoblast  to 
myofibers  in  tissue  culture  provides  a  model  for  the  study  of  cellular 
differentiation.   In  collaboration  with  S.  Scarpa  and  others  at  the  U. 
of  Rome,  we  have  examined  the  effect  of  DZA  on  myoblast  differentiation. 
We  have  found  that  treatment  of  myoblasts  with  DZA  greatly  stimulates 
the  degree  and  course  of  differentiation.   We  have  isolated  lines  of 
non-fusing  myoblast  that  upon  treatment  with  DZA  regain  the  ability  to 
undergo  differentiation  into  myofibers. 

These  observatioas  can  be  best  interpreted  by  proposing  that  DZA,  or 
actually  DZAHcy,  inhibits  the  methylation  of  one  or  more  genes  involved 
in  myoblast  differentiation.   We  are  at  the  present  time  in  the  process 
of  determining  if  we  can  document  this  interpretation. 

This  work  is  being  carried  out  in  Rome  and  in  Bethesda  by  S.  Scarpa,  P. 
Backlund,  R.  Aksamit  and  other  more  junior  investigators,  and  we  are 
fortunate  to  have  enlisted  the  collaboration  of  Dr.  A.  Razin. 

A  new  project  has  been  started  in  collaboration  with  Dr.  Aharon  Razin  a 
Fogarty  Scholar  in  Residence  between  September  1984  and  May  1985.   It  is  well 
established  that  the  pattern  of  DNA  methylation  in  vertebrates  may  undergo 
changes  and  this  gene  expression  is  often  associated  with  decrease  in  the 
abundance  of  the  meCpG  sequences  in  DNA.   No  biochemical  mechanism  has  been 
described  to  account  for  the  conversion  of  meCpG  to  CpG  sequences.   With  Razin 
we  have  been  searching  for  conditions  where  this  conversion  is  easily  demon- 
strated using  Friend  erytholeukemlc  cells  as  an  experimental  model.   These 

143 


cells  undergo  terminal  differentiation  in  tissue  culture  and  changes  in  the  DNA 
methylation  pattern  have  been  observed  concomitant  with  differentiation.   We 
have  obtained  preliminary  evidence  suggesting  that  DNA  demethylation  is  due  to 
an  active  biochemical  process  not  previously  described.   This  research  will  be 
continued  in  collaboration  with  Dr.  Razin  and  his  group  at  the  Hadassah  Medical 
School  in  Jerusalem;  Dr.  Razin  will  return  to  Bethesda  in  February  1986  to  resume 
his  residence  as  a  Fogarty  Scholar  and  take  again  an  active  part  in  the  project 
during  his  visit  to  the  Lab. 

In  the  Section  on  Alkaloid  Biosynthesis,  considerable  effort  was  devoted 
to  reduction  of  the  backlog  of  unpublished  observations.   As  a  result,  several 
manuscripts  have  appeared  and  several  are  in  press.   The  questionnaire  study 
of  cystathionine  g-synthase  deficiency  has  now  been  successfully  completed. 
Continuing  efforts  to  provide  deeper  understanding  of  factors  affecting  bio- 
synthesis of  the  essential  aminoacids  methionine,  threonine,  lysine,  and 
isoleucine  (upon  which  satisfactory  development  of  the  mammalian  brain  depends) 
have  taken  several  directions.   Experiments  with  radioactive  methionine  show 
that  about  four  times  as  many  methyls  are  utilized  for  methylation  reactions 
as  accumulate  in  total  plant  methionine,  (i)  Previous  studies  had  shown  that  in 
plants  methionine  participates  in  two  metabolic  cycles:   in  one,  it  serves  as 
the  ultimate  donor  of  the  3-carbon  moiety  of  spermidine;  in  the  other,  as  the 
ultimate  donor  of  a  methyl  group  to  various  compounds.   Phosphatidylcholine, 
pectin  methyl  ester,  chlorophyll  methyl  ester  and  carbon  moieties  of  neutral 
lipids  (possibly  cyclopropane  fatty  acids)  are  quantitatively  the  most  important 
end  products  of  transmethylation  and,  together,  accouat  for  virtually  all  of 
the  previously  determined  methyl  requirement.   Recognition  of  the  predominant 
role  of  phosphatidylcholine,  together  with  our  previous  demonstration  of  a 
very  specific  channel  for  uptake  of  choline,  suggested  external  choline  might 
regulate  methylneogenesis.   This  expectation  has  now  been  confirmed:  external 
choline  decreases  methyl  flux  into  phosphatidylcholine  by  90-95%,  and  reduces 
total  methylneogenesis  by  about  50%.   The  ability  to  manipulate  methylneogenesis 
furnishes  a  valuable  experimental  tool  which  sets  in  motion  a  complex  set  of 
major  metabolic  readjustments  which  we  are  now  beginning  to  define  and  explore. 
During  the  course  of  these  experiments  two  rather  unexpected  discoveries  were 
made:  (a)  Committment  of  methyl  groups  to  phosphatidylcholine  occurs  by  methyl 
transfer  to  phosphoethanolamine  rather  than  to  phosphatidylethanolamine,  as  is 
commonly  held  to  be  the  case  for  mammals  and  microorganisms.   (b)  Approximately 
10%  of  the  total  methyls  transferred  from  S-adenosylmethionine  are  utilized  to 
form  S-raethylmethionine  sulfonium.   The  quantitative  importance  of  this  reaction 
had  escaped  notice  previously  because  S-methylmethionine  does  not  accumulate 
commensurately ,  but  is  utilized  rapidly  (turnover  time  less  than  one  hour). 
We  are  currently  investigating  the  metabolic  fate  of  S-methylmethionine.   (ii) 
Ongoing  studies  of  threonine  biosynthesis  have  shown  that,  at  the  concentration 
of  inorganic  phosphate  measured  in  the  chloroplast,  the  activity  of  the  available 
threonine  synthase  would  fall  as  much  as  two  orders  of  magnitude  below  that 
required  to  provide  the  threonine  required  for  growth.   The  same  applies,  less 
strikingly,  to  cystathionine  y-synthase.   One  factor,  namely  sequestration 
and  concentration  of  0-phosphohoraoserine  within  the  chloroplast,  would  remedy 
this  apparent  difficulty.   Initial  studies  suggest  that  threonine  partially 
controls  the  rate  of  its  own  biosynthesis,  and  prove  conclusively  that  isoleucine 
regulates  the  conversion  of  threonine  to  isoleucine.   Further  studies  are  required 
to  confirm  and  determine  the  site  at  which  the  unexpected  regulation  by  threonine 
occurs. 


144 


Dr.  Cantoni  in  collaboration  with  Dr.  Razin  organized  a  Workshop  on  the 
Chemistry,  Biochemistry  and  Biology  of  DNA  Methylation  under  the  auspices  of  the 
Fogarty  International  Center.   The  meeting,  held  between  April  17-19  was  attended 
by  over  150  scientists  from  the  U.S  and  other  countries  and  was  very  successful. 
The  proceedings  of  the  workshop  are  being  published  by  Alan  A.  Liss  Publishers. 

Dr.  Cantoni  participated  as  an  invited  lecturer  in  a  workshop  on  Sensing 
and  Response  in  Microorganisms  at  Israel.   Drs .  Cantoni,  Aksamit  and  Backlund 
will  participate  as  invited  speakers  in  the  International  Symposium  on  The  Bio- 
chemistry of  S-adenosylmethionine  as  a  Basis  for  Drug  Design  at  Bergen,  Norway, 
where  Dr.  Cantoni  was  asked  to  present  the  key  note  address  at  the  banquet. 
The  title  of  his  lecture  will  be:  The  centrality  of  S-adenosylhoraocysteinase  in 
the  regulation  of  the  biological  utilization  of  S-adenosylmethionine. 

Drs.  Cantoni,  Aksamit,  Backlund  gave  seminars  at  University  of  Pennsylvania, 
Duke  University,  participated  in  Federation  of  American  Societies  for  Experimental 
Biology  Annual  Meeting  discussions. 


145 


Annual  Report  of  the  Laboratory  of  Molecular  Biology 
National  Institute  of  Mental  Health 
October  1,  1984  -  September  30,  1985 

David  M.  Neville,  Jr.,  M.D.,  Chief 

Introduction 

This  is  the  first  annual  report  of  the  Laboratory  of  Molecular  Biology  which 
was  created  from  personnel  previously  working  in  the  Laboratory  of  Neurochemis- 
try  and  the  Laboratory  of  General  and  Comparative  Biochemistry.  The  new  labora- 
tory brings  together  a  multidisciplinary  group  with  firm  roots  in  the  "new 
biology". 

The  Laboratory  of  Molecular  Biology  conducts  research  in  the  areas  of  molecular 
mechanisms  of  genetic  recombination,  transduction  of  membrane  receptor  hormone 
signaling  and  mechanisms  of  receptor-mediated  protein  transport  across  cell  mem- 
brane barriers.  This  research  is  performed  in  three  independent  sections 
headed  by  Dr.  Howard  Nash  (Molecular  Genetics),  Dr.  Werner  Klee  (Regulatory 
Proteins),  and  Dr.  David  Neville  (Biophysical  Chemistry).  Each  group  is  com- 
posed of  three  to  four  post-doctoral  fellows  and  one  to  two  support  personnel. 
These  small  groups  provide  daily  interchange  between  advisors  and  fellows  both 
at  the  level  of  laboratory  procedures  and  the  broader  overview  of  ongoing 
science.  Scientific  interchange  among  the  groups  is  encouraged  through  formal 
and  informal  arrangements;  bimonthly  meetings  are  held  with  other  basic  scien- 
tists within  building  36  or  the  rest  of  the  NIH  campus.  The  laboratory  strives 
to  combine  the  intellectual  stimulation  of  the  best  academic  centers  with  high 
level  research  production,  and  to  transmit  these  qualities  and  skills  to  post- 
doctoral colleagues. 

The  major  project  in  the  Section  on  Molecular  Genetics  is  the  study  of  molecular 
biology  of  site-specific  genetic  recombination.  In  this  research,  the  DNA  of 
E.  coli,  its  phages  and  plasmids  are  manipulated  both  by  classical  genetic  and 
by  cloning  techniques.  Special  emphasis  is  placed  on  chemical  and  enzymatic 
protection  experiments  that  reveal  the  details  of  protein-DNA  interaction  at  the 
nucleotide  level.  Current  studies  also  include  construction  of  novel  mutants 
by  oligonucleotide-directed  mutagenesis  and  primer  directed  incorporation  of 
nucleotide  analogs  at  specific  positions  in  DNA.  A  recently  initiated  project 
in  this  section  focuses  on  the  genetic  neurobiology  of  the  fruit  fly,  Drosophila 
melanogaster.  The  aim  is  to  use  classical  genetic  techniques  to  isolate  mutants 
that  affect  genes  for  important  components  for  the  nervous  system,  such  as  re- 
ceptors for  neurotransmitters.  In  addition,  mutants  will  be  sought  in  genes  that 
control  complex  neurological  processes,  such  as  the  response  to  anesthetics, 
whose  components  are  not  yet  known.  Molecular  genetic  techniques  will  then  be 
used  to  further  define  the  genes  and  their  gene  products. 

The  Section  on  Regulatory  Proteins  studies  receptors  for  polypeptide  hormones, 
such  as  the  enkephalins,  and  the  mechanisms  of  coupling  of  those  receptors  to 
other  membrane  proteins.  Coupling  mechanisms  currently  under  investigation  are 
those  linked  to  adenylate  cyclase  as  well  as  those  associated  with  phosphatidy- 
linositol  turnover.  These  studies  are  carried  out  with  cells  in  culture,  iso- 
lated membranes,  and  with  purified  components.  Current  studies  focus  on  the 
characterization  of  purified  membrane  proteins  and  their  assembly  into  func- 


147 


tional  assays.  Antibody  production  and  use  in  structure-function  studies  and 
gene  cloning  are  important  parts  of  the  program. 

The  Section  on  Biophysical  Chemistry  is  engaged  in  elucidating  the  mechanisms 
by  which  proteins  and  protein  toxins  such  as  ricin,  diphtheria,  and  tetanus 
toxins  gain  entrance  to  unique  cellular  compartments  by  receptor-mediated 
processes.  Diphtheria  toxin  and  ricin  effects  are  followed  by  assaying  the 
decline  in  protein  synthesis  in  cultured  cells.  Kinetic  modeling  of  the  data 
is  performed  to  delineate  rate  limiting  steps.  Monoclonal  antibodies  are 
generated  to  toxin  determinants  and  domains  involved  in  the  membrane  transport 
process  are  identified  and  correlated  with  protein  sequence  data.  Using  basic 
knowledge  of  toxin  mechanisms  this  group  has  pioneered  the  development  of 
immunotoxins.  Immunotoxins  are  constructed  by  linking  monoclonal  antibodies 
directed  at  unique  differentiation  antigens  to  toxins  thereby  creating  new 
biopolymers.  Clinical  applications  of  these  new  reagents  for  the  treatment  of 
malignancies  and  autoimmune  diseases  are  pursued  via  collaborative  studies. 

The  following  summaries  of  the  year's  progress  have  been  provided  by  the  Section 
Chiefs  of  the  Laboratory: 

SECTION  ON  MOLECULAR  GENETICS 

Howard  A.  Nash,  M.D.,  Ph.D.,  Chief 

The  Mechanism  of  Genetic  Recombination 

The  Section  on  Molecular  Genetics  continues  to  study  the  biochemistry  of  the 
site-specific  recombination  system  that  inserts  the  chromosome  of  bacteriophage 
lambda  into  the  DNA  of  its  host.  In  earlier  work,  we  identified  and  purified 
the  proteins  that  take  part  in  this  reaction  and  have  demonstrated  their  inter- 
actions with  the  target  DNA.  This  work  has  conclusively  showed  that  genetic 
exchange  occurs  by  physical  transfer  of  DNA  strands  between  the  recombining 
partners.  Such  transfer  is  accomplished  by  Int,  a  viral  protein  that  can  re- 
versibly  break  and  reseal  DNA.  Our  current  efforts  are  focused  on  how  each  DNA 
partner  is  activated  to  initiate  recombination  and  how  the  two  DNAs  are  brought 
together  so  that  the  breakage  of  DNA  by  Int  leads  to  exchange  of  genetic  infor- 
mation. 

An  early  result  of  the  biochemical  studies  was  that  the  substrate  DNA  for  lambda 
integrative  recombination  must  be  supercoiled  to  be  maximally  effective;  subse- 
quent studies  have  shown  that  such  supercoiling  is  essential  for  many  processes 
in  E.  coli.  Although  much  is  now  known  about  the  enzymology  of  supertwisting, 
the  reason  that  it  is  required  for  so  many  reactions  remains  obscure.  Our 
group  has  investigated  the  possibility  that  supercoiling  is  used  in  recombina- 
tion reactions  to  assist  the  melting  of  a  critical  stretch  of  DNA.  Such  a 
possibility  is  supported  by  theoretical  considerations  of  the  strain  introduced 
into  DNA  by  supertwisting  and  its  relief  upon  denaturation  of  DNA.  To  test 
this  hypothesis  we  reacted  DNA  with  bromoacetaldehyde,  a  reagent  that  covalently 
attacks  single-stranded  but  not  double-stranded  DNA.  We  found  that  supercoiling 
does  indeed  lead  to  a  bromoacetaldehyde-sensitive  structure  in  viral  DNA  but 
there  is  a  poor  correlation  between  the  melting  of  DNA  induced  in  the  recombi- 
nation site  (attP)  by  supercoiling  and  activation  of  attP  for  recombination. 


148 


Additional  studies  have  failed  to  reveal  any  new  sites  of  melting  in  response 
to  the  binding  of  recombination  proteins  to  attP.  The  findings  argue  against 
the  hypothesis  that  supercoiling  is  required  to  melt  segments  of  recombining 
DNA  and  one  must  look  elsewhere  for  an  important  consequence  of  this  mechanical 
strain. 

The  viral  attachment  site  (attP)  displays  a  bewilderingly  complicated  array  of 
binding  sites  for  recombination  proteins.  Int  binds  to  four  regions  of  attP 
and,  a  bacterial  recombination  protein,  IHF  binds  to  three  regions  that  are 
interdigitated  with  the  Int  binding  sites.  A  unique  DNA  sequence  is  repeated 
at  each  of  the  three  sites  of  interaction  with  IHF.  IHF  also  binds  to  this 
sequence  (or  close  relatives  of  it)  in  non-attachment  site  DNA.  We  are  using 
oligonucleotide-directed  mutagenesis  to  determine  the  features  of  this  consensus 
sequence  that  are  essential  for  IHF  binding.  In  this  method,  we  employ  a 
chemically  synthesized  stretch  of  DNA  that  has  one  or  two  base  changes  from 
the  sequence  found  at  an  IHF  binding  site.  This  oligonucleotide  is  used  as  a 
primer  for  DNA  synthesis  on  a  single-stranded  template  containing  the  wild-type 
site.  E.  coli  is  then  transfected  with  the  resulting  duplex  DNA  and  the  desired 
mutant  is  cloned.  In  this  way  we  have  introduced  specific  base  changes  into 
one  IHF  binding  site.  We  then  assay  the  mutant  sites  for  function  in  two  ways: 
DNAase  footprinting  to  determine  the  capacity  of  the  site  to  bind  IHF  and  in 
vitro  recombination  to  test  the  effect  of  the  mutant  on  the  function  of  the 
site.  Most  base  changes  have  an  effect  that  is  consistent  with  their  effect  on 
the  consensus  sequence  but  at  least  one  change  shows  that  the  binding  site  for 
IHF  has  some  flexibility  beyond  that  predicted  by  the  consensus  sequence.  We 
have  applied  the  information  gained  from  the  study  of  this  site  to  design 
changes  in  the  two  other  IHF  binding  sites  of  attP.  Preliminary  studies  show 
that  inactivation  of  these  sites  also  depress  recombination.  Thus,  we  have 
shown  for  the  first  time  that  (in  contrast  to  Int  where  there  is  redundancy 
in  the  binding  sites)  all  three  IHF  sites  are  needed  for  maximal  recombination. 

An  important  aspect  of  site-specific  recombination  concerns  the  reversibility 
of  the  reaction.  To  achieve  efficient  insertion  of  a  viral  genome,  integration 
of  lambda  DNA  must  not  be  rapidly  followed  by  excision  of  the  integrated  DNA. 
It  has  been  known  for  many  years  that  lambda  integration  is  irreversible;  ex- 
cision requires  the  participation  of  an  additional  viral  protein,  Xis,  that  is 
not  needed  for  integration  and  is  accordingly  not  made  early  after  infection. 
The  biochemical  basis  of  this  irreversibility,  i.e.,  the  role  for  Xis  protein, 
is  not  obvious.  The  attachment  sites  of  the  integrated  virus  (or  prophage) 
contain  all  the  sequences  needed  to  bind  Int  and  IHF  that  the  unintegrated 
virus  contains,  albeit  in  a  permuted  form.  We  do  not  know  if  the  permutation 
of  these  binding  sites  are  inconsistent  with  the  integration  pathway  and  demands 
a  substantially  different  mechanism  or  whether  Xis  simply  permits  the  integra- 
tion pathway  to  be  used  by  the  permuted  sites.  It  had  been  shown  earlier  that 
efficient  integration  requires  attB,  the  bacterial  recombination  site,  and  attP 
to  be  homologous  for  a  stretch  of  seven  base  pairs  around  the  point  of  crossing- 
over.  We  have  investigated  the  capacity  of  excisive  recombination  to  tolerate 
non-homologies  in  one  or  both  strands  of  an  attachment  site.  The  results  are 
identical  with  those  found  by  workers  at  the  University  of  Illinois  for  integra- 
tive recombination  -  if  at  least  one  strand  of  one  prophage  site  matches  a 
strand  of  its  partner  site,  recombination  is  maximally  efficient.  If  neither 
strand  of  prophage  site  is  identical  to  a  strand  of  its  partner,  recombination 
is  inefficient.  The  result  implies  that  the  search  for  homology  between  recom- 


149 


bining  partners,  a  critical  step  in  the  recombination  reaction,  uses  a  similar 
mechanism  during  integration  and  excision.  This  suggests  that  the  fundamental 
pathway  of  recombination  is  the  same  in  the  two  directions  and  that  irreversi- 
bility is  expressed  simply  at  the  initiation  of  recombination. 

Genetic  Neurobiology  of  Drosophila 

Our  group  is  also  initiating  a  research  program  to  investigate  the  neurobiology 
of  the  fruit  fly,  Drosophila  melanogaster ,  using  genetics  as  the  primary  tool. 
The  same  genetic  techniques  that  have  helped  unravel  pathways  in  intermediary 
metabolism.,  DMA  replication,  and  embryonic  development  in  this  organism  should 
be  applicable  to  the  study  of  behavior  and  the  nervous  system.  Indeed,  for  the 
past  fifteen  years  genetic  studies  of  the  fruit  fly  have  turned  up  interesting 
and  important  mutations  that  affect  sensory,  motor,  and  integrative  capacities 
of  the  organism.  With  the  advent  of  contemporary  genetic  techniques,  many  of 
the  Drosophila  genes  identified  by  these  mutations  are  now  being  studied  at  the 
molecular  level.  In  addition  to  its  small  genome  and  rapid  generation  time, 
Drosophila  offers  a  special  advantage  for  studying  complex  genetic  loci.  In 
the  past  three  years  techniques  have  been  developed  that  readily  permit  the  re- 
introduction  of  a  cloned  Drosophila  genes  back  into  the  fly's  genome,  permit- 
ting the  investigator  to  alter  the  gene  at  will  and  then  assess  its  function 
in  vivo.  We  want  to  isolate  mutants  that  will  identify  the  genes  for  important 
components  of  the  fly's  nervous  system  such  as  ion-specific  channels  and  recep- 
tors for  neurotransmitters.  In  addition,  we  want  to  identify  the  genes  that 
underlie  complex  neurological  processes  such  as  the  response  to  anesthetics 
whose  components  are  not  yet  known . 

SECTION  ON  REGULATORY  PROTEINS 

Werner  A.  Klee,  Ph.D.,  Chief 

Opiate  Receptor  Coupling  Mechanisms 

The  Section  on  Regulatory  Proteins  is  concerned  with  the  general  question  of 
understanding  the  mechanisms  by  which  information  is  transferred  from  cell  sur- 
face receptors  to  intracellular  enzyme  systems.  At  present,  the  efforts  of  our 
group  are  concentrated  mainly  on  those  receptors  which  are  coupled  to  adenylate 
cyclase  regulation,  in  particular  opiate  and  other  inhibitory  receptors.  Much 
of  this  work  is  performed  using  neuroblastoma  x  glioma  hybrid  NG1 08-15  cells 
which  are  richly  endowed  with  a  single  type  of  opiate  receptor.  The  receptors 
in  these  cells  are  coupled  to  inhibition  of  adenylate  cyclase  activity  in  a 
process  mediated  by  a  GTP  binding  regulatory  protein  (Ni).  Understanding  the 
nature  of  this  coupling  process  in  molecular  terms  is  a  major  goal  of  the  sec- 
tion. Although  the  actions  of  opiate  receptors  in  brain  are  undoubtedly  more 
varied  than  they  are  in  the  model  system  which  we  study,  it  is  abundantly  clear 
that  one  of  the  major  effects  of  opiate  receptor  occupancy  in  brain  is  that  of 
adenylate  cyclase  inhibition. 

The  work  of  the  section  in  recent  years  can  be  logically  grouped  into  three 
categories.  These  are  (1)  description  of  the  biochemical  actions  of  opiates 
and  of  the  proteins  involved,  (2)  isolation  and  characterization  of  each  of  the 
identified  components,  and  (3)  reconstitution  of  the  activities  of  the  coupled 
system  by  appropriate  integration  of  the  purified  components. 

150 


Opiate  inhibition  of  adenylate  cyclase  activity  is  accompanied  by,  and  may  be 
mediated  through  stimulation  of  an  associated,  low  Km,  GTPase  activity.  In 
following  up  this  observation,  members  of  the  section  have  studied  the  protein 
responsible  for  this  GTPase  activity,  currently  called  Ni.  This  protein  is  one 
of  several  members  of  a  family  of  GTP-binding  proteins,  the  N  proteins.  Several 
signal  transduction  processes  in  cell  membranes  are  mediated  by  receptor  coupl- 
ing to  one  of  a  family  of  GTP-binding  proteins  (N  proteins).  The  N  proteins 
are  all  closely  related  in  structure  and  are  composed  of  three  subunits 
(a,  3,  and  y) .  At  least  two  such  proteins  couple  receptors  to  adenylate 
cyclase:  Ns  is  required  for  stimulatory  receptor  activity,  and  Ni  (or  perhaps 
No)  for  inhibitory  receptors.  The  group  has  purified  a  mixture  of  Ni  and  No 
from  bovine  brain,  but  has  so  far  been  unable  to  separate  the  two  in  a  useful 
way.  Nonetheless,  this  preparation  will  restore  opiate  inhibition  of  adenylate 
cyclase  and  stimulation  of  low  Km  GTPase  activity  to  membranes  depleted  of 
these  activities  by  pertussis  toxin-catalyzed  ADP-ribosylation  of  Ni.  In 
collaborative  studies,  they  have  prepared  and  characterized  antibodies  which 
specifically  recognize  the  a  subunits  of  Ni  or  No.  (The  3  and  y  subunits  of 
the  proteins  are  apparently  identical.)  These  antibodies  are  being  used  to 
quantitate  the  tissue  distribution  of  the  two  proteins  after  their  electrophore- 
tic  separation.  Western  blots  of  membranes  prepared  from  cells  or  tissues  are 
analyzed  by  measuring  the  amount  of  l''25_protein  A  bound  to  them.  Some  tissues, 
such  as  liver  and  kidney,  contain  no  detectable  No  but  clearly  contain  Ni.  The 
brain,  on  the  other  hand,  is  a  very  rich  source  of  No  and  contains  lesser,  but 
still  appreciable  amounts  of  Ni.  The  functional  roles  of  No  and  Ni  are  not  yet 
completely  understood.  Because  they  are  both  substrates  for  pertussis  toxin- 
catalyzed  ADP-ribosylation  this  functional  probe  does  not  distinguish  between 
the  two.  We  are  currently  preparing  pure  Ni  from  liver  for  use  in  reconstitu- 
tive  assays  of  N  protein  function  to  examine  this  question. 

Another  of  the  components  of  the  system  which  has  been  purified  by  members  of 
the  section  is  the  opiate  receptor.  This  preparative  procedure  exploited  the 
availability  of  fentanylisothiocyanate  (FIT)  and  the  closely  related  derivative 
super-FIT  as  highly  selective  and  quantitative  affinity  labels  for  the  receptor. 
These  covalent  adducts  were  purified  to  homogeneity  in  three  steps,  the  most 
selective  of  which  used  antibodies  which  specifically  bind  to  FIT  and  super- 
FIT.  The  scale  and  efficiency  of  the  purification  procedure  has  been  increased 
so  that  nanomole  amounts  of  the  protein  are  now  available.  Antibodies  raised 
against  this  protein  effectively  bind  to  unmodified  opiate  receptors  in  solu- 
tion. Thus,  the  identification  of  the  58,000  Mr  protein  as  the  binding  subunit 
of  the  opiate  receptor  has  been  confirmed.  The  antibodies  also  recognize 
opiate  receptors  transferred  to  nitrocellulose,  and  other  matrices  after  elec- 
trophoretic  separation.  The  sera  are  being  purified  by  affinity  chromatography 
in  preparation  for  their  use  in  selection  of  those  clones  of  expression  vectors 
which  carry  the  opiate  receptor  genome.  We  also  anticipate  that  these  anti- 
bodies will  be  useful  histological  stains  and  will  facilitate  study  of  the 
biosynthesis,  processing,  and  breakdown  of  the  receptors.  The  receptor  can  be 
efficiently  converted  to  a  much  smaller  protein  by  cleavage  of  peptide  bonds 
following  some  of  the  methionine  residues  with  cyanogen  bromide.  The  fragment 
so  formed,  of  Mr  near  30,000  contains  both  the  carbohydrate  and  opiate  sites. 
This  material  is  currently  being  purified  for  amino  acid  sequence  studies.  The 
data  so  obtained  will  be  used  to  design  oligonucleotide  probes  for  gene  cloning. 


151 


Exciting  progress  has  recently  been  made  by  members  of  the  group  in  the  sphere 
of  opiate  receptor-adenylate  cyclase  reconstitution.  Recent  experiments  have 
successfully  demonstrated  the  reconstitutive  coupling  of  opiate  receptors,  Ni 
and  adenylate  cyclase  in  liposomes  prepared  from  solutions  of  these  proteins. 
The  experiments  were  carried  out  with  partially  purified  preparations  of  recep- 
tors and  of  adenylate  cyclase  added  to  a  highly  purified  Ni,  No  mixture. 
Availability  of  a  reconstitution  assay  should  allow  characterization  of  the 
activities  of  each  of  the  purified  components  of  the  membrane  signal  transduc- 
tion apparatus  as  well  as  identification  of  any  as  yet  unknown  components. 

SECTION  ON  BIOPHYSICAL  CHEMISTRY 

David  M.  Neville,  Jr.,  Chief 

Receptor-Mediated  Protein  Transport  to  the  Cytosol 

Cells  utilize  receptors  for  signaling  events,  for  uptake  of  necessary  materials 
and  to  internalize  materials  to  be  degraded.  Most  components  entering  cells 
from  the  environment  are  routed  through  the  cells  in  vesicles  and  never  cross 
the  membrane  barrier  and  gain  access  to  the  cytosol  or  nuclear  compartments 
where  the  vital  machinery  of  genetic  replication,  transcription  and  translation 
operates.  Certain  molecules  however  do  make  this  transition  and  in  a  highly 
specific  manner.  The  plant  and  bacterial  toxins  are  examples  of  this  group  and 
their  presence  is  known  because  each  contains  an  enzymatic  domain  which  alters 
a  crucial  cytosolic  substrate  leading  to  either  cell  death  or  dramatically 
altered  cell  function. 

The  study  of  toxin  transport  processes  and  toxin  substrates  asks  several  pro- 
found questions. 

1)  How  does  a  highly  charged  hydrophilic  macromolecule  cross  the  lipid  bi- 
layer? 

2)  What  is  the  natural  function  for  these  highly  specific  receptor -mediated 
transport  systems  and  finally; 

3)  does  the  toxin  enzymatic  derangement  of  cellular  substrates  have  a 
physiologic  counterpart? 

Over  the  past  two  years  Dr.  Thomas  Hudson  in  our  Section  has  performed  studies 
which  shed  considerable  light  on  how  diphtheria  toxin  crosses  the  membrane. 
Dr.  Hudson's  work  suggests  that  diphtheria  toxin  gains  entrance  to  the  cytosol 
compartment  by  destabilizing  the  vesicle  (or  a  derivative  of  this  vesicle)  into 
which  the  toxin  is  packed.  A  bolus  of  toxin  of  sufficient  size  to  rapidly  in- 
activate all  of  the  cell's  elongation  factor  II  (necessary  for  protein  synthesis) 
appears  to  enter  the  cell  in  a  concerted  process.  Intoxicated  cells  are  found 
to  consist  of  two  populations,  those  synthesizing  protein  and  those  not  synthe- 
sizing protein.  Over  the  time  course  of  intoxication  the  population  not  synthe- 
sizing protein  increases.  The  probability  that  a  bolus  of  toxin  will  be  re- 
leased into  a  cell  depends  on  receptor  occupancy.  This  view  of  diphtheria  toxin 
entry  is  radically  different  from  previous  models.  The  biochemical  mechanisms 


152 


responsible  for  this  kinetic  picture  should  provide  new  insights  for  the  con- 
struction of  efficient  immunotoxins  utilizing  the  diphtheria  toxin  entry  func- 
tion . 

The  Design  of  Immunotoxins  for  the  Treatment  of  Autoimmune  Diseases 

It  is  generally  believed  that  multiple  sclerosis,  rheumatoid  arthritis  and 
early  onset  diabetes  are  autoimmune  diseases.  Increasingly,  attention  is  being 
focused  on  provocative  reports  which  indicate  that  certain  subsets  of  mental 
illnesses  may  also  have  an  autoimmune  component  to  their  etiology.  Autoimmune 
diseases  occur  when  the  immune  system's  defense  mechanisms  are  turned  against  a 
person's  own  body  or  parts  of  the  body.  The  initiating  causes  of  these  diseases 
are  poorly  understood  but  both  environmental  factors  and  genetic  factors  seem 
to  be  involved. 

Current  therapy  for  autoimmune  diseases  aims  at  inhibiting  the  part  of  the 
immune  system  causing  the  damage.  Our  Section  is  working  to  develop  very  highly 
selective  drugs  to  inhibit  parts  of  the  immune  system.  To  do  this,  we  use  a 
totally  new  class  of  drug  -  immunotoxins.  Immunotoxins  consist  of  a  powerful 
toxin  linked  to  a  monoclonal  antibody  which  can  seek  out  and  bind  to  only  certain 
types  of  cells.  The  toxins  which  come  from  poisonous  plants  (ricin)  or  harmful 
bacteria  (diphtheria  toxin)  are  extremely  potent  and  have  specific  machinery 
for  entering  and  killing  cells. 

The  goal  of  our  program  is  to  understand  the  toxin  entry  machinery  sufficiently 
well  so  that  we  can  make  therapeutically  useful  selective  reagents.  Already  we 
and  our  colleagues  at  the  University  of  Minnesota  have  been  able  to  do  this 
when  cells  are  treated  outside  of  the  body  with  anti-T  cell  monoclonal  antibody- 
ricin  conjugates.  This  has  been  useful  in  depleting  immune  system  T  cells  from 
donor  bone  marrow  and  thereby  reducing  the  incidence  of  graft-versus-host 
disease.  We  are  presently  working  to  extend  these  findings  for  the  treatment 
of  human  autoimmune  diseases.  To  this  end  we  have  identified  the  variables 
which  lead  to  efficient  entry  of  the  toxin  portion  of  immunotoxins  into  the 
targeted  cell;  affinity,  receptor  number,  and  an  entry  function  located  in  part 
on  the  toxin  B  chain.  Manipulating  this  entry  function  to  only  operate  on 
targeted  cells  is  the  goal  of  current  research. 


153 


ANNUAL  REPORT  OF  THE  LABORATORY  OF  NEUROCHEMISTRY 

NATIONAL  INSTITUTE  OF  MENTAL  HEALTH 

OCTOBER  1,  1984  THROUGH  SEPTEMBER  30,  1985 

Seymour  Kaufman,  Chief 

During  the  last  year,  one  of  the  major  efforts  of  the  Laboratory  of 
Neurochemistry  has  continued  to  be  a  study  of  the  mechanism  of  regulation  of  the 
activity  of  hepatic  phenylalanine  hydroxylase.  These  regulatory  studies  have  been 
extended  to  include  characterization  of  kidney  phenylalanine  hydroxylase  which  has 
been  found  to  have  distinctive  characteristics.  One  of  the  reasons  for  this  emphasis  is 
that  it  is  known  that  a  normal  functioning  phenylalanine  hydroxylating  system  is 
essential  for  normal  brain  development.  Another  reason  is  that  this  enzyme  serves  as  a 
useful  model  for  understanding  the  regulation  of  the  other  two  aromatic  amino  acid 
hydroxylases,  tyrosine  and  tryptophan  hydroxylases.  In  addition  to  the  studies  on  the 
regulatory  properties  of  phenylalanine  hydroxylase,  we  have  during  the  last  12  months 
answered  several  important  questions  about  the  structure  of  this  enzyme. 

We  have  recently  demonstrated  the  existence  of  a  novel  mechanism  by  which  the 
activity  of  hepatic  phenylalanine  hydroxylase  is  regulated.  This  control  system  involves 
opposing  effects  of  the  enzyme's  substrate,  phenylalanine,  and  its  coenzyme, 
tetrahydrobiopterin  (BH4).  We  have  found  that  at  physiological  levels  (micromolar 
range)  BH4  dramatically  inhibits  the  phosphorylation-mediated  activation  of 
phenylalanine  hydroxylase,  whereas  phenylalanine  stimulates  this  reaction.  Moreover, 
phenylalanine  can  completely  overcome  the  inhibition  by  BH4.  These  findings  provide 
another  example  of  the  redundant  control  of  the  activity  of  this  enzyme  by  its  substrate. 
This  newly  discovered  mechanism  converges  with  other  previously  described 
mechanisms  to  make  certain  that  the  activity  of  phenylalanine  hydroxylase  correlates 
with  tissue  levels  of  phenylalanine,  i.e.,  it  is  high  when  blood  and  tissue  levels  of 
phenylalanine  are  high  (e.g.,  after  the  ingestion  of  a  protein-containing  meal)  and  it  is 
low  when  levels  of  phenylalanine  are  low.  It  seems  most  likely  that  the  former  aspect 
of  this  control  mechanism  functions  in  the  organism  to  protect  the  fetal  and  neonatal 
brain  from  the  potential  harmful  effects  of  elevations  in  blood  phenylalanine  levels, 
whereas  the  latter  aspect  functions  to  prevent  the  lethal  depletion  of  the  organism's 
stores  of  phenylalanine  by  the  uncontrolled  conversion  to  tyrosine,  a  process  that  could 
potentially  occur  if  the  activity  of  phenylalanine  hydroxylase  were  not  tightly  regulated 
by  the  tissue  levels  of  phenylalanine. 

Although  it  has  been  known  for  more  than  20  years  that  the  kidney  contains 
significant  amounts  of  phenylalanine  hydroxylase,  the  enzyme  from  that  tissue  has  been 
largely  neglected,  probably  because  the  activity  of  the  kidney  hydroxylase  was  believed 
to  be  trivial  compared  to  that  of  the  liver  enzyme.  We  have  now  shown  that  this  view 
is  erroneous.  When  assayed  with  BH4,  the  kidney  enzyme  appears  to  be  in  an  unusual 
super-activated  state  compared  to  the  liver  enzyme.  Based  on  these  assays  it  can  be 
estimated  that  the  BH^-dependent  hydroxylase  activity  of  the  kidney  enzyme  may 
contribute  as  much  as  20  to  30%  of  the  organism's  total  phenylalanine  hydroxylase 
activity.  We  have  found  that  this  unique  activated  state  of  the  kidney  hydroxylase  is 
readily  lost  during  attempts  to  purify  the  enzyme.  We  have  succeeded,  however,  in 
purifying  the  enzyme  so  that  it  retains  the  characteristic  property  and  are  now  in  a 
position  to  study  the  structural  basis  of  the  super-activated  state. 


155 


We  have  recently  answered  two  longstanding  questions  about  the  structure  of 
hepatic  phenylalanine  hydroxylase.  It  has  been  known  for  many  years  that  the  N- 
terminal  amino  acid  is  blocked.  Despite  the  fact  that  major  portions  of  the  enzyme 
have  been  indirectly  sequenced,  the  identiy  of  the  N-terminal  amino  acid,  as  well  as  the 
nature  of  the  blocking  group  have  remained  elusive.  We  have  now  shown  that  the  N- 
terminal  amino  acid  is  N-acetylalanine.  This  finding  raises  questions  about  the  function 
of  the  blocking  group,  questions  that  can  be  addressed  experimentally.  A  second 
structural  question  that  we  have  recently  answered  is  whether  hepatic  phenylalanine 
hydroxylase  is  comprised  of  different  subunits.  Despite  the  published  evidence  that  had 
previously  suggested  the  possibility  that  there  are  different  types  of  subunits,  our 
structual  studies  of  pure  hepatic  phenylalanine  hydroxylase  from  Sprague-Dawley  rats 
have  shown  conclusively  that  the  enzyme  from  this  source,  at  least,  is  composed  of 
identical  subunits. 

We  have  made  significant  progress  in  our  studies  of  the  diagnosis  and  treatment  of 
the  variant  form  of  phenylketonuria  that  is  caused  by  defects  in  BH^  synthesis.  As  far 
as  treatment  is  concerned,  we  have  shown  that  when  given  subcutaneously,  only  10%  of 
the  oral  dose  of  BH/^  is  required  to  achieve  comparable  blood  levels  of  BHz^.  We  have 
been  maintaining  a  BH4-deficient  child  on  the  lower  subcutaneous  dose  of  BH4  for 
about  six  months  and  both  the  biochemical  and  neurological  responses  appear  to  be 
comparable  to  those  seen  on  the  10-fold  higher  oral  dose  of  BH4.  Since  the  treatment 
of  patients  with  BH4  deficiency  costs  about  $15,000  to  $45,000  per  year,  our  findings 
indicate  that  the  cost  of  this  type  of  treatment  can  be  lowered  to  the  point  where  BH4 
replacement  therapy  can  be  more  widely  used. 

In  the  area  of  diagnosis,  we  have  described  two  patients  with 
hyperphenylalaninemia  caused  by  a  defect  in  BH4  biosynthesis  in  which  the  metabolic 
block  appears  to  be  restricted  to  peripheral  tissues.  These  children  have  normal  CSF 
levels  of  BH4  as  well  as  of  biogenic  amine  metabolites,  and  show  no  neurological 
symptoms.  They,  therefore,  do  not  require  BH4  supplementation.  These  results 
indicate  that  the  demonstration  of  abnormal  BH4  metabolism  in  blood  or  urine  may  not 
be  an  adequate  basis  for  instituting  BH4  replacement  therapy.  Such  treatment  should 
be  based  on  a  demonstrated  lack  of  BH4  in  CSF. 


156 


Annual  Report  of  the  Laboratory  of  Neurophysiology 
National  Institute  of  Mental  Health 
Edward  V.  Evarts,  M.D. ,  Chief,  deceased 
Steven  P.  Wise,  Ph.D.,  Acting  Chief 
October  1,  1984  —  September  30,  1985 

The  broad  goal  of  research  in  the  Laboratory  of  Neurophysiology  (LNP)  is  the  elu- 
cidation of  the  linkages  between  brain  areas  involved  in  controlling  mood,  move- 
ment and  preparatory  set:   CNS  control  of  the  most  flexible,  least  automatic 
behaviors.   The  way  in  which  these  linkages  operate  can  be  discovered  only  by  a 
combined,  interdisciplinary  approach  that  uses  the  techniques  of  neurochemistry, 
neuroanatomy,  experimental  psychology  and  neurophysiology.   LNP  research  is  pri- 
marily directed  toward  a  group  of  subcortical  brain  structures  referred  to  col- 
lectively as  the  basal  ganglia  and  the  cortical  areas  most  directly  linked  with 
basal  ganglia  function.   Inputs  to  the  basal  ganglia  arise  from  many  different 
parts  of  the  cerebral  cortex,  including  motor,  sensory,  and  association  areas. 
But,  in  addition,  the  basal  ganglia  receive  input  from  the  limbic  system.   Thus, 
the  basal  ganglia  provide  a  junction  point  for  limbic  inputs  underlying  mood  and 
cortical  inputs  that  provide  signals  related  to  preparatory  set  and  movement. 

The  LNP  currently  consists  of  two  groups  engaged  in  an  analysis  of  the  basal 
ganglia  and  associated  cortical  regions,  one  headed  by  Miles  Herkenham,  the  other 
by  Steven  Wise.   A  third  part  of  the  laboratory  consists  of  Ichiji  Tasaki,  whose 
work  focuses  on  mechanical  and  thermal  events  associated  with  neural  activity. 
Herkenham  and  his  group  are  investigating  the  Functional  Neuroanatomy  of  the 
basal  ganglia,  with  an  emphasis  on  the  distribution  of  receptors  that  mediate 
the  function  of  the  endogenous  opioid  peptides.   In  addition,  this  work  includes 
an  analysis  of  compartmental  organization  in  the  basal  ganglia,  the  degree  to 
which  these  compartments  could  functionally  interact,  and  the  localization  of 
function  within  the  limbic  and  sensorimotor  areas  of  the  basal  ganglia.   The 
second  major  group  within  the  LNP,  headed  by  Wise,  is  studying  the  Cerebral 
Physiology  of  parts  of  the  neocortex  involved  in  the  sensory  guidance  of  volun- 
tary movements.   These  cortical  areas,  referred  to  collectively  as  the  somatic 
sensorimotor  cortex,  are  intimately  involved  in  basal  ganglia  function  since 
they  supply  the  bulk  of  its  input.   Thus  the  physiological  aspects  of  the  LNP 
program  are  critically  dependent  upon  and  interrelated  with  its  neuroanatomical 
studies  on  the  basal  ganglia.   There  are  two  approaches  to  cerebral  physiology 
currently  being  pursued  in  the  LNP.   The  first  main  physiological  approach  is 
aimed  at  areas  involved  in  higher-order  aspects  of  behavioral  control.   It  is 
now  known  that  the  major  output  target  of  the  basal  ganglia  in  the  primate  brain 
is  a  cortical  region  known  as  the  supplementary  motor  cortex  (Mil).   Mil  is  one 
of  two  nonprimary  motor  cortical  fields  that  have  been  the  subject  of  study  by 
Wise  and  his  colleagues  in  the  laboratory.   In  addition  to  improving  the  defini- 
tion of  these  fields.  Wise  has  been  developing  behavioral  methods  for  studying 
the  specialized  activity  of  each  part  of  the  nonprimary  motor  cortex.   In  his 
ejcperiments,  the  critical  dimension  is  the  preparation  and  readiness  for  move- 
ment rather  than  the  actual  execution  of  movement.   Future  work  will  be  aimed 
at  comparing  different  nonprimary  motor  areas  with  an  aim  of  determining  how 
the  one  receiving  inputs  from  basal  ganglia  (MIX)  differs  from  those  with  cere- 
bellar inputs.   The  second  main  physiological  approach  is  more  directly  aimed 
at  understanding  the  cortrol  of  motor  execution.   This  aspect  of  the  laboratory's 
program  was  headed  by  Evarts  until  his  death  in  July  and  now  is  being  guided  by 


157 


wise.   The  work  deals  with  the  primary  motor  and  somatic  sensory  areas,  which 
are  directly  involved  in  the  execution  of  movement,  as  well  as  with  cell  dis- 
charge in  the  basal  ganglia.   This  work  serves  as  a  bridge  between  studies  of 
the  Functional  Neuroanatomy  of  the  basal  ganglia  and  studies  of  the  Cerebral 
Physiology  of  areas  controlling  higher-order  aspects  of  behavioral  control, 
areas  that  provide  many  of  the  inputs  to  the  basal  ganglia  and  receive  its 
major  output. 

I.  Functional  Neuroanatomy; 

Work  in  this  area  is  guided  by  Herkenham  and  carried  out  in  collaboration  with 
Stafford  McLean  and  Charles  Gerfen.   The  projects  now  in  progress  may  be  divided 
into  two  areas,  one  broad-based  and  directed  toward  an  understanding  of  the 
roles  that  neurotransmitters  and  receptors  play  in  dynamic  brain  function  and 
the  other  concerning  the  neurochemical  and  connectional  organization  of  the 
basal  ganglia. 

Neurotransmitter/Receptor  Localization  Studies;  Over  the  last  decade  a  major 
thrust  of  neuroscience  research  has  been  the  identification  of  neurotransmitter, 
neuromodulator  and  hormone  receptors  in  the  brain.   An  understanding  of  receptor 
function  requires  knowledge  of  the  biochemistry  and  pharmacology  as  well  as  the 
neuroanatomical  localization  of  receptors.   Identification  of  receptors  by 
pharmacological  criteria  has  been  carried  out  in  collaborative  studies  with 
Dr.  C.  B.  Pert,  Clinical  Neuroscience  Branch,  and  Dr.  R.  B.  Rothman,  Laboratory 
of  Preclinical  Pharmacology,  both  of  the  NIMH,  IRP.   Herkenham  and  his  col- 
leagues are  seeking  to  identify  the  neuronal  circuitry  that  is  associated  with 
these  receptors  by  comparison  with  known  anatomical  pathways  and  by  immunohisto- 
chemical  identification  of  transmitter-specific  connections.   Other  main  objec- 
tives are  to  understand  the  role  of  a  receptor  or  various  receptor  subtypes 
in  any  given  region  by  determining  normal  receptor  density,  plasticity  of  re- 
ceptors as  a  function  of  developmental  time  course  or  pharmacological  manipula- 
tion, and  altered  distribution  in  pathological  states. 

To  permit  these  analyses,  Herkenham  and  his  associates  have  successfully  develop- 
ed an  in  vitro  autoradiographic  technique  for  visualizing  drug  and  neurotransmit- 
ter receptors  in  slide-mounted  tissue  sections.   Using  emulsion  autoradiography, 
the  receptor  distributions  can  be  compared  with  underlying  morphology  seen  in 
the  Nissl  stained  sections.   Stains  for  other  features  of  brain  chemistry  and 
functional  heterogeneity  can  be  applied  to  the  same  or  adjacent  sections.   Using 
film  autoradiography,  the  receptor  patterns  can  be  analyzed  by  a  densitometer 
for  computer-assisted  quantification  of  receptor  densities. 

The  group  has  been  particularly  successful  at  detecting  subtle  differences  in 
the  distributions  of  receptor  subtypes  and  precise  correlations  with  other 
anatomical  and  chemical  markers  such  as  cell  and  fiber  stains,  immunohisto- 
chemistry  of  transmitters  and  transmitter-specific  enzymes,  labeled  pathways, 
and  catecholamine  fluorescence.   The  technique  is  well-suited  to  study  fragile 
tissues  and  tissues  that  require  suboptimal  binding  conditions,  and  so  the 
group  has  succeeded  in  studying  receptor  development  in  the  rat  fetuses  and 
the  binding  of  several  "novel"  peptides. 


158 


studies  of  rat  neostriatum  have  shown  that  the  rate  of  development  of  dopamine 
islands  and  opiate  receptor  patches  is  influenced  by  dopamine  and  opiate  receptor 
blockers  (antagonists)  administered  via  osmotic  minipumps  to  pregnant  rats  during 
a  critical  prenatal  period.   In  another  study,  sex  differences  in  opiate  receptor 
distributions  in  the  preoptic  area  were  noted.   Both  studies  also  showed  that 
the  adult  patterns  could  be  altered  by  hormonal  manipulations  in  the  early  post- 
natal period. 

Herkenham's  group  has  prepared  detailed  maps  of  several  neurochemical  systems 
using  the  techniques  of  autoradiography  and  immunohistochemistry .   This  work 
has  permitted  the  first  careful  test  of  the  commonly  held  hypothesis  that  the 
maps  of  transmitter  distribution  (at  nerve  terminals  where  release  occurs)  will 
closely  resemble  maps  of  associated  receptor  distributions.   A  clear  violation 
of  that  hypothesis  was  provided  in  the  substance  P  system.   The  density  of  this 
putative  transmitter  in  terminals  within  the  substantia  nigra  is  the  highest  in 
the  rat  brain.   Yet  binding  of  [■'-^^I]  substance  P  (SP)  and  [-"-^^I]  eledoisin  to  two 
subtypes  of  the  SP  receptor  did  not  occur  in  substantia  nigra.   In  fact,  the 
distribution  patterns  of  these  receptors  were  not  correlated  at  all  with  the 
distribution  pattern  of  substance  P  in  the  brain:  in  general,  where  there  is 
substance  P  there  are  few  or  no  SP  receptors,  where  there  are  SP  receptors  there 
is  little  or  no  substance  P.   A  similar,  though  less  dramatic,  picture  emerged 
after  comparing  the  distributions  of  the  opioid  peptides  —  enkephalin  and 
dynorphin  —  with  the  distributions  of  the  major  opiate  receptor  subtypes  —  mu, 
delta  and  kappa.   These  mismatches  are  pervasive;  they  are  present  throughout 
all  well-characterized  and  mapped  neurotransmitter/receptor  systems  and  cannot 
easily  be  escplained  in  terms  of  technical  failure  (such  as  antibody  recognition 
problems  or  inadequate  characterization  of  receptor  subtypes).   Herkenham's  group 
has  emphasized  several  more  innovative  explanations:  low-affinity  synaptic  recep- 
tors for  neurotransmission,  high-affinity  nonsynaptic  receptors,  transmitter  ex- 
cesses in  storage  pools,  and  diffusion  of  transmitter  to  distant  targets.   These 
ideas  have  implications  for  our  understanding  of  drug  actions  on  brain  function, 
since,  in  view  of  Herkenham's  findings,  it  may  be  hypothesized  that  exogeneously 
administered  drugs  mimic  normal  physiology  more  than  previously  believed. 

Organization  of  the  Basal  Ganglia:   Studies  of  the  neuroanatomical  and  neuro- 
chemical organization  of  the  basal  ganglia  are  headed  by  Gerfen  and  serve  to 
provide  a  bridge  between  the  functional  neuroanatomical  and  physiological  ap- 
proaches to  brain  mechanisms  involved  in  controlling  mood,  movement  and  prepara- 
tory set.   The  neuroanatomical  correlates  subserving  the  linkage  of  these  func- 
tions within  the  basal  ganglia  have  been  elusive  in  part  because  of  this  brain 
system  in  the  absence  of  distinct  cytoarchitectural  features  that  have  aided  in 
similar  studies  of  the  cortex  and  thalamus.   Gerfen  has  developed  a  methodolog- 
ical approach  for  examining  the  interrelations  of  biochemically  defined  neural 
circuits  in  the  brain.   This  methodology  is  particularly  well  suited  to  study 
of  the  basal  ganglia  since  it  has  biochemically  rather  than  cytoarchitecturally 
distinct  systems  underlying  its  functional  organization. 

The  central  technique  is  the  Phaseolus  vul gari s - 1 euca ggluti ni n  (PHA-L)  antero- 
grade axonal  tract  tracing  method  developed  by  Gerfen  and  P.  E.  Sawchenko  at 
the  Salk  Institute.   Immunohistochemical  localization  of  PHA-L,  which  has  been 
injected  into  the  brain,  incorporated  by  neurons  at  the  site  of  injection  and 
transported  specifically  in  the  anterograde  direction  within  axons,  demonstrates 
morphological  details  of  labeled  neurons'  cell  bodies,  dendrites  and  axon  pro- 
jections, including  terminal  specializations.   Concurrent  immunohistochemical 

159 


localization  of  biochemical  markers  and  PHA-L  in  labeled  axons  provides  a  means 
of  tracing  chemically  defined  projections.   The  connections  of  neurons  that  are 
targets  of  PHA-L  labeled  afferents  can  then  be  determined  with  retrograde  axonal 
transport  of  fluorescent  dyes.   The  relationship  of  such  biochemically  and  ana- 
tomically characterized  neural  systems  to  autroradiographically  labeled  receptor 
systems  provides  further  information  regarding  the  functional  neuroanatomy  of 
the  basal  ganglia. 

Previous  studies  had  established  the  neurochemical  heterogeneity  of  the  striatum 
by  the  demonstration  of  the  uneven  distribution  of  mu  opiate  receptor  binding 
and  acetylcholinesterase  histochemical  staining.   The  morphological  basis  of 
striatal  heterogeneity  was  provided,  in  part,  by  Gerfen's  demonstration  that 
somatostatin  fiber-immunoreactivity  and  striatonigral  projection  neurons  that 
contain  calcixim  binding  protein  (CaBP)immunoreactivity  are  selectively  distri- 
buted in  a  "matrix"  that  is  complementary  to  opiate  receptor  rich  "patches". 
These  patches  themselves  contain  a  distinct  population  of  striatonigral  projec- 
tion neurons.   Taken  together  these  two  types  of  striatal  systems  fill  the  stria- 
tum in  a  mosaic  fashion.   Gerfen's  studies  showed  that  these  compartments  serve 
to  segregate  paralled  striatal  throughout  systems  from  each  other.   Specifically, 
the  matrix  receives  inputs  from  somatic  sensory  and  motor  areas  of  cortex,  and 
cells  in  the  matrix  project  to  the  GABAergic  neurons  in  the  substantia  nigra. 
This  is  a  major  pathway  through  which  the  basal  ganglia  directly  affect  move- 
ments.  Conversely,  the  striatal  patches  receive  inputs  from  the  prelimbic  cor- 
tex, a  cortical  area  linked  with  limbic  brain  areas.   Cells  in  the  patches  pro- 
ject to  the  dopaminergic  neurons  in  the  substantia  nigra.   This  is  a  pathway 
which  provides  feedback  regulation  of  the  striatum.   Thus,  neuroanatomical 
pathways  thought  to  subserve  motor  and  affective  functions  are  segregated  in 
different  striatal  compartments.   Interactions  of  these  systems  might  occur 
through  at  least  two  types  of  pathways:   directly  via  an  intrinsic  somatostatin 
striatal  system  that  links  the  patches  and  matrix  and  indirectly  via  the  nigro- 
striatal  feedback  system. 

These  studies  are  now  being  extended  to  the  monkey  and  preliminary  results  show 
that  the  compartmentalization  of  striatal  input,  output  and  intrinsic  systems  is 
comparable  to  that  in  the  rat.   The  physiological  basis  of  this  organizational 
scheme  will  be  examined  in  the  primate  by  Melvyn  Heyes  who  will  work  under  the 
direction  of  Gerfen  and  Wise  in  the  upcoming  year. 

II.  Cerebral  Physiology; 

The  second  major  area  of  research  within  the  Laboratory  of  Neurophysiology  is 
being  carried  out  by  Wise  and  involves  monitoring  the  activity  of  nerve  cells 
in  association  with  voluntary  movement.   The  data  obtained  provide  information 
about  the  mechanisms  underlying  control  of  the  least  automatic  animal  behaviors 
by  the  brain.   As  with  the  studies  of  Functional  Neuroanatomy,  those  of  Cerebral 
Physiology  may  be  divided  into  two  main  areas.   The  first  area  of  research  is 
conducted  by  Wise  and  his  colleagues  within  the  LNP,  Von  Jennings,  Andrew  Mitz 
and  Kiyoshi  Kurata,  and  is  directed  toward  higher-level  control  of  behavior. 
The  second  main  area  was  headed  by  Evarts  and  is  conducted  by  his  colleagues, 
Seth  Pullman,  Ray  Watts,  and  Jerome  Sanes.   This  work  is  also  devoted  to  a  study 
of  cerebral  physiology  as  it  relates  to  the  motor  system  but  is  more  directly 
concerned  with  the  execution  of  movement  than  its  higher-level  control.   The 
work  in  both  of  these  areas,  comprising  five  projects,  is  aimed  at  improving  the 


160 


understanding  of  specializations  and  commonalities  of  function  among  the  distinct 
cortical  fields  of  the  somatic  sensorimotor  cortex  and  the  basal  ganglia. 

One  of  the  major  goals  of  the  work  being  carried  out  in  Cerebral  Physiology  has 
been  to  improve  the  understanding  of  the  functional  significance  of  sensory 
input  to  motor  outputs,  especially  in  the  cerbral  cortex.   Only  in  recent  years 
has  it  become  clear  that  there  are  a  number  of  motor  cortical  fields  acting  along 
with  the  better-studied  primary  motor  cortex  (area  4)  in  the  control  of  sensorial- 
ly  guided  movements.   For  example,  when  a  visual  stimulus  located  at  a  particular 
point  in  space  is  the  target  for  a  visually  guided  limb  movement,  a  number  of  cor- 
tical areas  are  involved  in  different  aspects  of  the  control  of  the  limb  to  the 
target.   The  visual  information  must  be  used  to  select  patterns  of  muscle  dis- 
charge that  will  guide  the  limb  towards  that  target,  and  it  seems  likely  that 
transformations  occur  during  the  passage  of  nerve  impulses  from  visual  cortex  to 
the  premotor  cortex  and  thence  to  the  precentral  (primary)  motor  cortex,  as  well 
as  between  these  cortical  areas,  the  basal  ganglia  and  the  supplementary  motor 
cortex.   Wise  has  developed  experimental  paradigms  that  enable  him  to  examine 
the  changes  in  discharge  patterns  of  neurons  in  the  premotor,  supplementary  and 
precentral  motor  cortex  during  a  variety  of  visually  guided  movements.   Similarly, 
somatic  sensory  inputs  are  important  in  the  guidance  of  voluntary  movement,  and 
Jennings,  Pullman,  and  Watts  are  conducting  several  experiments  aimed  at  exami- 
ning the  role  of  somesthetic  input  to  precentral  motor  and  somatic  sensory  cor- 
tex.  And  since  these  sensory  inputs  are  ultimately  directed  toward  the  control 
of  motor  output,  Mitz  has  been  examining  the  efferent  organization  of  the  supple- 
mentary motor  cortex  and  the  primary  motor  cortex  with  a  modified  intracortical 
electrical  stimulation  method  that  he  has  developed.   Further,  it  is  a  major 
goal  of  the  laboratory  to  improve  the  understanding  of  these  cortical  areas  in 
relation  to  the  basal  ganglia.   Accordingly,  Pullman  and  Evarts  studied  the 
activity  of  single  neurons  in  the  putamen  and  globus  pallidus,  as  well  as  somatic 
sensorimotor  cortex.   Applying  the  lesions  learned  from  non  human  primates  to 
studies  of  the  human  motor  system  in  health  and  disease  has  been  the  goal  of  the 
fifth  project,  that  headed  by  Sanes.   Thus  the  work  on  Cerebral  Physiology  con- 
sists of  five  interrelated  projects: 

1 )  Nonprimary  Motor  Cortex  and  the  Cerebral  Control  of  Movement 

2)  Somatic  Sensory  and  Motor  Activity  of  Cortical  Cells 

3)  Efferent  Organization  of  the  Motor  Areas  of  Cortex 

4)  Cerebral  Control  of  Voluntary  Movements 

5)  Motor  Function  in  Patients  with  Neuropsychiatric  Disease 

The  work  on  Nonprimary  Motor  Cortex  is  a  combined  neuropsychological,  neuro- 
anatomical,  and  neurophysiological  approach  that  is  designed  to  provide  inform- 
ation about  the  way  in  which  frontal  lobe  nerve  cells  control  complex,  highly 
skilled  voluntary  movements  and  the  preparation  for  such  movements.   The  studies 
of  Somatic  Sensory  and  Motor  Activity  use  neurophysiological  techniques  to  ex- 
amine information  processing  in  somatic  sensory  and  motor  areas,  and  probe  the 
functional  significance  of  somatic  sensory  inputs  to  motor  cortex.   These  studies 
involve  a  detailed  examination  of  single  cell  activity  in  the  cerebral  cortex  as 
well  as  muscle  activity  in  response  to  perturbations  of  the  limb.   The  work  on 
Efferent  Organization  seeks  to  apply  sophisticated  electroanatomical  techniques 
to  the  areas  of  motor  cortex  for  which  several  questions  remain  concerning  their 
topographic  organization.   The  study  of  the  Cerebral  Control  of  Voluntary  Move- 
ments  includes  direct  study  of  cell  activity  in  the  basal  ganglia  as  well  as  the 


161 


cerebral  cortex.   Thus  this  project  complements  the  above-mentioned  projects  in 
that  it  serves  as  a  bridge  between  the  work  on  cortical  sensorimotor  areas  and 
the  work  on  the  anatomical  and  chemical  organization  of  the  basal  ganglia.   The 
fifth  line  of  investigation.  Motor  Function  in  Patients  with  Neuropsychiatric 
Disorders  attempts  to  examine  the  interactions  between  central  motor  programming 
of  movements  and  afferent  inputs.   This  project  also  seeks  to  learn  how  normal 
motor  control  functions  are  disordered  in  patients  with  cerebellar  disease. 

Nonprimary  Motor  Cortex  and  the  Cerebral  Control  of  Movement:   This  work,  and 
that  of  the  next  three  projects  described,  involves  utilization  of  single  neuron 
recording  and  operant  conditioning  techniques  in  behaving  monkeys  to  study  corti- 
cal and  basal  ganglia  mechanisms  underlying  voluntary  movement.   One  general  ob- 
jective of  this  project  is  an  improved  understanding  of  the  organization  of  the 
entire  motor  cortex  and  its  role  in  the  cerebral  control  of  movement.   The  entire 
motor  cortex  includes,  in  addition  to  its  "core,"  the  MI  cortex,  a  surrounding 
neocortical  "belt"  containing  two  or  more  representations  of  the  motor  periphery, 
namely  the  premotor  cortex  and  supplementary  motor  cortex.   This  project  is  de- 
voted to  gaining  a  better  understanding  of  the  cortical  fields  involved  in  the 
sensory  guidance  of  movements  and  the  preparation  for  motor  acts. 

The  initial  focus  of  Wise's  study  of  the  nonprimary  motor  cortex  is  the  premotor 
cortex  (PM).  In  view  of  recent  anatomical  findings  linking  another  nonprimary 
motor  field,  the  supplementary  motor  cortex  (Mil),  to  the  basal  ganglia,  increas- 
ed effort  will  be  directed  toward  this  cortical  field  in  the  immediate  future. 
In  this  work,  monkeys  are  conditioned  to  make  a  variety  of  visually  guided  limb 
movements.   The  monkey  is  given  a  visual  cue  that  serves  as  an  instruction  for 
the  next  movement  to  be  rewarded.   However,  the  monkey  is  not  allowed  to  execute 
the  movement  at  the  time  the  instruction  is  delivered,  but  instead  must  delay  its 
response  until  a  trigger  stimulus  is  presented  several  seconds  later.   Thus, 
neuronal  activity  can  be  examined  during  a  period  when  the  monkey  is  set  to  make 
a  particular  movement,  but  must  refrain  from  executing  the  movement. 

The  results  of  this  study  have  provided  the  first  clear  demonstration  of  "set- 
related"  (in  contrast  to  movement-related)  activity  in  premotor  cortex.   It  was 
found  that  whereas  many  neurons  in  premotor  cortex  markedly  change  activity  be- 
fore the  onset  of  a  voluntary  movement,  a  substantial  population  of  neurons  are 
clearly  related  to  planned  motor  activity.   Several  experiments  have  been  and 
are  being  designed  to  further  test  this  proposition  and  to  elucidate  the  prop- 
erties of  these  neurons  under  a  variety  of  experimental  conditions.   Recent 
evidence  confirms  the  role  of  the  set-related  neurons  in  higher-order  aspects 
of  behavioral  control  and  suggests  additional  roles  for  some  of  these  cells 
in  anticipation  of  predictable  environment  events.  Future  work  will  be  directed 
toward  a  study  of  the  topographic  organization  of  PM  and  testing  hypotheses 
concerning  the  functional  distinctions  between  cerebellum-related  premotor 
cortex  and  the  basal  ganglia-related  supplementary  motor  cortex. 

Somatic  Sensory  and  Motor  Activity  of  Cortical  Cells:   This  phase  of  the  work 
involves  a  study  of  the  role  of  sensory  inputs  to  the  cerebral  cortex  in  the 
control  of  motor  behavior  in  primates.   Work  by  Jennings  during  the  past  year 
has  involved  a  detailed  examination  of  muscle  activity  (EMG)  in  relation  to 
perturbations,  such  as  stopping  limb  movements.   Parallel  investigations  have 
been  completed  concerning  single-unit  activity  in  the  primary  motor  and  somato- 
sensory cortex  during  the  same  behavioral  conditions.   These  experiments  should 

162 


provide  important  clues  concerning  the  role  of  peripheral  inputs  to  MI  in  the 
initiation  and  control  of  movement. 

Much  work  in  the  past  year  has  been  devoted  to  an  analysis  of  the  neural  and 
muscular  events  that  follow  the  interruption  of  a  voluntary  movement.   It  has 
been  found  that  cortical  and  EMG  responses  to  perturbation  signal  the  distance 
remaining  to  the  target  of  the  movement.   Thus,  the  further  from  its  target  a 
movement  is  interrupted,  the  larger  is  the  cortical  and  EMG  response  caused 
by  the  perturbation.   Since  larger  responses  will  lead  to  faster,  larger  limb 
movements  as  soon  as  the  interruption  ceases,  the  data  suggest  that  the  target 
is  acquired  faster  (than  it  would  otherwise  be)  as  a  result  of  the  sensorially 
mediated  response  to  perturbation.   These  findings  and  conclusions  add  new  in- 
sight into  the  mechanisms  of  goal  achievement  in  volitional  movement. 

Another  approach  to  the  study  of  sensory  inputs  to  the  cortex  has  been  taken  by 
Evarts,  Pullman  and  Watts.   They  examined  the  responses  of  single  cortical  neu- 
rons to  cerebellar  inputs  and  peripheral  sensory  inputs  during  a  period  of  re- 
laxed immobility.   They  found  that  neurons  in  the  superficial  layers  of  the  cor- 
tex receive  a  stronger  somatic  sensory  and  cerebellar  drive  than  neurons  in  the 
deeper  cortical  layers,  cells  identified  as  cortical  output  neurons.   Thus, 
during  periods  of  relaxed  immobility  there  appears  to  be  a  gating  of  sensory 
and  cerebellar  information  between  the  point  at  which  it  enters  the  cortex,  the 
superficial  layers,  and  the  point  at  which  it  exits  the  cortex,  the  deep  layers. 
The  same  fundamental  findings  were  obtained  in  the  primary  motor  cortex  and  the 
primary  somatic  sensory  areas.   Future  studies  will  be  directed  toward  an  analy- 
sis of  the  behavioral  situations  in  which  the  relative  sensitivity  of  superficial 
and  deep  cortical  neurons  to  cerebellar  and  peripheral  input  changes  from  that 
seen  during  relaxed  immobility. 

Efferent  Organization  of  the  Motor  Areas  of  Cortex;   Despite  decades  of  effort 
directed  at  "mapping"  the  motor  areas  of  cerebral  cortex,  several  questions 
remain.   Many  of  the  questions  can  now  be  profitably  approached  through  a  multi- 
disciplinary  attack  including  variations  of  an  electroanatomical  technique, 
intracortical  microstimulation,  and  its  combination  with  modern  neuroanatomical 
methods.   The  initial  studies  on  the  supplementary  motor  area  (Mil)  with  this 
approach  have  already  shed  new  light  on  its  organization.  In  contrast  to  recent- 
ly published  studies  that  were  interpreted  as  showing  that  Mil  lacks  a  somato- 
topic  pattern  of  organization,  work  by  Mitz  and  Wise  has  demonstrated  a  clear 
somatotopic  organization,  one  that  includes  a  representation  of  the  extraocular 
as  well  as  somatic  musculature.   Future  study  will  be  directed  to  determining 
the  precise  cytoarchitectonic  correlate  of  Mil  as  defined  by  the  modified  elec- 
troanatomical techniques,  and  determining  the  nature  of  its  efferent  connectivity 
with  the  basal  ganglia. 

Cerebral  Control  of  Voluntary  Movement:  The  work  on  the  cerebral  cortex  associa- 
ted with  this  project  has  already  been  discussed  in  the  section  on  somatic  sen- 
sory and  motor  activity  in  cortical  cells  (see  above).   The  aspect  of  this  pro- 
ject concerned  with  the  basal  ganglia  is  of  particular  significance  in  connection 
with  clinical  disorders,  since  it  provide  clues  for  discovering  how  toxic  sub- 
stances produce  Parkinson's  disease  and  how  therapeutic  agents  exercise  benefi- 
cial effects  in  Parkinson's  disease.   It  is  becoming  increasingly  well-documented 
that,  in  addition  to  the  motor  control  function,  the  basal  ganglia  play  an  im- 
portant role  in  cognitive  function  and  ellucidation  of  its  role  in  either  move- 
ment or  cognition  will  promote  understanding  of  both. 

163 


Previous  neurophysiological  studies  in  monkeys  have  shown  that  in  one  part  of 
the  basal  ganglia,  the  putamen,  neurons  related  to  voluntary  movement  are  silent 
in  the  absence  of  movement  and,  conversely,  that  tonically  active  putamen  neurons 
appear  unrelated  to  movement.   Work  in  the  LNP  by  Evarts  and  Pullman  has  confirm- 
ed that  tonically  active  putamen  neurons  are  unrelated  to  body  movements  per  se, 
but  has  shown  that  such  neurons  may  exhibit  highly  reliable  responses  to  external 
events.   An  auditory  stimulus  (a  click)  elicited  short-latency  responses  in  this 
type  of  putamen  neuron  when  the  click  was  a  cue  for  juice  delivery  and  its  con- 
sumption, but  such  a  stimulus  failed  to  elicit  responses  when  juice  delivery  had 
repeatedly  failed  to  follow  the  sound.   Tonically  active  putamen  neurons  that 
had  been  responsive  to  the  click  lost  their  responses  within  a  few  repetitions 
of  the  unrewarded  click,  showing  that  the  characteristic  responses  of  these  cells 
to  the  click  depended  on  the  "set"  of  the  animal  towards  consumption  of  the  re- 
ward.  Though  dependent  on  set,  the  responses  in  the  tonically  active  neurons 
were  not  related  to  licking  movement  per  se,  i.e.  the  cell  did  not  change  its 
discharge  rate  in  relation  to  movements  not  preceded  by  the  auditory  stimulus. 
These  neurons  responded  to  the  click  with  single  impulses  well  in  advance  of 
the  first  movements  in  a  sequence  of  licking  movements  and  showed  no  apparent 
relation  to  the  subsequent  successive  licks.   By  contrast,  typical  movement-re- 
lated neurons  had  bursts  of  discharge  with  each  of  the  series  of  self-paced  arm 
movements  or  licking  movements. 

Tonically  discharging  putamen  cells  with  set-dependent  responses  were  observed 
throughout  the  putamen,  and  unlike  the  movement-related  neurons  they  appeared 
to  be  independent  of  any  somatotopic  organization.   It  may  therefore  be  inferred 
that  the  single  impulses  evoked  in  tonically  active  cells  about  60  ms  after  the 
click  resulted  in  a  synchronous  event  within  a  large  part  of  the  putamen.   These 
findings  point  the  way  for  neurochemical  and  and  morphological  identification 
of  the  tonically  active  putamen  neurons  and  a  further  analysis  of  their  set- 
dependent  responses. 

All  of  the  studies  on  cerebral  physiology  are  higly  dependent  on  computer  tech- 
nology and  increasing  computational  capabilities  open  several  new  possibilities 
for  neurophysiological  research.   Karl  Arrington,  of  the  LNP  support  staff,  has 
developed  within  the  past  year  a  new  neurophysiological  analysis  program  that 
allows  user  interaction  in  a  detailed  qualitative  and  quantitative  analysis  of 
both  single-unit  data  and  analog  signals  associated  with  subject  behavior. 

Motor  Function  in  Patients  with  Neuropsychiatric  Disorders:  Sanes  and  his  col- 
leagues have  recorded  muscle  activity  and  kinematics  of  limb  position  while  hu- 
man subjects  manually  match  a  target  display  with  either  a  rapid  or  slow  movement 
or  maintain  postures  when  limb  position  is  passively  changed.   Sanes  has  found 
that  large  movements  can  be  made  accurately  in  the  presence  or  absence  of  visual 
feedback  concerning  limb  position  after  disturbances  of  the  subject's  movements, 
and  after  changes  in  sensory  input.   However,  fine,  small  movements  are  highly 
dependent  on  an  uninterupted  task  performance.   These  normal  subjects  can  be  com- 
pared in  motor  performance  with  patients  with  cerebellar  disorders  and  peripheral 
sensory  neuropathy.   Sanes  found  that  patients  with  peripheral  sensory  neuropa- 
thy have  a  severely  impaired  sense  of  muscular  effort,  i.e.  they  cannot  perceive 
how  much  force  they  are  generating  with  their  muscles.   Together,  the  studies 
emphasize  the  importance  of  somatic  sensory  inputs  in  the  guidance  of  fine  move- 
ments or  force  adjustments  and  suggest  that  the  neural  representation  of  motor 
commands  degrades  quickly  in  the  absence  of  peripheral  feedback.   With  the  de- 


164 


parture  of  Sanes  from  the  LNP,  this  aspect  of  the  program  will  be  curtailed, 
although  we  anticipate  continued  collaboration  with  Sanes  in  his  new  position 
in  the  NINCDS/IRP. 

III.  Unit  on  Neurobiology; 

The  goal  of  the  research  activity  of  Tasaki,  in  the  Unit  on  Neurobiology,  is 
to  elucidate  the  nature  of  synaptic  and  sensory  processes  in  the  nervous  system 
by  analyzing  mechanical,  thermal  and  optical  signs  of  excitation.   Excitatory 
processes  in  the  frog  spinal  cord  and  retina  were  examined  by  using  piezoelectric, 
pyroelectric  and  special  optical  detectors  developed  or  devised  in  the  laboratory. 

In  the  spinal  cord,  an  increase  in  the  turbidity  of  the  dorsal  column  evoked  by 
the  arrival  of  afferent  nerve  impulses  was  detected.  A  rise  in  the  temperature 
was  also  demonstrated.  Attempts  were  made  to  show  changes  in  the  absorbance  of 
cytochromes  in  the  spinal  cord;  but  technical  difficulties  associated  with  this 
study  have  not  yet  been  solved. 

Tasaki ' s  investigation  into  the  sensory  and  synaptic  processes  in  the  frog  eye 
began  with  a  successful  demonstration  of  mechanical  responses  of  the  retina  to 
brief  light  stimuli.   He  found  that  the  dendrites  of  the  amacrine  and  ganglion 
cells  exhibit  a  contractile  motion  when  the  dark-adapted  eye  is  exposed  to  brief 
light  pulse.   A  sign  of  swelling  of  the  outer  segment  of  the  rods  was  recorded. 
Using  a  thermal  detector  constructed  with  polyvinylidene  fluoride,  the  sequence 
of  events  in  the  retina  initiated  by  a  brief  light  pulse  was  analyzed  by  taking 
heat  production  as  an  index.   A  detailed  analysis  of  these  thermal  responses  of 
the  retina  is  now  completed  and  should  shed  light  on  the  mechanisms  of  normal 
nerve  cell  function  in  the  CNS. 

IV.  Outlook; 

The  foregoing  sections  of  this  summary  have  described  the  major  areas  of  re- 
search within  the  Laboratory  of  Neurophysiology  (LNP).   Much  of  this  research  is 
newly  undertaken  and  has  involved  recently  recruited  scientists  whose  skills  and 
training  equip  them  to  take  maximum  advantage  of  the  many  promising  neurobiologi- 
cal  techniques  that  have  developed  in  the  last  decade.   For  instance,  Herkenham 
and  Wise  have  each  incorporated  two  new,  younger  workers  in  their  groups  within 
the  past  year.   The  LNP  has  evolved  from  a  laboratory  that  was  almost  exclusively 
oriented  toward  the  electrophysiology  of  the  motor  system  ten  years  ago  to  one 
that  now  applies  a  number  of  interdisciplinary  neuroscience  techniques  to  the 
study  of  general  brain  organization  and  mechanisms  of  higher  brain  functions. 
Our  work  is  now  directed  to  questions  much  broader  in  scope  than  those  tradition- 
ally addressed  by  specialists  in  the  motor  system.   Much  of  the  thinking  behind 
the  approach  of  the  laboratory  to  the  study  of  these  higher  brain  functions  was 
elaborated  in  a  monograph  entitled  Neurophysiological  Approaches  to  Higher  Brain 
Functions,  published  in  1984.  This  monograph  was  written  by  Evarts  and  Wise,  in 
collaboration  with  Yoshikazu  Shinoda,  a  former  member  of  the  LNP,  to  strengthen 
the  conceptual  foundations  for  neurophysiological  investigations  of  the  most 
flexible  animal  behaviors.   The  evolution  toward  more  general  problems,  approach- 
ed with  a  broader  methodology,  is  still  taking  place,  and  the  formulation  of 
new  experiments  that  focus  on  mechanisms  of  higher  brain  function  is  now  begin- 
ning to  yield  results.   Future  work  employing  fundamental  approaches  to  questions 


165 


about  higher  brain  function  can  be  expected  to  yield  significant  insight  into 
the  normal  functions  of  the  brain,  and  this  work  will  ultimately  be  of  value  in 
understanding  disorders  of  CNS  function. 


166 


Annual  Report  of  the  Research  Services  Branch 

National  Institute  of  Mental  Health 

National  Institute  of  Neurological  and  Communicative  Disorders  &  Stroke 

October  1,  1984  -  September  30,  1985 

The  Research  Services  Branch  (RSB)  provides  broad  technical  support  for  the  Intramural 
Research  Programs  of  NIMH  and  NINCDS  through  (1)  research  and  development  in  advanced 
biomedical  instrumentation  techniques  and  systems;  (2)  evaluation,  specification  and  management 
of  computer  systems;  (3)  direction  of  a  program  of  laboratory  animal  medicine  and  care  (NIMH 
only);  and  (4)  provision  of  other  technical  services  in  support  of  the  research  program. 

The  Branch  is  comprised  of  three  sections: 

1.  Section  on  Instrumentation  and  Computers 

2.  Section  on  Laboratory  Animal  Medicine  and  Care 

3.  Section  on  Research  Support  (St.  Elizabeth's  Hospital) 

SECTION  ON  INSTRUMENTATION  AND  COMPUTERS 

The  Section  on  Instrumentation  and  Computers  (ICS)  provides  technical  support  for 
investigators  by  (1)  assessing  the  instrumentation  and  computer  needs  of  the  investigator;  (2) 
designing,  developing  and  constructing  special-purpose  electronic  and  mechanical  instrumentation 
and  systems  not  commercially  available;  and  (3)  designing,  specifying  and  managing  laboratory 
computer  systems  for  data  acquisition  and  processing. 

Additional  services  provided  by  the  Section  include  consultation  on  measurement 
techniques,  signal  processing,  noise  and  electro-magnetic  interference  in  data  measurement 
systems,  and  equipment  purchases.  Several  formal  and  informal  courses  for  investigators  are 
taught  by  ICS  personnel;  topics  include  electrical  circuit  theory,  operational  amplifier  applications, 
digital  logic  design,  and  computer  applications. 

Due  to  manpower  limitations  and  economic  considerations,  the  Section  is  unable  to  provide 
the  following  services:  repair  of  commercial  instruments,  duplication  of  off-the-shelf  commercially 
available  equipment,  and  fabrication  of  non-instrument  items  (shelves,  bookcases,  etc.). 

When  an  investigator  requires  the  services  of  the  Section,  he  first  meets  with  the  Section 
Chief  and  other  personnel  as  needed  to  discuss  his  requirements.  On  the  basis  of  this  meeting,  a 
decision  is  made  as  to  whether  ICS  will  take  on  the  project.  If  a  commercially  produced 
instrument  will  satisfy  the  investigator's  requirements,  he  is  advised  to  purchase  it.  If  custom 
instrumentation  is  needed,  ICS  wiU  accept  the  project  unless  we  lack  the  appropriate  expertise,  or 
our  current  work  backlog  is  excessive.  In  these  cases  the  project  may  be  contracted  to  a  private 
firm,  or  the  investigator  may  be  directed  to  the  Biomedical  Engineering  and  Instrumentation 
Branch  (BEIB). 

When  the  Section  Chief  or  the  Assistant  to  the  Branch  Chief  agree  to  accept  a  project,  the 
investigator  submits  a  standard  work  request  form  (available  from  ICS),  signed  by  his  Lab  Chief. 
This  form  will  state  the  nature  of  the  instrument  or  service  requested,  and  wUl  contain  as  many 


167 


details  and  specifications  as  the  investigator  can  provide. 

The  project  is  then  assigned  to  an  engineer,  who  will  confer  with  the  investigator  to 
formulate  a  set  of  engineering  specifications  and  a  timetable  and  cost  estimate  for  the  project.  The 
ICS  does  not  charge  for  services,  but  the  investigator  will  be  billed  for  the  cost  of  the  components 
used.  Upon  delivery  of  the  completed  instrument,  a  memo  is  sent  to  the  investigator  Usting  the 
component  costs  and  asking  permission  to  have  the  Administrative  Officer  transfer  funds  from  his 
CAN  to  the  Section's  CAN. 

TNSTRI JMENTATION 

The  Section  has  a  staff  of  five  engineers  and  five  technicians  to  design,  develop,  and  fabricate 
electronic  and  mechanical  instruments.  The  major  effort  is  in  the  production  of  electronic 
instruments  for  basic  neurophysiological  research,  and  for  clinical  studies  involving  affective 
disorders.  The  following  are  brief  descriptions  of  representative  projects,  chosen  from  a  total  of 
238  projects  undertaken  tiiis  year. 

(1)  Patient  Activity  Monitoring  System.  The  Section  has  continued  to  develop  the  Patient 
Activity  Monitor  (PAM)  and  the  support  hardware  and  software  which  forms  the  system. 

(a)  Monitor.  The  current  version  of  the  PAM  has  a  memory  capacity  of  1024 
locations  and  is  in  its  third  year  of  production.  Fabrication,  testing,  and  calibration  of  a  set  of  60 
units  begun  last  year  has  been  completed  and  another  set  of  30  monitors  is  now  in  the  final  phase 
of  fabrication.  Most  of  the  older  versions  of  tiie  PAM  have  now  been  retired.  Approximately  100 
monitors  are  in  use,  with  the  Section  providing  battery  changes  and  repairs  as  needed.    The 
injection-molded  plastic  case  developed  for  the  monitor  last  year  is  now  in  wide  use.  Early  field 
tests  revealed  problems  with  static  electricity  interference.  Coating  the  exterior  of  the  case  and  end 
cap  with  a  metalic  conductive  paint  eliminated  this  problem.  Compared  to  tiie  older  metal  case,  the 
plastic  case  reduces  the  overall  weight  of  the  monitor  by  30%,  provides  a  more  water-resistant 
cover,  and  is  less  expensive  and  easier  to  produce. 

(b)  Telecommunications.    A  remote  readout  terminal  for  tiie  patient  activity 
monitor  has  been  developed  and  is  now  ready  for  field  evaluation.   The  terminal  has  tiie  capability 
to  be  used  in  the  home  of  a  subject  or  in  an  office/laboratory  environment.  Using  its  internal 
modem,  the  terminal  first  dials  a  remote  computer  facility,  then  reads  the  contents  of  an  activity 
monitor,  sends  the  activity  data  over  the  phone  lines,  clears  tiie  monitor's  memory,  and  hangs  up. 
Initially,  the  VAX  computer  managed  by  the  Section  will  be  the  remote  (recipient)  computer. 
Software  has  been  written  to  reformat  the  data  from  the  VAX  into  standard  PDP-1 1  activity  files 
for  further  analysis. 

(c)  Computer  Support.  A  PDP-1 1/23  minicomputer  with  a  20  Mbyte  hard  disk  is 
being  prepared  as  a  second  PAM  readout  station  for  PAM  users  in  Bldg.  10.  This  powerful 
system  will  handle  all  of  the  PAM  software,  eliminating  the  present  dependency  on  the  Bldg.  36 

1 1/34  system  for  some  of  the  more  complex  analysis  programs.  Software  is  also  being  developed 
to  reformat  activity  files  to  allow  direct  transfer  into  WYLBUR  files  for  statistical  analysis.  A  new 
PAM  computer  interface  using  the  RS-232-C  serial  data  format  is  under  development.  This 
interface  will  allow  an  inexpensive  personal  computer  with  a  serial  port  to  serve  as  a  readout 
device.  The  Section  will  develop  software  for  the  Apple  Macintosh  personal  computer  to  support 
this  readout  method.  This  combination  will  allow  individual  laboratory  readout  stations  and  will 
also  facihtate  collaboration  with  groups  outside  the  IRP. 


168 


(2)  Neurophysiological  Data  Preprocessor.    A  microprocessor  system  has  been  developed 
to  replace  the  custom  logic  circuitry  presently  used  by  the  Laboratory  of  Neurophysiology  Data 
Acquisition  System.  THs  preprocessor  records  the  times  of  occurrences  of  64  different  events 
and  eight  different  pulses.  This  information  is  transmitted  to  the  main  processor  (a  PDF- 1 1 
minicomputer)  through  a  parallel  interface  and  the  information  is  coded  in  such  a  form  as  to  ensure 
compatibility  with  existing  software  that  is  used  for  analysis  and  display  of  the  data.    The 
preprocessor  decreases  response  time  to  events  and  pulses  and  it  frees  the  main  processor  for 
experiment  control.  Following  complete  lab  testing  of  the  first  unit  fabricated  last  year,  the  Section 
constructed  five  additional  units  for  the  LNF/NIMH  and  two  units  for  the  LNLC/NEMCDS. 

(3)  Rodent  Activity  System.   A  system  was  completed  and  is  currently  being  used  to 
monitor  the  running  wheel  activity  of  72  rodents.  Running  wheel  activity  is  important  in  circadian 
rhythm  studies  involving  light  response  to  free-running  hamsters.  Six  surplus  tissue  culture 
boxes  were  modified  to  hold  12  cages  each  (2  on  each  of  6  shelves).  The  wheel  activity  in  each 
cage  is  recorded  with  a  simple  microswitch  and  interface  logic  controlled  by  a  16-bit  Plessey  6100 
laboratory  computer.  In  addition  to  tlie  72  running  wheels,  36  fluorescent  lights  present  a 
programmed  light  stimulus  to  each  shelf  (two  cages).  The  computer  monitors  each  light  by  means 
of  a  photodetector  to  provide  verification  that  the  lights  were  on  at  the  proper  time.  The  activity 
and  light  data  are  stored  on  a  10  megabyte  Winchester  disk  and  also  on  two  1  megabyte  floppy 
disks.  In  addition,  data  from  aU  108  channels  is  continuously  plotted  in  15-minute  intervals  on  a 
printer/plotter  in  a  strip  chart  format.  The  data  for  each  cage  is  stored  in  continuous  files  to  permit 
analysis  using  existing  activity  profile  programs. 

(4)  Microprocessor-based  Rotometer.    A  third  generation  animal  rotation  monitor  was 
completed  that  utilizes  an  inexpensive  microcomputer  board  to  determine  the  clockwise  or 
counter-clockwise  rotations  of  one  to  four  rodents  in  cylindrical  cages  and  to  hold  this  data  for 
input  into  the  serial  port  of  a  Macintosh  computer.  The  computer  board  uses  the  16-bit  Intel  8088 
microprocessor  and  has  an  on-board  BASIC  interpreter  for  fast  program  development.  A  second 
logic  control  board  designed  by  ICS  collects  the  rotational  data  using  FIFO  buffers  until  it  is 
processed  by  the  8088.  Software  is  being  written  which  will  use  the  capabilities  of  the  Macintosh 
to  store  the  data  on  disk,  and  to  display  the  data  in  real-time  histogram  form. 

(5)  Biotelemetry  Temperature  Measurement.  A  microprocessor-based  instrument  is  being 
developed  to  continuously  monitor  the  body  temperature  telemetered  from  laboratory  rodents. 
Data  from  an  implanted  commercial  miniature  transmitter  is  converted  by  a  standard  AM  receiver 
into  a  series  of  pulses  whose  period  is  inversely  related  to  temperature.  The  instrument  will  derive 
the  actual  temperature  values  from  a  memory  calibration  table  and  then  display  the  result  with  0. 1 
degree  centigrade  resolution. 

(6)  EEG  Amplifier  System.  A  second  32-channel  EEG  amplifier  system  has  been 
completed  for  use  in  several  ongoing  research  projects  including  topographic  brain  mapping.  The 
design  incorporates  state-of-the-art  integrated  circuit  components  and  printed  circuit  board  layouts 
to  produce  a  rehable,  compact,  low-cost-per-channel  unit.  Each  channel  consists  of  a 
preamplifier,  amplifier,  and  a  selectable  antialiasing  filter.  A  flexible  design  and  front  panel 
switches  aUow  control  over  signal  bandwidth,  monitoring  by  a  tape  recorder  and  a  16-channel 
Grass  polygraph,  and  digitizing  and  analysis  of  the  EEG  signal  by  a  computer.  A  related  project 
involved  the  design  and  construction  of  three  EEG  calibrators.    By  generating  an  8  hertz, 
100|ivolt  signal  simultaneously  on  each  of  the  32  channels,  this  device  allows  system  calibration 
and  verification  that  all  channels  are  working  prior  to  a  recording  session. 

(7)  32-Channel  Analog  Interface.    Speech  pathology  studies  will  utiUze  a  multiplexed  A/D 
converter  controlled  by  a  PDF- 1 1/73  minicomputer  to  digitize  speech,  muscle,  and  neuronal 


169 


analog  signals.  To  maximize  the  digitized  signal-to-noise  ratio,  a  compact  32-channel  analog 
conditioning  instrument  has  been  developed.  Each  channel  provides  adjustable  gain/attenuation,  a 
selectable-bandwidth  antialiasing  filter,  and  a  sample/hold  amplifier.  Each  signal  level  is  also 
displayed  on  a  10-segment  LED  VU  meter  so  that  its  amplitude  can  be  optimized  before  the  A/D 
conversion.  Printed  circuit  board  construction  and  a  modular  packaging  system  were  used  to 
simplify  fabrication  and  to  increase  reliability. 

(8)  Data  Acquisition  Computer  Interface.    A  third  generation  interface  device  for  data 
acquisition  and  control  has  evolved  in  several  IRP  laboratories.  This  instrument  provides  the 
interface  between  the  experiment  and  the  A/D  and  D/A  boards  within  a  PDP- 1 1  minicomputer. 
The  Section  has  designed  and  fabricated  six  of  these  interfaces  this  year.  A  companion  4-channel 
signal-conditioning  system  was  deUvered  with  three  of  these  units.  The  companion  instrument 
provides  four  decades  of  adjustable  gain,  selectable  high  and  low  frequency  filtering,  and 
adjustable  input  offset  capabiUty. 

(9)  Pulse  Generator  System.  A  multi-channel  timing  instrument  (pulse  generator  system) 
is  a  vital  part  of  many  neurophysiological  experiments.  Instruments  used  within  the  IRP  that  were 
purchased  about  15  years  ago  are  no  longer  manufactured  and  have  become  somewhat  unreliable. 
Newer,  commercially  available  units  lack  the  flexibility  and  convenience  of  the  older  devices.  Last 
year  ICS  designed  a  five-channel  pulse  generator  system  to  fill  this  void.  By  employing  both 
analog  and  CMOS  digital  design  techniques,  an  instrument  with  both  the  required  technical 
specifications  and  a  high  degree  of  operator  convenience  was  realized.  Six  of  these  instruments 
were  fabricated  last  year  and  an  additional  five  units  are  currently  under  construction. 

(10)  Ambulatory  Lux  Monitor.    An  ambulatory  data  acquisition  system  (Vitalog  PMS-8) 
is  being  used  to  monitor  the  temperature  of  manic  depressive  patients.  To  allow  simultaneous 
recording  of  the  ambient  light  levels  experienced  by  these  patients,  a  small,  micro-power  lux  meter 
is  being  developed  as  an  input  transducer  for  the  PMS-8.  Several  photodiode/  logarithmic 
amphfier  combinations  are  being  evaluated  to  obtain  a  five  decade  photometric  response.  The 
microprocessor  data  processing  algorithms  employed  in  the  PMS-8  and  in  the  readout  Apple  II 
computer  are  being  modified  to  collect,  convert,  and  store  the  light  intensity  data. 

(11)  Microdensitometer.    A  standard  split- viewing  Zeiss  compound  microscope  has  been 
converted  into  a  microdensitometer  that  produces  a  density  reading  from  a  small  central  spot 
(selectable  as  either  .25,  .63  or  1.6mm  dia.)  within  the  18mm  diameter  viewing  area.  A  linear 
photodiode/amplifier  combination  converts  the  light  transmission  value  within  the  spot  into  a 
proportional  voltage  for  a  microprocessor-controlled  A/D  converter.  Corresponding  to  each 
transmission  value,  a  logarithmic  density  value  is  obtained  from  a  memory  look-up  table. 
Transmission  and  density  values  are  simultaneously  displayed  and  the  density  value  may  be 
printed  to  facilitate  recording  of  numerous  successive  readings.  The  split-viewing  ability  of  the 
microscope  allows  precise  areas  on  the  autoradiographic  film  to  be  identified  by  simultaneously 
viewing  the  film  and  a  stained  slide  of  the  same  brain  slice  section. 

(12)  30-Channel  Electrode  Array  Amplifier  System.    A  complete  system  for  ampUfying 
and  processing  signals  from  a  micro-miniature  array  of  30  gold  electrodes  is  being  used  in  a 
variety  of  experiments  to  record  cultured  nerve  tissue  cell  interactions.  The  neural  signals  are 
preamplified  on  a  circuit  board  that  also  serves  as  a  base  for  the  electrode  array  holder.  The 
preamplifiers  connect  to  the  main  amplifier/discriminator  units  which  provide  three  settings  for 
overall  gains  of  100,  1,000,  or  10,000.  Each  amplified  signal  is  fed  to  a  comparator  with  a  front 
panel  adjustable  threshold  level.  The  comparator  output  triggers  a  one-shot  which  is  latched  and 
sampled  by  a  computer.  Additionally,  a  multiplexer  is  provided  to  display  the  amplified  signal, 
comparator  level,  and  one-shot  output  on  a  single  output  of  an  oscilloscope.  Use  of  printed  circuit 


170 


cards  for  both  the  preampUfier  and  amplifier  units  and  a  modular  packaging  system  greatly 
simplified  the  fabrication  and  increased  the  system  reliability. 

COMPUTERS 

Small  computers  are  ideally  suited  for  laboratory  research  in  neurophysiology  and 
psychology.  They  are  used  in  the  laboratory  for  on-line,  real-time  interactions,  process  control, 
and  data  acquisition.  Recorded  data  may  be  stored,  combined  with  other  data,  reduced 
statistically,  transferred  to  larger  computers  for  further  analysis,  transformed  for  presentation 
graphically  or  mathematically,  and  the  results  may  be  printed  or  plotted,  hicreasing  use  is  being 
made  of  the  small  computer  for  processing  the  text  of  scientific  papers  and  communications.  Data 
base  management  is  now  available  for  the  small  computer,  as  are  limited  management  information 
systems. 

Techniques  have  been  developed  for  image  processing  which  are  applicable  to  many 
diverse  experimental  systems,  ranging  from  autoradiographs  of  brain  tissue  sections  to  the 
analysis  of  two-dimensional  electrophoresis  gels. 

Larger  minicomputers,  the  so-called  super-mini's,  have  been  reduced  in  price  and  are  now 
available  for  functions  formerly  performed  by  larger  time-shared  systems.  These  systems  allow 
applications  in  modeling,  curve  fitting  and  statistical  treatment  that  would  be  prohibitively 
expensive  on  large  systems. 

Inexpensive  personal  computers  are  proving  useful  for  dedicated  appUcations.  Many 
scientists  are  developing  software  for  these  computers,  which  they  offer  to  the  scientific 
community  at  low  cost.  PCs  will  become  increasingly  useful  in  the  laboratory  and  their  potential 
should  be  exploited. 

Microcomputers  incorporated  in  the  design  of  biomedical  instrumentation  provide  a 
savings  in  design  and  fabrication  time  for  instruments,  and  a  more  flexible  system  than  one  based 
on  discrete  components. 

The  Section  on  Instrumentation  and  Computers  is  actively  involved  in  the  applications  of 
small  computers  in  the  IRP.  By  integrating  the  functions  of  biomedical  instrument  design  and 
laboratory  computer  systems  with  software  designed  specifically  for  the  research  community,  the 
Section  offers  computer  support  services  for  a  broad  range  of  scientific  disciplines. 

LABORATORY  COMPUTERS 

The  design  goal  for  the  laboratory  instrument  computer  is  to  provide  maximum  function, 
tailored  to  the  specific  experimental  design,  with  minimum  cost.  ICS  provides  consultation  on  the 
specification  and  selection  of  laboratory  computers  for  new  appUcations;  conducts  systems  studies 
in  collaboration  with  the  scientist;  and  helps  the  scientist  in  the  procurement,  installation  and 
maintenance  of  the  equipment. 

In  support  of  these  efforts,  RSB  has  maintained  two  PDP- 1 1  central  computers,  one  in 
Bldg.  36,  and  one  in  the  Clinical  Center.  The  functions  previously  provided  by  these  computers 
are  now  being  largely  obtained  by  newer  computer  systems.  The  multi-user  VAX- 1 1/750 
managed  by  RSB  in  Bldg.  36  provides  high-capacity  data  storage,  and  efficient  data  processing, 
including  graphic  functions  with  plotting  and  printing  on  a  high-resolution  laser  printer. 


171 


Additionally,  large  price  reductions  have  permitted  individual  ERP  laboratories  to  acquire  higher 
performance  minicomputer  systems  which  are  self-supporting.  Due  to  these  developments,  the 
central  PDF- 1 1/40  in  the  Clinical  Center  has  been  retired  and  the  PDF- 1 1/34  in  Bldg.  36  is  being 
replaced  with  a  more  powerful  LSI-1 1/73  with  significantly  reduced  maintenance  costs.  In 
addition  to  use  for  program  development  and  training,  the  1 1/73  will  be  equipped  as  a  video  image 
processing  system. 

TRAINING  AND  SOFTWARE  SUPPORT 

ICS  provides  training  for  the  scientist  or  support  personnel  who  will  be  programming  and 
maintaining  the  system.  Personnel  limitations  make  it  difficult  for  ICS  to  provide  complete 
programming  for  specific  individual  applications,  so  such  programming  must  be  supplied  by  the 
laboratory.  ICS  computer  personnel  are  always  available  for  consultation,  training,  and  help  in 
debugging,  as  well  as  assistance  in  the  selection  of  part-time  programmers  or  consultants. 
Commercial  software  packages  or  applications  from  other  research  labs  are  often  available,  and 
ICS  will  evaluate  such  systems. 

ICS  develops  and  maintains  a  Ubrary  of  procedures  which  are  written  specifically  for  the 
laboratory  computers  used  in  the  intramural  community.  These  procedures  are  designed  to  be 
incorporated  into  the  users'  programs.  In  addition,  ICS  will  aid  the  investigator  in  writing  the 
difficult  time  and  data  dependent  sections  of  real-time  programs.  ICS  also  develops  some 
application  programs  which  will  have  wide  use  within  one  or  more  laboratories  or  will  support 
data  acquisition  hardware  developed  by  ICS. 

PROGRAM  MAINTENANCE 

There  are  now  more  than  60  minicomputers  in  the  program;  many  of  these  systems  have 
been  in  use  for  years.  A  significant  number  of  library  procedures  and  general-purpose  application 
programs  are  used  on  these  machines.  As  experimental  protocols  develop  and  change,  software 
changes  are  often  required,  so  program  maintenance  is  a  continual  and  time-consuming  function  of 
the  Section.  This  effort  is  aided  by  structured  programming  techniques  and  standardization  of 
laboratory  computers  and  peripheral  equipment 

VAX  COMPUTER  SYSTEM 

The  Section  manages  a  multi-user  VAX- 1 1/750  computer  system  that  is  available  for  use 
by  all  investigators  in  the  IRP.  The  VAX  is  located  in  Bldg.  36,  in  space  furnished  by  the 
Laboratory  of  Cerebral  Metabolism,  NIMH.  Potential  users  in  Bldg.  36  may  request  installation  of 
hard  wired  cable  connections,  or  the  VAX  may  also  be  used  on  a  dial-up  basis. 

A  device  independent  graphics  package  (PLOTLEB)  has  been  developed  on  the  VAX  that 
permits  plots  to  be  generated  on  numerous  display  terminals  and  hardcopy  devices.  A  terminal 
emulation  program  (TEM)  was  developed  which  permits  small  PDP-1 1  laboratory  computers  to 
function  as  graphics  terminals  when  using  the  VAX.  TEM  also  supports  file  transfers  in  both 
directions.  A  similar  terminal  emulation  program  is  available  for  the  Macintosh  personal  computer. 

A  TALARIS  laser  printer  has  been  installed  on  the  VAX  which  now  permits  pubUcation 
quality  plots  and  documents  to  be  quickly  and  easily  generated.  The  PLOTLIB  graphics  package 
was  updated  to  support  the  laser  printer  and  a  program  (FPRINT)  has  been  written  to  allow 


172 


documents  incorporating  superscripts,  subscripts,  and  Greek  letters  to  be  printed  on  the  laser 
printer. 

IMAGE  PROCESSING 

The  Section  on  Instrumentation  and  Computers  maintains  a  general  purpose  image 
processing  system  consisting  of  an  Optronics  rotating  drum  film  scanner,  a  Gould/DeAnza  image 
array  processor,  and  a  PDP-1 1/60  computer.  Images  to  be  processed  may  be  obtained  by  scanning 
autoradiographs,  x-ray  fihn,  or  photographic  negatives,  or  by  using  images  generated  by  CAT  or 
ECAT  scanners.  A  camera  station  is  available  to  generate  color  hardcopy  using  Polaroid  SX-70  or 
35mm  film. 

Software  packages  that  are  easy  to  learn  and  use  have  been  developed  to  provide  an 
extensive  and  expandable  repertoire  of  basic  image  processing  functions.  Special  purpose 
functions  can  be  developed  to  meet  specific  user  requirements.  The  facility  is  useful  for  numerous 
applications  involving  evaluation  and  quantification  of  biomedical  images  .  The  two  primary 
applications  of  the  system  are  the  densitometric  analysis  of  autoradiographs  of  brain  or  tissue 
sections  and  the  analysis  of  two-dimensional  electrophoresis  gels. 

The  Section  is  developing  a  new  PDP-1 1/73  based  image  processing  system  that  will  be 
capable  of  using  these  software  packages.   This  system  will  use  a  TV  camera  for  digitizing 
images  instead  of  the  rotating  drum  film  scanner.    Unlike  the  drum  scanner  which  can  only 
digitize  transparencies,  the  TV  digitizer  will  permit  any  object  that  can  be  placed  under  a  camera  to 
be  digitized. 

PERSONAL  COMPUTERS 

The  Section  has  evaluated  Apple  Macintosh  personal  computers  for  potential  use  in  both 
scientific  and  administrative  appUcations.  The  Macintosh  was  chosen  for  its  ease  of  learning, 
advanced  design,  and  high  quality  graphics.  It  has  proven  to  be  useful  in  a  number  of  areas  and  is 
remarkably  easy  to  use. 

The  most  popular  use  of  the  Macintosh  has  been  for  scientific  word  processing.  It  has 
proven  to  be  a  very  cost-effective  alternative  to  expensive  and  inflexible  dedicated  word 
processors.  It  can  easily  produce  text  containing  equations,  Greek  letters,  superscripts,  and 
subscripts.  In  addition,  it  can  also  produce  posters  or  camera-ready  charts  for  slides.  When  used 
with  the  new  Apple  LaserWriter  printer,  print  quality  is  as  good,  or  better,  than  that  produced  by  a 
dedicated  word  processor. 

The  Macintosh  has  also  proven  useful  as  a  graphics  workstation  for  use  with  the  VAX.  An 
inexpensive  program  (VERSATERM)  allows  the  Macintosh  to  function  as  either  a  VTIOO 
compatible  full  screen  editing  terminal  or  as  a  Tektronix  4014  compatible  graphics  display 
terminal.  Both  text  and  graphics  generated  by  the  VAX  can  be  printed  on  the  Macintosh  printer. 
In  addition,  text  files  can  be  transferred  in  both  directions.  The  Macintosh  also  functions  well  as  a 
terminal  with  other  host  computers  such  as  WYLBUR,  DECSystem-10,  and  MEDLINE. 

The  Macintosh  is  being  used  in  three  Section  projects  for  low-speed  laboratory  data 
acquisition  and  control.  The  first  project  involves  presenting  stimuli  (various  words  or  geometric 
designs)  to  Alzheimer's  patients  with  recording  of  patient  responses.  A  second  project  uses  the 
Macintosh  to  control  and  collect  data  from  an  HP  8450  Spectrophotometer.  The  third  project  uses 


173 


the  Macintosh  to  log  data  generated  by  a  four-channel  rodent  rotometer  developed  by  ICS. 

MTCROPROCESSORS 

ICS  also  maintains  a  microprocessor  development  system  for  the  software  and  hardware 
development  of  microprocessor-based  instrumentation  at  both  the  chip  and  single  board  computer 
level.  The  system  currently  supports  three  common  microprocessors;  one  16-bit  processor,  and 
two  8-bit  processors.  These  microprocessors  and  their  associated  peripheral  chips  are  now 
available  in  CMOS  low  power  versions.  This  development  allows  the  design  of  both  smaller, 
more  reliable  bench  instruments  and  more  intelligent  portable  instrumentation.  The  Section  is 
evaluating  a  computer  board  which  uses  the  16-bit  processor  (Intel  8088)  and  comes  with  an 
on-board  BASIC  interpreter.  This  combination  allows  rapid  software  development  and  has 
already  proved  useful  in  low- speed  data  acquisition  applications. 


174 


ENGINEERING.  COMPUTER  AND  FABRICATION  SERVICES 

This  table  shows  the  distribution  of  the  Section's  workload  among  the  various  laboratories  and 
branches.  We  have  listed  only  the  major  users. 

LABORATORY  OR  BRANCH 

Clinical  Psychobiology,  NIMH 

Neurophysiology,  NINCDS 

Neurophysiology,  NIMH 

Experimental  Therapeutics,  NINCDS 

Medical  Neurology,  NINCDS 

Clinical  Neuroscience,  NIMH 

Biophysics,  NINCDS 

Neuropsychiatry,  NIMH 

Cerebral  Metabolism,  NIMH 

Psychology  &  Psychopathology,  NIMH 

Neural  Control,  NINCDS 

Biological  Psychiatry,  NIMH 

Neuropsychology,  NIMH 

Child  Psychiatry,  NIMH 

Molecular  Biology,  NIMH 

Neurochemistry,  NINCDS 

Cell  Biology,  NIMH 

Molecular  Biology,  NINCDS 

Molecular  Genetics,  NINCDS 

Surgical  Neurology,  NINCDS 

Neurobiology,  NINCDS 

Preclinical  Pharmacology,  NIMH 

Clinical  Science,  NIMH 

Clinical  Neuroscience,  NINCDS 

CUnical  Neurogenetics,  NIMH 

Neuropathology  &  Neuroanatomical  Sciences,  NINCDS 

*NIMH  (TOTAL) 
*NINCDS  (TOTAL) 
*NICHD  (TOTAL)  ** 

*These  figures  represent  our  total  effort;  they  include  time  for  labs  not  listed  individually. 
**NICHD  loans  the  Section  one  position,  and  is  thus  entitled  to  1700  hours  of  service. 


HOURS 

PERCENT 

2981 

14.07 

1493 

7.05 

1322 

6.24 

1251 

5.91 

1181 

5.58 

1140 

5.38 

1139 

5.38 

1030 

4.86 

992 

4.68 

987 

4.66 

909 

4.29 

817 

3.86 

765 

3.61 

702 

3.31 

404 

1.91 

362 

1.71 

321 

1.52 

308 

1.45 

301 

1.42 

287 

1.36 

269 

1.27 

231 

1.09 

228 

1.08 

152 

0.72 

138 

0.65 

114 

0.54 

12,066 

56.97 

7,830 

36.97 

1,284 

6.06 

21,180 

100.00 

175 


SECTION  ON  LABORATORY  ANIMAL  MEDICINE  AND  CARE 

The  Section  on  Laboratory  Animal  Medicine  and  Care  provides  a  comprehensive  animal 
care  and  use  program  for  the  NIMH  Intramural  Research  Program.  The  average  daily  animal 
inventory  is  3,500  animals,  including  mice,  rats,  hamsters,  guinea  pigs,  rabbits,  and  nonhuman 
primates.  The  Section  has  responsibility  for  25  rooms,  comprising  approximately  10,000  sq.ft. 
The  staff  includes  a  laboratory  animal  veterinarian,  16  laboratory  animal  care  technicians,  and  one 
part-time  secretary.  Approximately  250  ERP  investigators  are  involved  in  animal  research,  and 
depend  on  the  Section  for  animal  supervision  and  advice  on  animal  care  and  use  issues.  Specific 
LAMC  activities  include  the  following. 

Animal  programs  required  by  ADAMHA  and  NIH  regulations: 

Clinical  care  of  animals.  7  day  clinical  care  is  provided  for  all  IRP  animals. 

Animal  use  forms.  The  Section  monitors  the  documentation  and  approval  process  for 
animal  usage. 

NIH  "Guide  for  the  Care  and  Use  of  Laboratory  Animals".  The  Section  is  responsible  for 
assuring  IRP  compliance  with  the  NIH  regulations  in  the  guide. 

Animal  facility  site  visits.  Formal  site  visits  are  conducted,  and  appropriate  documentation 
of  findings  is  provided. 

Animal  Research  Committee  meetings.  The  Section  is  responsible  for  assuring  that  the 
Committee  meets  regularly,  and  for  providing  fuU  documentation  of  the  deliberations. 

Animal  facility  accreditation.  A  plan  for  AAALAC  accreditation  of  the  faciUties  at  NIH,  St. 
EUzabeths,  and  Poolesville  is  being  developed. 

Surgical  facilities.  The  Section  provides  a  fully  equipped  surgical  facility,  and  assists 
investigators  with  anesthesia  and  surgical  techniques. 

Experimental  rooms.  The  Section  provides  and  maintains  several  nonhuman  primate 
experimental  rooms. 

Clinical  records.  A  system  of  maintaining  clinical  records  for  all  experimental  animal 
subjects  is  being  developed. 

Employee  health  system.  The  Section  maintains  a  system  for  monitoring  the  health  of 
employees  who  have  regular  contact  with  animals. 

Animal  room  sanitation.  Adequate  sanitation  of  all  animal  facilities  must  be  provided. 

Provide  attending  veterinary  services  for  St.  Elizabeths  and  Poolesville  facilities. 

Prepare  annual  FDA  report  of  animal  usage. 

Prepare  Annual  Report  of  Research  Facility  as  required  by  ADAMHA. 

Provide  space  for  storage  of  animal  care  equipment. 


176 


Other  programs  and  activities: 

Animal  and  space  inventory  system.  Define  equitable  distribution  of  animal  holding  space, 
and  monitor  usage  by  maintaining  a  comprehensive  and  current  inventory  of  animal  holdings. 

Animal  ordering  system.  Maintain  a  centralized  system  for  ordering  animals. 

Provide  a  stable  source  of  supply  of  nonhuman  primates. 

Provide  adequate  security  for  the  animal  facilities,  to  guard  against  unauthorized 
intrusions. 

Maintain  membership  and  active  involvement  in  the  following  committees:  NIH  Animal 
Research  Committee;  Interagency  Animal  Research  Committee;  ADAMHA  Animal  Research 
Committee;  and  BID  Veterinarians  Committee. 


SECTION  ON  RESEARCH  SUPPORT  rST.  ELIZABETHS  HOSPITALS 

The  Section  on  Research  Support  (SEH)  consists  of  eight  individual  functions  (animal 
care,  Ubrary,  instrumentation,  graphics,  electronics,  glassware,  photography  and  receiving) 
providing  support  services  for  the  biopsychiatric  and  biomedical  research  laboratories  and 
administrative  staff. 

The  animal  care  unit  provides  housing,  maintenance  and  post-surgical  care  for  3,200 
research  animals  ranging  from  rodents  to  nonhuman  primates.  Recently  completed  improvements 
and  modifications  to  the  animal  facility  are  expected  to  result  in  AAALAC  accreditation  being 
awarded  in  the  near  future. 

The  glassware  unit  processes  an  average  of  151,000  pieces  of  apparatus  yearly.  The 
library  maintains  13,000  volumes  and  journals  and  is  the  focal  point  for  literary  resources  for  both 
NIMH  and  SEH  medical  staff. 

The  electronics  and  instrumentation  shops  were  combined  to  allow  for  greater  flexibility 
and  integration  of  job  skills  providing  a  wider  service  offering  while  decreasing  the  elapsed  time 
from  submission  to  job  completion. 

The  graphics  and  photography  units  continue  to  perform  in  an  exemplary  fashion, 
producing  the  highest  quality  professional  work  product  for  over  200  publications  and  scientific 
presentations  this  year. 

The  diversified  and  often  unique  requests  for  services  present  an  ongoing  challenge  to  the 
talents  of  the  support  team  members  who  have  provided  imaginative  and  creative  solutions  to 
research  demands. 


177 


Report  of  the  Associate  Director  for  Research  at  Saint  Elizabeths  Hospital 

and  Chief  of  the  Neuropsychiatry  Branch,  Intramural  Research  Program, 

National  Institute  of  Mental  Health 

During  the  past  reporting  year  the  Adult  Psychiatry  Branch  changed  its  name. 
We  are  now  the  Neuropsychiatry  Branch.  Our  new  name  better  reflects  the  work 
performed  by  our  group.  Enconpassed  in  the  Neuropsychiatry  Branch  is  an  array 
of  bionedical  research  integrating  neurochemistry,  neuropsychiatry,  urmunology, 
psychopharmacology,  neuroanatomy  and  neurophysiology.  These  efforts  fit  into 
the  interface  between  psychiatry  and  neurology  that  we  believe  will  be 
important  for  understanding  sane  of  psychiatry's  major  problems.  The  multiple 
skills  and  talents  of  our  researchers  coordinate  these  interrelated 
subspecialties  in  their  investigations  of  the  etiologies,  mechanisms  and 
treatment  of  the  schizophrenia  syndrotie  and  the  major  psychiatric  disorders  of 
aging. 

When  our  name  changed  officially,  the  creation  of  sections  within  the  Branch 
became  official  as  well.  William  Freed,  Ph.D.  is  now  Chief  of  our  Section  on 
Preclinical  Neurosciences,  Joel  Kleirman,  M.D.,  Ph.D.  is  now  Chief  of  our 
Section  on  Clinical  Brain  Studies,  and  Daniel  Weinberger,  M.D.,  is  now  Chief  of 
our  Section  on  Neuropsychiatry  and  Neuro  Behavior.  The  sections  have  been 
organized  and  functioning  for  almost  a  full  year  and  have  added  substantially 
to  the  smooth  running  and  productivity  of  our  work. 

Other  administrative  changes  have  also  taken  place.  Mr.  Richard  Staub  has 
become  our  new  Administrative  Officer,  filling  the  vacancy  left  by  Carl 
Pergler.  Mrs.  Mollie  Strotkamp,  after  ten  years  as  my  secretary,  has  moved 
across  the  hall  into  the  position  of  Administrative  Supervisor,  succeeding  Mrs. 
Cathy  Bowie.  And  although  Mollie  is  badly  missed,  Mrs.  Nancy  Bryant,  her 
successor,  is  doing  an  excellent  job  and  learning  quickly.  Throughout  the 
transition,  Mrs.  Evan  DeRenzo  seemed  to  be  everywhere  at  once,  doing  the  work 
of  about  ten  people.  Special  praise  goes  also  to  Ms.  Theresa  Hoffman  who  types 
as  if  she  has  a  dozen  arms  and  always  has  a  kind  word,  even  the  night  before 
APA  abstracts  are  due.  Also  a  warm  welcome  to  Ms.  Gail  Miller. 

But  just  because  our  internal  disruptions  seem  to  be  settling  down,  we  have  no 
reason  to  be  conplacent.  Vfe  continue  to  swim  in  the  midst  of  currents  of 
other's  comings  and  goings.  Fortunately  Dr.  Goodwin  has  decided  to  stay  at  the 
NIMH,  rather  than  go  to  Yale.  Dr.  Costa,  who  has  been  a  vital  part  of  the 
Intramural  Program  at  Saint  Elizabeths  Hospital,  has  been  offered  an 
exceptional  position  in  industry  in  association  with  Georgetown  University. 
While  on  a  very  personal  basis  I  hope  that  Dr.  Costa  stays  with  us,  as  I  write 
this  we  remain  unsure. 

In  large  part  because  of  the  planned  District  of  Columbia  takeover  of  Saint 
Elizabeths  Hospital,  the  plans  to  move  us  to  the  main  NIH  campus  have  been  more 
actively  discussed  than  at  any  previous  time.  Although  optimists  say  it  might 
be  accomplished  in  as  short  as  three  years,  realistic  appraisal  appears  to  be 
at  least  five.  While  these  more  distant  plans  go  on,  I  am  very  pleased  to 
report,  the  US  Office  of  Management  and  Budget  has  approved  expenditure  of  the 
necessary  funds  to  renovate  our  building  to  proper  Life  Safety  standards, 
ensuring  the  safety  and  welfare  of  our  patients  and  staff.  Scientifically, 
this  has  been  a  year  of  considerable  outside  recognition  for  current  laboratory 

179 


members  as  well  as  some  of  our  alumni.  Dr.  William  Freed,  won  the  1985 
Arthur  S.  Flemmlng  Award.  Drs.  Karen  Berman  and  David  Shore  received  the 
ADAMHA  Administrator's  Award  for  Meritorious  Achievement  and  our  Clinical 
Director,  Dr.  Lewellyn  Bigelow,  received  the  Public  Health  Service  Meritorious 
Service  Award,  from  ADAMHA.  I  received  the  first  Mary  Byrd  Rawlings  Award 
from  Tri-Services ,  Incorporated  and  Dr.  Daniel  Weinberger  won  the  Young 
Investigators  Award  from  the  National  Alliance  for  the  Mentally  111.  Dr. 
Peter  Bridge,  now  the  Scientific  Director  of  ADAMHA,  received  the  1985  New 
Investigator  Award  in  Neuroscience  from  the  American  Geriatric  Society.  Dr. 
Bridge  received  this  honor  for  research,  with  Dr.  Betsy  Parker  from  NIAAA, 
performed  while  still  a  member  of  our  Branch.  Finally,  I  want  to  congratulate 
another  colleague,  formerly  of  our  Branch,  Dr.  Henry  Nasrallah.  We  wish  him 
much  success  as  he  becomes  Chairman  of  Psychiatry  at  Ohio  State  University. 

Meanwhile,  the  Branch  remains  as  active  as  ever.  Sharing  some  highlights  with 
you,  we  have  published  ,  or  in  press,  almost  200  papers,  enjoyed  26  invited 
seminar  speakers  and  have  had  28  guest  researchers  working  with  our  regular 
staff. 

We  reported  the  installation  of  our  regional  cerebral  blood  flow  machine  In 
last  year's  Annual  Report  and  we  can  now  announce  that  in  the  first  year  of 
operation  Drs.  Berman  and  Weinberger  have  amassed  a  tremendous  amount  of  data. 
Almost  1000  tests  have  been  run.  With  the  help  of  Dr.  Richard  Coppola  of  the 
NIMH  Laboratory  of  Psychology  and  Psychopathology,  several  programs  for  data 
analysis  have  been  developed  and  important  preliminary  findings  have  emerged. 

It  appears  that  schizophrenic  patients  have  difficulty  activating  their 
frontal  cortex  when  challenged  by  frontal  cortex-specific  cognitive  tasks. 
The  task  used  for  our  studies,  the  Wisconsin  Card  Sort,  is  significantly  more 
difficult  to  perform  for  our  schizophrenic  patients  compared  with  normal 
controls.  We  are  continuing  to  analyze  the  voluminous  data  and  will  perform 
further  testing  to  investigate  this  finding  of  hypof rontality. 

Dr.  David  Shore,  who  still  has  one  foot  in  our  Branch  and  one  foot  in  the 
Extramural  Program,  has  been  involved  in  research  in  collaboration  with  the 
Secret  Service.  Analyzing  detailed  national  arrest  records  of  White  House 
cases.  Dr.  Shore  has  shown  that  males  are  more  likely  to  have  violent  crime 
arrests  after  hospital  discharge. 

In  other  work  Dr.  Shore  has  shown  that  fenfluramine  improved  activation  and 
global  BPRS  ratings  of  our  schizophrenic  patients,  while  worsening  BPRS 
negative  symptoms.  Also,  blood  serotonin  concentrations  for  each  subject 
declined  by  at  least  50  percent  on  active  fenfluramine. 

Dr.  Dilip  Jeste  has  continued  his  tardive  dyskinesia  (TD)  investigations 
measuring  plasma  dopamine-beta-hydroxylase  (DBH)  activity,  norepinephrine, 
dopamine  and  their  metabolites  in  cerebrospinal  fluid,  and  indices  of  brain 
atrophy  on  CT  scans  in  patients  with  and  without  TD.  Patients  with  TD  had 
greater  plasma  DBH  activity  and  higher  CSF-norepinephrine  than  non-TD 
patients.  The  two  groups  were  similar  in  CSF-dopamine  and  its  metabolites. 
TD  patients  with  low  plasma-DBH  activity  had  greater  ventricular  enlargement 
and  high  bif rontal-bicaudate  ratios  compared  with  non-TD  patients.  This 
finding  seems  to  suggest  that  TD  is  a  heterogeneous  syndrome,  with  subgroups 
characterized  by  noradrenergic  hyperactivity  or  subcortical  atrophy. 


180 


Dr.  Darrell  Kirch's  guidance  of  our  long-standing  haloperidol  research  has  born 
fruit  this  year  and  some  important  findings  directly  related  to  present 
treatment  patterns  have  emerged.  It  seems  that  clinical  response  to 
haloperidol  plateaus  and  is  not  enhanced  by  administration  of  increasingly 
higher  concentrations.  This  finding,  if  sufficiently  replicable,  will  have 
significant  impact  on  the  treatment  of  schizophrenia. 

Dr.  Jack  Grebb  has  presented  the  first  basic  and  clinical  reports  of  his 
investigations  of  behavioral  and  biochemical  effects  of  calcium  channel 
inhibitors  (CCI's).  This  work,  performed  in  collaboration  with  Drs.  Costa  and 
Hanbauer,  has  produced  preliminary  results  indicating  that  in  animals 
nifedipine-like  CCI's  block  PCP  stimulation.  Nifedipine  or  flunarizine  blocks 
amphetamine- induced  stimulation,  and  co-administration  of  delitazem  or 
verapamil  (but  not  nifedipine)  with  chronic  haloperidol  treatment  inhibits  the 
development  of  neuroleptic-induced  aponorphine  supersensitivity.  Clinical 
studies  investigating  verapramil  and  nifedipine,  as  treatments  for 
schizophrenia,  are  now  underway. 

Dr.  Craig  Karson  has  begun  research  investigating  the  neuropathology  and 
neurochemistry  of  teenage  suicide  and  violent  behavior  and  has  completed  work 
subtyping  the  schizophrenias.  Given  the  recent  findings  that  teen  suicide  may 
be  related  in  some  way  to  maternal  illness,  prenatal  hypoxia  and  labor 
complications.  Dr.  Karson  is  beginning  to  collect  the  brains  of  teen  age 
suicides  from  the  New  Mexico  Medical  Examiner  as  well  as  fran  the  District  of 
Columbia  Medical  Examiner,  to  investigate  possible  neuropathological 
disturbances.  These  neuropathology  studies  will  be  complimented  by  companion 
studies  following  previously  delineated  neurochemical  lines  of  investigation 
concerning  possible  serotonergic  abnormalities.  Parallel  studies  will  be 
performed  with  the  brains  of  violent  patients  and  convicted  murderers. 

Dr.  Karson' s  efforts  to  delineate  schizophrenic  subgroups  have  been  to  quantify 
psychological,  behavioral  and  jiiysiological  variables.  He  has  developed  a 
quotient  constructed  from  items  on  the  Brief  Psychiatric  Rating  Scale  that  may 
differentiate  paranoid  schizophrenic  patients  from  other  schizophrenic 
patients  and  nonnals. 

Dr.  Farouk  Karoum  has  been  investigating  the  central  and  peripheral  disposition 
of  catecholamines  and  biogenic  amines  such  as  E±ienylethylamine  and  p-tyramine 
in  depression.  These  investigations  revealed  a  close  association  between 
depression  and  a  tendency  for  an  increase  in  total  body  turnover  of 
norepinephrine  as  compared  to  those  of  dopamine  and  serotonin.  Consistent  with 
these  observations,  five  different  forms  of  anti-depressants  were  found  to 
preferentially  reduce  norepinephrine  turnover  both  in  the  periphery  and  in  the 
brain. 

Dr.  Frank  Putnam's  work  on  multiple  personality  disorder  has  continued  apace 
over  the  past  reporting  year.  His  groundbreaking  investigations  of  this  little 
understood  disorder  have  been  recently  reported  in  both  the  New  York  Times  and 
the  Washington  Post.  One  aspect  of  this  work  has  been  Dr.  Putnam's  efforts  to 
better  delineate  the  clinical  f^enomenology  of  multiple  personality  disorder. 
One  hundred  recent  cases  were  collected  on  a  386-item  questionnaire,  completed 
by  f^iysicians  treating  multiple  personality  patients.  This  work  documents  the 
clinical  syndrome,  characterized  by  a  core  of  depressive  and  dissociative 
symptoms  and  a  childhood  history  of  significant  trauma,  primarily  child  abuse. 


181 


Also,  Dr.  Putnam  has  made  progress  in  developing  and  testing  a  psychonetric 
instrument  to  measure  the  degree  and  type  of  dissociative  psychopathology  in 
patients  and  normal  controls. 

Like  Dr.  Putnam,  Dr.  Charles  Kaufmann  has  devoted  much  attention  to  a 
relatively  unstudied  population,  the  honneless.  Dr.  Kaufmann  has  been  guiding 
an  epidemiologic  study  of  honeless  wonen  in  the  District  of  Columbia.  The 
study  looks  at  the  interaction  of  psychopathology,  cognition,  life  stressors 
and  social  supports  as  they  effect  subjects'  ability  to  maintain  stable  shelter 
and  ongoing  psychiatric  treatinent.  Within  the  laboratory.  Dr.  Kaufmann  has 
continued  his  studies,  with  Dr.  Janice  Stevens,  investigating  the  possibility 
of  transmissible  agents  in  the  pathogenesis  of  schizophrenia  and  examining 
immune  dysfunction  in  schizophrenic  patients.  Although  no  consistent 
behavioral  abnormalities  have  been  noted,  three  of  six  squirrel  monkeys  have 
developed  comparable  neurologic  disturbances  after  inoculation  of  brain 
suspension  from  schizophrenic  patients.  Dr.  Kaufmann  is  now  attempting 
reisolation  and  blind  passage  of  the  presumptive  transmissible  agent. 

CXir  molecular  biology  laboratory,  unfinished  at  printing  of  last  year's  report, 
is  up  and  producing  results.  Dr.  Anita  Feenstra  has  begun  a  sonatostatin 
project  looking  at  RNA  distribution  in  the  brains  of  schizophrenic  and 
Huntington's  Disease  patients  and  normals.  Dr.  Anne-Marie  Duchemin  has  been 
studying  neurotrophic  factors  in  degenerative  brain  disorders  and  has  been 
looking  at  the  expression  of  gene  coding  for  cholecystokinin  (CCK)  with  a 
prepro  CCK  specific  cDNA  probe  to  quantify  mRNA.  In  the  project  encoding  for 
neurotrophic  factors.  Dr.  Duchemin  has  progressed  through  the  developnent  of 
the  assays  to  test  for  neurotrophic  activity  and  its  products  and  various 
techniques  have  been  applied  to  the  identification  of  lesioned  brain-specific 
mRNAs.  In  Dr.  Duchemin's  gene  coding  work,  she  has  identified  a  single  band  of 
mRNA  corresponding  to  800  bases  and  continues  to  investigate  specific  mPNA 
synthesis. 

The  multiple  roots  of  our  neural  regeneration  research  have  continued  to 
lengthen  and  take  hold  during  the  last  reporting  year.  Dr.  William  Freed  has 
continued  his  research  of  brain  tissue  transplantation  in  non-primate  animals. 
Over  the  past  year  progress  has  been  made  applying  grafting  techniques  to 
models  other  than  rat.  Studies  using  two  models,  one  involving  cortical  injury 
and  one  involving  blindness,  have  been  started  to  further  investigate  the 
generality  of  functional  effects  of  brain  grafting.  Data  are  being  collected 
and  we  should  have  sanne  news  of  this  work  by  next  year.  We  have  achieved 
moderate  success  in  our  initial  attempts  to  replicate  this  work  in  primates. 
We  have  now  transplanted  tens  of  thousands  of  adrenal  medulla  cells  that  have 
survived  into  the  brains  of  our  host  monkeys. 

Dr.  Luis  deMedinacelli,  in  addition  to  continuing  to  perfect  his  surgical 
techniques  of  peripheral  nerve  repair,  has  developed  a  mathematical  model 
predicting  outcontie  of  peripheral  nerve  injuries.  Dr.  deMedinacelli  and  his 
colleagues  hypothesized  that  considering  the  individual  regrowth  of  the 
elementary  components  of  a  nerve  rather  than  global  organ  regeneration  could 
lead  to  a  better  understanding  of  the  mechanisms  of  nerve  repair.  To  test  this 
hypothesis  they  have  designed  a  probability  model  describing  the  prospects  of 
regrowth  for  nerve  composed  of  several  types  of  fibers.  The  model  is  now  being 
tested  in  pre-determined  situations  to  judge  its  validity.   His  work  is  now 


182 


proceeding  to  the  spinal  cord.  The  principles  learned  fron  this  work  should 
have  widespread  implications. 

My  microchip  research  with  Mr.  Paul  Oliver,  also,  has  advanced  successfully 
throughout  the  year.  I  am  happy  to  report  that  we  now  have  neurons  growing  on 
the  surface  of  the  microchip,  fabricated  by  Dr.  Martin  Peckarer  of  the  Naval 
Research  Laboratory.  Further,  this  nervous  tissue  has  survived  several  months 
and  the  microchip  is  clearly  recording  the  neuron's  firing. 

Last  but  not  least,  I  want  to  mention  the  new  machines  that  make  many  of  these, 
and  other  studies  performed  over  the  past  year,  possible.  With  the  able 
management  by  our  Computer  Specialist,  Ivan  Waldman,  we  have  purchased  and 
installed  a  new  Digital  Corporation  Vax  system  for  the  Branch.  To  make  maximum 
use  of  this  equipiient,  we  have  automated  many  areas  and  laboratories.  Our 
scientists  and  staff  have  newly  expanded  access  to  terminals  and  word 
processors  and  these  devices  are  greatly  enhancing  both  the  quality  and 
quantity  of  our  research  as  well  as  increasing  the  quality  of  patient  care. 

Thus,  it  has  been  a  busy  and  successful  year  in  the  Neuropsychiatry  Branch. 
And  as  always,  the  high  levels  of  productivity  and  excellence  have  been 
achieved  through  the  conbined  efforts  of  a  superior  cadre  of 
clinician-scientists  and  our  support  staff.  As  in  past  years,  sane  of  our 
colleagues  and  friends  have  left  and  some  have  joined  us  to  take  their  places. 
This  year  we  say  good-bye  to  Dr.  Bill  Lawson  who  now  holds  the  dual  title  of 
Research  Coordinator  of  Metropolitan  State  Hospital  in  Norwalk,  California  as 
well  as  Assistant  Professor  of  Psychiatry  at  the  University  of  California  at 
Irvine.  Dr.  Dieter  Naber  has  returned  to  Germany  to  take  up  his  post  as 
Assistant  Professor  of  Psychiatry  at  Munich  University,  Dr.  Jack  Grebb  has  left 
for  his  position  as  Assistant  Professor  of  Psychiatry  at  New  York  University 
and  Dr.  Rick  Shelton  has  gone  to  Vanderbilt  where  he  has  a  dual  appointment  to 
the  Departments  of  Psychiatry  and  Pharmacology.  Dr.  Allen  Church  is  now  with 
the  Drug  Enforcement  Agency,  Dr.  Ron  Zee  is  at  Southern  Illinois  University, 
Dr.  Andrei  Jaeger  has  taken  an  appoint  as  Assistant  Professor  of  Psychiatry  at 
Columbia  University  and  Dr.  H.  Stephan  Bracha  is  leaving  for  the  Department  of 
Psychiatry  at  the  University  of  California  at  San  Diego.  Dr.  Shore,  while  not 
gone  quite  yet,  expects  to  soon  be  moving  over  to  the  Extramural  Program  in  the 
Institute  as  Deputy  Director  of  the  Center  for  Studies  of  Schizophrenia. 
Finally,  the  Veteran's  Administration  is  gaining  two  of  our  members.  Dr.  Grant 
Ko  is  going  to  the  Veteran's  Administration  Hospital  in  Seattle,  Washington  and 
Dr.  John  Morihisa  has  been  named  Psychiatrist-in-Chief  of  the  Veteran's 
Administration  Medical  Center  here  in  Washington,  D.C.  Vfe  wish  all  of  our 
colleagues  great  success  in  their  new  positions  and  look  forward  to  continuing 
our  relationship  with  them  in  the  years  to  cone. 

As  they  leave,  we  welcone  Drs.  Myles  Jaffe,  Gregory  Straw,  George  Jaskiw, 
Barbara  Illowsky  and  Terry  Goldberg  into  the  fold.  Vie  look  to  our  newconers  to 
settle  in  smoothly  and  infuse  our  remaining  scientists  and  staff  with  new  ideas 
and  creativity.  I  look  forward  to  reporting  on  all  of  our  progress  in  next 
year's  Annual  Report. 


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Annual  Report 

Laboratory  of  Preclinical  Pharmacology 

October  1,  1984  thru  September  30,  1985 

Chief:  Erminio  Costa,  M.D. 

This  is  my  last  report  on  the  activities  of  the  Laboratory  of  Preclinical 
Pharmacology  which  I  have  headed  since  its  activation  in  June  1968.  For  this 
reason,  I  believe  it  is  appropriate  to  begin  this  report  by  high  lighting  the 
research  and  training  activities  carried  out  in  this  laboratory  during  its  life 
span.  The  laboratory  initiated  its  activities  with  a  staff  of  11  positions 
which  were  increased  gradually  to  a  peak  of  21,  and  now  includes  17  positions. 
The  other  than  permanent  positions  have  oscillated  periodically  from  6  to  10 
within  the  years.  We  have  trained  154  researchers,  each  for  an  average  period 
of  2  years  who  are  now  in  leading  positions  in  various  departments  of 
pharmacology  and  psychiatry  in  this  and  in  several  foreign  countries.  Forty- 
eight  trainees  came  to  us  with  their  own  salaries.  During  the  seventeen  years 
the  laboratory  was  given  in  addition  to  the  salaries  about  seven  million  dollars 
in  other  object  money.  Using  these  resources,  the  laboratory  has  carried  out 
research  which  was  reported  in  850  publications.  These  publications  mainly 
concern  original  contributions  rather  than  extensions  of  work  in  the  literature 
and  a  good  number  of  these  reports  opened  up  new  research  areas  or  pioneered 
significant  conceptual  developments  in  neurochemical  pharmacology.  It  is  well 
recognized  by  peers  that  the  laboratory  has  opened  up  and  contributed  to  the 
following:  1)  development  of  methods  to  measure  turnover  rate  of 
catecholamines,  serotonin,  GABA,  and  to  assess  the  dynamic  state  of  neuronal 
stores  of  peptides,  2)  long  and  short  term  regulation  of  tyrosine  hydroxylase 
including  the  discovery  of  the  role  of  cyclic  AMP  in  the  transsynaptic 
regulation  of  medullary  tyrosine  hydroxylase;  3)  molecular  mechanisms  leading 
to  up  and  down  regulation  of  transmitter  receptors;  4)  pioneered  the  concept  of 
multiple  signals  in  synaptic  transmission  and  of  supramolecular  organization  of 
postsynaptic  receptors;  5)  first  proposed  that  opiate  peptides  function  as 
neuromodulators  in  structures  that  are  not  involved  in  pain  threshold 
regulation,  such  as  caudate  nucleus,  adrenal  medulla  and  sympathetic  ganglia; 
6)  pioneered  the  concept  that  GABAergic  transmission  plays  an  important  role  in 
the  action  of  benzodiazepines,  7)  discovered  several  endogenous  ligands  for  the 
recognition  sites  of  psychoactive  drugs,  9)  studied  DBI  as  a  biochemical  marker 
in  affective  disorders;  8)  elucidated  endogenous  mechanisms  operative  in  opiate 
tolerance,  10)  introduced  mass  fragmentography  as  a  quantitative  method  to 
study  the  dynamic  of  transmitter  steady  state  in  brain  nuclei. 

On  July  1,  1985,  the  laboratory  staff  includes  17  permanent  positions;  10  other 
than  permanent  positions  and  11  guest  researchers. 

During  the  current  year  two  major  discoveries  were  made,  perhaps  one  of  them  may 
bring  about  a  better  understanding  of  the  mechanism  whereby  morphine  tolerance 
occurs,  and  the  other  concerns  the  mode  of  action  of  benzodiazepines  and  the 
endogenous  regulation  of  GABAergic  transmission.  The  latter  turned  out  to  be  of 
potential  significance  also  as  a  departing  point  to  reveal  a  new  putative 
biochemical  marker  for  affective  disorders  and  ethyl  alcohol  chronic 
intoxication.  With  regard  to  morphine  tolerance  a  new  family  of  brain 
neuropeptides  was  discovered,  two  fragments  were  purified  from  bovine  brain  to 
physical  homogeneity  and  sequenced.  Both  peptides  albeit  with  different 
potencies  counteract  morphine  analgesia  by  activating  a  receptor-receptor 
mechanism  influencing  opiate  receptors.    This  peptide  family  may  well 


185 


correspond  in  mammals  to  the  family  of  cardioexcitatory  tetrapeptides 
discovered  in  molluscs.  While  in  molluscs  the  active  group  is  Phe-Met-Arg-Phe- 
NHp,  perhaps  in  the  mammals  it  is  Pro-Gln-Arg-Phe-NH2.  The  two  peptides 
purified  from  bovine  brain  have  several  homologies,  for  instance,  they  share  the 
carboxy  terminal  tetrapeptide  but  they  have  8  and  18  amino  acid  residues, 
respectively.  They  antagonize  morphine  induced  increase  in  the  threshold  of 
nociception  and  they  lower  this  threshold.  Presently,  we  are  determining  the 
structure  of  the  precursor  of  this  new  family  of  peptides  using  the  recombinant 
DNA  technology  in  collaboration  with  Dr.  S.  Sabol  of  NHBLI. 

The  complete  amino  acid  sequence  of  the  peptide  that  regulates  GABA  receptors 
and  displaces  ligands  from  the  benzodiazepine/beta-carboline-3-carboxylate  (B- 
pc)  recognition  sites  located  in  synaptic  membranes,  has  already  been 
determined  in  human  and  rat  hypothalamus,  as  a  result  of  a  collaboration  with 
Dr.  P.  Seeburg  from  Genentech  (San  Francisco,  CA).  Because  it  displaces 
ligands  from  B-pC  recognition  sites,  this  peptide  has  been  termed  DBI  (diazepam 
binding  inhibitor).  The  sequence  of  the  peptide  extracted  from  rat  and  human 
brain  does  not  appear  to  be  entirely  homologous.  The  epicenter  of  the 
biological  activity  of  the  peptide  extracted  from  the  brain  of  these  two  species 
appears  to  be: 
Human: 

...Gln-Ala-Thr-Va-Gly-Asp-Ile-Asn-Glu-Arg-Pro-Gly-Met-Leu-Asp-Phe-Thr-Gly-Lys.. 
Rat: 

. . .61n-Ala-Thr-Va-Gly-Asp-Val-Asn-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys-Gly-Lys.. 
51  68 

The  DBI  fragment  51-68  (ODN)  of  the  rat  was  synthesized  and  it  was  confirmed 
that  it  is  endowed  with  biological  activity.  It  was  also  found  to  be  three 
times  more  active  than  DBI  in  the  Vogel  test  run  in  the  modality  that  detects 
proconflict  activity.  DBI  also  displaces  preferentially  B-BC  esters  from  B-BC 
recognition  sites,  a  similar  activity  is  possessed  also  by  ODN  which  is  more 
active  than  DBI  but  still  requires  uM  concentrations.  Finally,  in  collaboration 
with  Dr.  J,  Borman  (Max  Planck  Institute,  Gottingen,  Germany),  it  was  found  that 
DBI  reduces  the  Cl~  currents  generated  by  GABA  in  primary  cultures  of  mouse 
spinal  neurons  measured  when  the  cells  are  patch-clamped  with  a  holding 
potential  of  70  mV,  Since  the  activities  of  DBI  can  be  inhibited  by  the 
imidobenzodiazepine,  RO  15  1788,  a  weak  partial  agonist  of  the  B-pC  recognition 
site,  it  can  be  argued  that  DBI  is  the  precursor  of  the  endogenous  ligand  for  B- 
6C  recognition  site  modulating  GABAergic  transmission.  The  exact  sequence(s) 
of  the  fragment(s)  that  functions  as  the  ligand  is  not  known.  An  ODN  like 
sequence  is  present  in  rat  and  human  brain  but  it  is  still  debated  whether  this 
peptide  is  the  fragment  DBI  51-68  or  OBI  51-70.  The  fragments  DBI  62-68,  61-68 
and  63-68  of  the  rat  sequence  have  been  synthesized  and  found  to  be  almost  as 
active  as  ODN  in  the  Vogel  test  and  in  binding  to  B-pC  recognition  sites.  The 
ODN  amide  of  the  alpha-carboxy  group  of  the  Lys  in  position  DBI  68  was 
synthesized  and  tested  in  the  Vogel  model  and  as  a  displacer  of  beta-carboline- 
3-carboxylate,  but  this  molecule  was  found  to  be  completely  inactive  at 
concentration  100  juM.  Thus,  a  free  alpha  carboxyl  terminal  in  the  terminal  Lys 
residue  of  ODN  appears  to  be  essential  for  activity.  Whether  a  carboxy  terminal 
extended  form  of  ODN  such  as  DBI  51-70  is  more  active,  is  currently  being 
studied.  DBI  is  present  also  in  spinal  fluid  and  can  be  detected  and  measured 
in  0.1  ml  of  fluid  with  a  human  DBI  antibody  we  have  prepared. 

In  collaboration  with  Drs.  F,  Goodwin  and  S.  Paul  of  the  NIMH,  we  have  found 

186 


that  male  spinal  fluid  contains  a  higer  amount  of  DBI  than  in  females,  in  both 
sexes  DBI  increases  with  age  but  is  not  higher  in  old  people  with  Alzheimer's 
disease  or  in  schizophrenic  patients.  It  is  increased  in  depressed  patients 
with  high  levels  of  anxiety.  The  significance  of  the  increases  in  spinal  fluid 
DBI  cannot  be  assessed  at  this  time  because  we  have  to  know  whether  the 
biologically  active  fragment  of  DBI  is  increased.  These  studies  are  currently 
in  progress. 

Group  on  Molecular  Biology  (Head:  Dr.  J.  Schwartz,  Ph.D.) 

Drs.  J.  Schwartz,  I.  Mocchetti  (Guest  Researcher)  and  J.  Naranjo  (Guest 
Researcher)  have  developed  a  methodology  with  which  to  estimate  changes  in  the 
dynamic  state  of  neuropeptide  stores  which  occur  as  a  result  of  pharmacological 
treatments.  The  original  method,  utilizing  specific  mRNA  measurements 
hybridizing  with  cDNA  probes  for  specific  peptide  precursors  and  the 
measurement  of  peptide  levels  by  radioimmunoassay,  has  been  significantly 
improved  in  several  ways.  Quantitation  of  specific  neuropeptide  precursor 
mRNAs  is  expressed  relative  to  the  amount  of  two  mRNAs  which  are  invariant  from 
tissue  to  tissue.  Chromatographic  separation  of  immunoactivity  provides  an 
analysis  of  precursor  proteins  or  peptides  as  well  as  the  biologically  active 
low  molecular  weight  peptides  themselves.  The  combination  of  these 
measurements  can  thus  determine  whether  a  drug  that  changes  the  steady  state  of 
a  neuropeptide  acts  by  modifying  the  amount  of  precursor  mRNA,  the  rate  of 
translation  or  processing  of  the  precursor,  or  the  utilization  of  the 
neuropeptide  itself. 

Using  this  methodology.  Dr.  Mocchetti  has  measured  changes  in  the  dynamic  state 
of  proenkephalin  (PE)  and  proopiomelanocortin  (POMC)  stores  in  specific  brain 
regions  following  treatment  of  rats  with  a  variety  of  neuroactive  drugs.  The 
enkephal in-containing  neurons  of  the  striatum  are  under  tonic  inhibitory 
control  by  nigral  dopamine:  removal  of  dopamine  either  by  depletion  with 
reserpine  or  intra-nigral  6-hydroxydopamine  or  by  blockade  with  the  antagonist 
haloperidol  results  in  an  increase  of  PE  mRNA,  PE  and  enkephalin  peptides. 
Haloperidol  and  other  dopamine  receptor  antagonists  by  attenuating  the  dopamine 
inhibition  of  PE  synthesis  turn  on  the  PE  synthesis.  In  contrast,  fenfluramine, 
pCl  phenylalanine  and  5,7-dihydroxytryptamine  which  by  various  mechanisms 
reduce  brain  serotonin  content,  increase  hypothalamic-beta-endorphin  and  the 
hypothalamic  and  striatal  content  of  enkephalin,  but  they  fail  to  affect  the 
content  of  either  PE  or  POMC  mRNAs  or  of  the  precursors  for  enkephalin  (PE)  and 
for  beta  endorphin  (POMC).  Hence,  these  changes  elicited  by  serotonin  depletion 
must  occur  via  a  modification  in  the  rate  of  peptide  utilization  while 
translation  and  peptide  processing  remain  unchanged.  There  are  no  changes  in 
the  PE  system  in  rats  made  tolerant  to  and  dependent  on  morphine:  however,  the 
POMC  mRNA  content  of  hypothalamus  is  decreased  by  50%  without  changes  in  either 
POMC  or  p-endorphin  content. 

Dr.  Naranjo  has  found  that  a  reserpine  treatment,  capable  of  increasing  PE  mRNA, 
and  enkephalin  contents  of  striatum,  has  a  different  effect  on  the  PE  system  in 
the  adrenal  medulla,  presumably  because  it  depletes  directly  the  catecholamine 
content  of  the  chromaffin  cells  containing  the  enkephalins  rather  than  acting  on 
the  enkephalin  store  transsynaptically,  as  in  the  striatum.  Reserpine 
administration  to  rats  results  in  a  loss  of  PE  mRNA  in  adrenal  medulla,  and  in 
an  increase  of  the  conversion  of  HMW  enkephal in-containing  peptides  to  LMW 
enkephalins:  there  is  thus  an  initial  increase  of  enkephalin  content,  which 

187 


starts  to  decline  after  several  days  as  the  precursor  pool  is  depleted. 
Comparable  changes  have  been  obtained  by  treating  bovine  adrenal  chromaffin 
cells  in  culture  with  reserpine,  thus  confirming  that  the  effects  of  reserpine 
occur  as  a  result  of  a  direct  action  on  the  chromaffin  cells.  Catecholamines 
are  also  depleted  by  the  addition  of  appropriate  reserpine  concentrations  to  the 
cultured  cells.  Exposure  of  the  cells  to  dexamethasone  results  in  an  increase 
in  the  content  of  PE  mRNA  and  enkephalin  peptides  but  this  hormone  has  no 
significant  effect  on  tyrosine  hydroxylase  mRNA  or  catecholamine  content. 
These  studies  demonstrate  independent  regulation  of  the  synthesis  of  the 
catecholamines  and  enkephalin  peptides  coexisting  in  the  same  granules  of  the 
chromaffin  cells. 

In  order  to  understand  the  role  that  nerve  growth  factor  and  related 
neuronotrophic  factors  may  play  in  disease  states,  Drs.  Schwartz  and  T.T. 
Quach  (Visiting  Associate)  are  cloning  the  gene  for  a  neuronal  trophic  factor 
which  is  produced  in  lesioned  brain.  This  factor  is  assayed  by  its  ability  to 
support  neuron  survival  in  dissociated  cultures  of  12-day  chick  embryo  sensory 
or  sympathetic  ganglia.  The  mRNA  prepared  from  either  lesioned  brain  or  intact 
brain  is  screened  by  in  vitro  translation  in  oocytes  in  the  presence  of  the  mRNA 
for  the  neuronotrophic  factor.  The  mRNA  will  be  cloned  from  a  subtraction 
library  representing  those  messages  specific  to  the  lesioned  brain.  The  types 
of  messages  present  in  the  lesioned  brain  cDNA  library  will  be  of  general 
interest  in  terms  of  understanding  the  molecular  events  involved  in  brain 
injury. 

Group  on  Immunochemistry  (Head:  Dr.  P.M.  Chuang,  Ph.D.) 

Dr.  Chuang  and  Ms.  Dillon-Carter  continued  their  studies  on  the  internalization 
of  beta-adrenergic  receptor  (BAR)  during  desensitization.  These  internalized 
BAR  sites  are  present  in  the  30,000  x  g  supernatant  associated  with  inside-out 
endocytotic  vesicular  structures  excluded  by  Sepharose  4B  column 
chromatography.  Using  crude  detergent  extract  of  purified  erythrocyte  plasma 
membranes  as  the  antigen  they  have  produced  a  monoclonal  antibody  whose 
antigenic  determinant  is  a  membrane  component  which  reacts  with  BAR.  Based  og 
several  criteria,  they  have  concluded  that  the  antigenic  protein  (MW=  4.3x10 
daltons  and  pl=  6.2)  is  distinct  from  BAR  site  and  from  the  Ns  subunit  of  the  GTP 
dependent  coupler  protein  and  have  suggested  that  the  monoclonal  antibody  is 
against  an  unidentified  membrane  component  of  the  BAR-adenylate  cyclase  system. 

Since  clathrin-coated  vesicles  (CVs)  have  been  implicated  in  both  endocytotic 
and  intracellular  transport  of  a  variety  of  receptors.  Dr.  Chuang  in 
collaboration  with  Dr.  C.  J.  Coscia  at  St.  Louis  Univ.,  Mo.,  have  plurified  CVs 
from  bovine  brain  and  examined  them  for  the  presence  of  BAR  binding  and 
adenyl-jcU^  cyclase  activities.  These  CVs  contain  BAR  binding  sites  as  assessed 
with  I-cyanopindolol  as  ligand  in  Sepharose  column  assays  (Bmax=  32+3 
fmol/mg  protein).  Their  data  suggest  that  BAR  and  adenylate  cyclase  present  in 
brain  CV  preparation  may  be  molecular  entities  undergoing  endocytosis  or 
intracellular  transport. 

Drs.  T.  Nakaki  (Visiting  Fellow)  and  B.  Roth  (Guest  Researcher)  are  studying 
the  molecular  mechanisms  of  rat  aorta  contraction  induced  by  5-HT.  A 
phosphoinositide  specific  phospholipase  C  in  rat  thoracic  aorta  is  stimulated 
by  5HT  (ECcQ=  10  /jM)  and  appears  to  be  operative  in  5HT  induced  contractions. 
This  activation  is  blocked  by  various  5-HT2  receptor  antagonists  (ketanserin, 

188 


metergoline,  pizotifen  and  mianserin).  Dr.  Nakaki  found  that  5-HT-induced 
contractions  have  a  f^^asic  and  a  tonic  component.  Nifedipine,  an  inhibitor  of 
voltage-dependent  Ca  channels,  inhibits  only  the  phasic  component  of  the 
contraction  while  totally  blocking  the  KCl-induced  contraction.  2-Nitro-4- 
carboxyphenyl-N-N-diphenylcarbamate,  an  inhibitor  of  phosphol ipase  C,  inhibits 
the  tonic  component  of  contractions  as  well  as  the  5-i|J-induced  phosphol ipase  C 
activity  in  rat  aorta.  In  the  presence  of  a  Ca  ionophore  A23187,  this 
component  of  contraction  can  be  mimicked  by  a  protein  kinase  C  activator,  12-0- 
tetradecanoy^^horbol-13-acetate  (TPA).  These  data  suggest  that  voltage- 
dependent  Ca  channel  activation  participates  in  the  phasic  component  of  the 
contraction,  whereas  breakdown  products  of  phosphatidyl inositides  which 
activate  protein  kinase  C  play  a  physiologically  important  role  in  the  tonic 
component  of  the  aortic  contraction.  Recently  Dr.  Nakaki  found  that  in  the 
cell-free  system  of  rat  aorta,  the  phosphorylation  of  at  least  two  proteui  (MW= 
20  and  92.5  kilodaltons)  can  be  increased  by  the  presence  of  Ca  and 
phosphatidylserine  or  by  the  addition  of  protein  kinase  C  activators  (such  as 
TPA  and  1,2  diolein).  Preliminary  results  show  that  5-HT  increases  also  the 
phosphorylation  of  these  two  protein  species  in  the  smooth  muscle  primary 
culture  prepared  from  rat  aorta.  These  results  are  compatible  with  the  view 
that  stimulation  of  5-HTp,  receptors  in  rat  aorta  triggers  a  cascade  mechanism 
resulting  in  an  activation  protein  kinase  C  and  phosphorylation  of  certain 
specific  proteins;  this  protein  phosphorylation  appears  to  play  a  key  role  in 
causing  the  tonic  phase  of  contraction. 

Section  on  Neuroendocrinology  (Chief:  A.  Guidotti,  M.D) 

The  activities  of  this  section  have  been  focussed  on  the  study  of  the  molecular 
mechanisms  of  GABA/benzodiazepine  interaction  as  a  model  to  investigate 
possible  biochemical  markers  of  anxiety  and  affective  disorders  in  general.  A 
major  effort  has  been  devoted  to  identify  specific  putative  neuromodulators 
acting  on  high  affinity  recognition  sites  for  B-6C  and  thereby  regulate  the 
duration  of  GABA  dependent  Cl~  gate  opening  bursts.  Previous  work  from  this 
section  has  suggested  that  GABAergic  synapses  contain  at  least  two  polypeptides 
involved  in  the  modulation  of  GABA  receptor  function.  One  of  them  is  a  membrane 
polypeptide  termed  GABAmodulin  while  the  other  is  a  polypeptide  present  in  axon 
terminals  and  neuronal  cell  bodies  and  was  termed  DBI  because  of  its  ability  to 
displace  ligands  from  B-pC  recognition  sites. 

Both  of  these  polypeptides  were  purified  to  homogeneity.  Dr.  F.  Vaccarino 
(Guest  Researcher)  has  found  that  the  one  located  in  brain  synaptic  membranes 
GABAmodulin,  appears  to  be  operative  in  modulating  the  shift  in  the  abundance  of 
high  and  low  affinity  recognition  sites  for  GABA.  When  the  amino  acid 
composition  of  rat  GABA  modulin  was  studied  it  was  found  that  it  resembled  that 
of  small  molecular  weight  myelin  basic  proteins  of  rat  brain.  However,  Dr. 
Vaccarino  has  shown  that  GABAmodulin  can  be  differentiated  from  the  myelin  basic 
protein  by  several  criteria  including  the  polypeptide  fragmentation  pattern 
after  partial  proteolysis;  currently  the  subcellular  location  is  determined  by 
immunocytochemical  techniques.  The  other  brain  peptide  (DBI)  appears  to 
participate  in  the  modulation  of  GABAergic  transmission.  Recent 
electrophysioloigical  studies  in  collaboration  with  Dr.  J.  Bormann  (Max  Planck 
Inst.Gottingen,  W.  Germany)  indicate  that  DBI  shortens  the  burst  of  Cl~  channel 
opening  elicited  by  GABA  thus  acting  like  a  B-pC  derivative.  This  peptide  may 
represent  an  endacoid  acting  at  GABAergic  synapses  involved  in  the  modulation  of 
anxiety  states.  DBI,  when  injected  intracerebroventricularly  into  rats  elicits 

189 


a  pharmacological  response  with  a  profile  similar  to  that  of  anxiogenic  drugs 
structurally  related  to  B-pC.  In  order  to  elucidate  the  possible  physiological 
role  of  DBI  Dr.  P.  Ferrero  (Guest  Researcher)  has  raised  antibodies  against  DBI 
extracted  from  human  and  rat  brain.  These  antisera  show  high  sensitivity  and 
specificity  for  human  or  for  rat  DBI  they  have  been  used  to  study  brain 
distribution,  localization,  and  processing  of  DBI  in  the  two  species.  Dr.  M. 
Fujimoto  (Visiting  Fellow)  has  studied  the  distribution  of  DBI  in  32  areas  of 
the  rat  brain  and  found  that  DBI  is  highest  in  the  hypothalamic  nuclei  and 
lowest  in  the  pituitary.  Moreover,  Dr.  H.  Alho  (Visiting  Associate)  studying 
the  location  of  DBI  in  rat  brain  with  immunohistochemical  techniques  has  shown 
that  DBI  is  highly  concentrated  in  selected  neuronal  populations.  In  order  to 
optimize  conditions  to  study  this  distribution,  rats  must  be  pretreated  with 
colchicine  to  inhibit  neurotubular  transport  and  cause  the  accumulation  of 
immunoreactivity  in  cell  bodies.  For  example,  high  levels  of  immunoreactivity 
were  found  in  nerwe  terminals  in  hypothalamic  and  amygdaloid  nuclei,  in  cell 
bodies  of  pyramidal  cells  in  the  hippocampus  and  in  layer  VI  of  the  cerebral 
cortex  and  in  a  layer  of  cells  (perhaps,  basket  cells)  surrounding  the  Purkinje 
cells  of  cerebellum.  In  order  to  ascertain  whether  DBI  is  colocalized  with  GABA 
in  GABAergic  interneurons.  Dr.  Vaccarino  and  Dr.  Alho  have  studied  the 
immunoreactivity  for  DBI  and  GAD  in  primary  culture  of  hipppocampal  cells.  The 
results  indicate  that  OBI  coexists  with  GABA  in  some  but  not  in  all  GABA 
neurons.  Dr.  Ferrero  has  detected  and  characterized  DBI  in  human  brain  and 
found  that  immunochemical  ly  is  not  identical  to  rat  brain  DBI.  A  difference 
between  DBI  of  the  two  species  has  been  confirmed  by  recent  amino  acid  sequence 
established  by  Dr.  P.  Seeburg  of  Genentech  (San  Francisco,  CA)  using  DBI-cDNA. 

Dr.  Ferrero  has  also  shown  that  DBI  can  be  found  and  measured  in  human  spinal 
fluid.  Based  on  this  observation.  Dr.  M.L.  Barbaccia  (Guest  researcher)  Drs. 
S.  Paul  and  F.K.  Goodwin  (NIMH)  have  initiated  a  collaborative  study  to  answer 
the  question  of  whether  DBI  or  DBI-like  substances  change  in  human  CSF  as  a 
consequence  of  physiological  or  pathological  conditions.  In  an  initial 
analysis  of  83  CSF  samples  obtained  from  normal  subjects  or  patients  suffering 
from  different  psychiatric  disorders,  it  appears  that  the  content  of  DBI  may 
change  with  age  and  mood  disorders.  Dr.  M.  Miyata  (Visiting  Fellow)  is 
extending  these  studies  to  the  CSF  of  rats  in  order  to  elucidate  the 
relationships  between  the  changes  in  DBI  steady  state  in  brain  and  the  increase 
in  DBI  spinal  fluid  content.  To  tackle  the  problem  of  whether  DBI  is  a 
precursor  of  a  smaller  molecular  weight  peptide  functioning  as  a  physiological 
modulator  at  GABAergic  synapses.  Dr.  Ferrero  studied  the  proconflict  action  of 
several  peptide  fragments  obtained  by  trypsinization  of  rat  DBI.  It  could  be 
shown  that  the  proconflict  action  of  DBI  resides  in  DBI  51-68  a  characteristic 
octadecaneuropeptide  (ODN).  This  peptide  was  synthesized  by  Peninsula  and 
injected  intracerebroventricularly.  It  mimicked  the  proconflict  action  of  DBI 
and  its  effect  was  blocked  by  the  imidobenzodiazepine,  RO  15  1738,  which  is  a 
specific  antagonist  of  the  B-^C  sites.  Antibodies  were  raised  against  the  DBI 
51-68  and  using  immunochromatography  with  these  antibodies  combined  with  HPLC 
and  RIA,  it  could  be  found  that  rat  brain  contains  ODN-like  material.  These 
data  support  the  contention  that  DBI  may  indeed  act  as  a  precursor  of  a  smaller 
molecular  weight  peptide  endowed  with  physiological  function.  In  order  to 
detail  the  pharmacological  action  of  DBI  51-68  and  to  delineate  the  minimal 
structure  requirement  within  DBI  51-68  amino  acid  sequence  that  is  required  to 
modulate  GABAergic  function  the  following  peptides  were  synthesized  by 
Peninsula:  the  amidation  of  the  terminal  Lys  of  ODN-Lys-amide  (DBI  51-68),  DBI 
60-68,  DBI  61-68,  DBI  62-68.  Injected  intraventricularly  into  the  rat,  the  DBI 


190 


fragments  60-68,  61-68,  and  62-68  behave  as  proconflict  agonists  and  in  large 
doses  can  induce  tonic  clonic  convulsions.  The  ODN-Lys  amide  peptide  is  not 
convulsant  and  does  not  induce  proconflict  action,  l^wever,  wiien  we  decided  to 
study  the  ability  of  these  peptides  to  displace  H-B  or  H--pC  from  their 
specific  binding  sites  located  on  crude  brain  synaptic  membranes  we  discovered 
that  the  action  of  this  peptide  was  only  marginal.  Since  synaptic  membranes 
contain  proteolytic  enzymes  including  an  abundance  of  exopeptidases  (amino  and 
carboxypeptidases)  we  decided  to  develop  an  intact  neuronal  preparation  to 
investigate  the  effect  of  the  various  DBI  fragments  peptides  on  benzodiazepine- 
beta-recognition  sites. 

A  suitable  preparation  for  these  studies  appeared  to  be  the  primary  culture  of 
cerebellar  granule  cells.  This  culture  established  in  our  laboratory  with  the 
assistance  of  Dr.  V.  Gallo  (Guest  Researcher)  appears  to  be  an  ideal  model 
system  to  study  in  intact  cells  the  regulation  of  GABA/benzodiazepine/Cl 
ionophore  receptor  complex.  Using  this  model  Dr.  M.R.  Santi  (Visiting  Fellow) 
and  Dr.  Ferrero  were  able  to  show  that  DBI  61-68,  60-68,  52-68  all  displace^  H- 
ec  with  an  affinity  (pM  range)  higher  than  that  required  to  displace  H-B 
Moreover,  an  important  aspect  highlighted  by  the  binding  studies  is  that  the 
active  site  of  ODN  must  contain  the  carboxyl  terminal  sequence  intact  because 
the  alpha-amidation  of  the  terminal  Lys  of  ODN  fails  to  displace  both  H-B  or 
H-BC.  Taken  together  these  observations  suggest  that  ODN  or  an  ODN-like 
endogenous  neuropeptides  may  represent  the  physiologically  occurring  ligand 
resulting  from  DBI  processing. 

There  is  increasing  evidence  that  GABAergic  neurons  are  not  exclusive  of 
mammalian  brain  but  they  play  a  role  in  peripheral  mammalian  neurotransmission. 
Drs.  Y.  Kataoka  (Visiting  Fellow)  and  Y.  Gutman  (Visiting  Scientist)  have  made 
the  original  observation  that  adrenal  chromaffin  cells  in  culture  contain, 
synthesize,  release  and  take  up  GABA.  Moreover,  they  found  that  chromaffin 
cells  contain  GABA  receptors  coupled  to  B  recognition  sites  and  J.  Bormann  et 
al.  (PNAS  82:  2168,  1985)  have  recently  published  that  these  GABA  receptors  like 
those  in  brain  are  coupled  with  Cl~  channel.  Dr.  Fujimoto  has  shown  that  a 
GABAergic  system  similar  to  that  found  in  cow  adrenal  medulla  is  also  present  in 
dog,  rat  and  guinea  pig  adrenal  glands.  Moreover,  Dr.  Alho  has  shown  by  using 
the  immunocytochemical  technique  that  GAD  and  GABA  are  not  only  localized  in 
chromaffin  cells,  but  also  in  specialized  nerve  fibers  which  impinge  on  the 
chromaffin  cells. 

Based  on  these  data  Drs.  I.  Hanbauer  (NHLBI),  Fujimoto  and  Kataoka  studied  the 
effect  of  GABA  and  GABA  mimetics  on  the  release  of  catecholamines  and  opiate 
peptides  from  the  dog  adrenal  medulla  in  vivo.  The  in  vivo  study  show  that  in 
the  dog  appropriate  doses  of  GABA  and  other  GABA  mimetic  drugs  (THIP,  muscimol, 
but  not  baclofen)  release  catecholamines  and  opiate  peptides  into  the 
circulation.  This  release  was  not  blocked  by  hexamethonium,  naloxone  or 
splanchnicectomy  but  was  prevented  by  bicuculline  pretreatment.  These  data 
suggest  that  GABA  induced  catecholamines  and  peptide  release  was  not  the 
consequence  of  an  activation  of  transynaptic  mechanisms  involving  either 
acetylcholine  or  enkephalin,  but  rather  the  consequence  of  chromaffin  cell 
depolarization  following  direct  stimulation  of  GABA^  receptors  located  on 
chromaffin  cell  membranes. 


191 


Section  on  Neuropeptides  (Chief:  H.-Y.T.  Yang,  Ph.D.) 

Cardioexcitatory  peptide,  phe-met-arg-phe-NH^  (FMRF-NHp)  is  a  neuropeptide  of 
clam  origin,  however,  existence  of  FMRF-NH^- rike  immunoreactivity  in  mammalian 
CNS  is  now  well  known.  Previously,  we  have  found  that  FMRF-NH^  as  well  as 
endogenous  mammalian  FMRF-NH^-like  immunoreactive  material  are  capable  of 
attenuating  the  analgesic  effect  of  opiates.  In  order  to  explore  whether 
mammalian  FMRF-NHp-1 ike  peptides  play  a  physiological  role,  we  have  decided  to 
purify  these  peptides  from  mammalian  brains  and  determine  their  structures  and 
biological  activities.  Two  FMRF-NHp-like  immunoreactive  peptides  were  purified 
to  physical  homogeniety  from  bovine  brains  by  combination  of  affinity  column 
chromatography  and  successive  HPLC  steps.  Subsequently,  these  two  purified 
peptides  were  sequenced  by  Applied  Biosystem.  The  amino  acid  sequence  of  the 
two  purified  peptides  was  proposed  to  be,  1)  Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro- 
Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NHp  (Ala-lS-Phe-NH^;  2)  Phe-Leu-Phe- 
Gln-Pro-Gln-Arg-Phe-NH^  (Phe-S-Phe-NH^) .  These  peptides  were  synthesized  by 
Peninsula  and  compared  with  the  isolated  endogenous  FMFR-NHj;,-like 
immunoreactive  peptides  by  HPLC.  The  synthesized  peptides  and  the  endogenous 
peptides  were  found  to  have  the  same  HPLC  retention  times  thus  confirming  the 
proposed  structures.  Rats  receiving  the  synthesized  peptides  in  the  cerebral 
ventricles  were  found  to  have  a  lower  basal  tail  flick  latencies.  Phe-8-Phe-NHp 
was  more  potent  than  the  Ala-lS-Phe-NH^.  Furthermore,  Phe-S-Phe-NHp  injecteO 
intraventricularly  in  nmolar  doses  was  round  to  attenuate  the  analgesia  induced 
by  morphine.  The  mechanism  underlying  this  antagonism  will  be  investigated  in 
the  future;  however,  preliminary  data  indicate  that  a  receptor-receptor 
interaction  may  be  operative  causing  an  allosteric  modification  of  the  opiate 
receptors. 

The  FMRF-NH„  like  immunoreactivity  is  high  in  the  spinal  cord,  thus  Dr.  C. 
Ferrarese  tvisiting  Associate)  investigated  the  location  of  FMRF-NH^ 
immunoreactive  neurons  in  the  spinal  cord  by  lesion  s^tudies.  These  studies 
indicate  that  FMRF-NHj,-like  immunoreactive  peptides  in  the  spinal  cord 
originate  from  sensory  ganglia. 

NPY  is  a  36  amino  acid  neuropeptide  found  to  coexist  with  catecholamines  in  some 
neurons  and  also  in  adrenal  glands.  It  has  been  suggested  that  NYP  may  have  a 
modulatory  role  on  the  release  and  action  of  catecholamines  at  receptors.  In 
order  to  further  explore  the  role  of  NPY  in  sympathetic  function  we  have  studied 
the  NPY  distribution  in  bovine  adrenal  glands  radiochemically  and 
immunohistochemically.  NPY  is  unevenly  distributed  in  bovine  adrenal  glands 
with  the  highest  level  in  chromaffin  granules.  Immunohistochemically  NPY 
immunoreactivity  was  detected  in  norepinephrine  containing  chromaffin  cells  and 
also  in  nerve  fibers  crossing  through  adrenal  cortex  and  medulla.  NPY  and 
enkephalin  immunoreactivities  did  not  appear  to  coexist  in  the  same  chromaffin 
cells.  Biochemical  characterization  by  HPLC  revealed  two  major  molecular  forms 
of  NPY  immunoreactivity:  authentic  NPY  and  another  molecular  form  which  was 
more  hydrophilic  than  NPY.  Both  ^oleculaK^  forms  of  NPY  were  released  from 
cultured  chromaffin  cells  by  45  mMK  in  a  Ca  dependent  manner.  In  the  adrenal 
gland,  NPY  is  primarily  stored  in  chromaffin  granules  which  similarly  to  other 
chromaffin  cell  constituents  are  greatly  affected  by  the  splanchnic  nerve 
activity.  Because  of  this,  the  effect  of  the  splanchnic  denervation  on  adrenal 
content  of  NPY  was  studied  by  Dr.  H.  Higuchi  (Visiting  Fellow). 
Interestingly,  NPY  immunoreactivity  in  the  rat  adrenal  gland  was  found  to 
increase  markedly  with  age;  an  increase  of  40-fold  was  observed  from  6  week  to 
24  week  old  rats.  This  age  dependent  increase  of  adrenal  NPY  was  abolished  by 
splanchnic  nerve  transection.    An  additional  molecular  form  of  NPY-like 

192 


immunoreactivity  was  detected  in  the  adrenal  glands  of  older  rats.  The 
biological  and  chemical  nature  of  this  molecular  form  of  NPY  immunoreactivity 
has  not  yet  been  studied.  Thouyh  NPY  is  found  in  .._,  ena'i  ylands  of  many  species 
in  rather  high  concentrations,  the  biological  significance  of  this  peptide  in 
the  adrenal  gland  is  yet  to  be  explored.  Dr.  Higuchi  found  that  NPY  (10  M)  can 
inhibit  significantly  the  nicotinic  receptor-activated  catecholamine  release 
from  bovine  chromaffin  cells.  Therefore,  the  change  in  adrenal  NPY  content 
during  aging  may  have  consequences  on  the  release  of  catecholamines  from  adrenal 
medulla.  Since  the  catecholamine  release  induced  by  K  depolarization  is  not 
inhibited  by  NPY  the  possibility  that  the  action  of  NPY  is  mediated  by 
mechanisms  other  than  inhibition  of  catecholamine  autoreceptors  can  be 
considered. 

c  fi    7 

It  has  been  demonstrated  that  the  met  -enkephalin-arg  -phe  is  metabolized  by 

dipeptidyl  carboxypeptidase  and  aminopaptidase.  Because  of  this.  Dr.  B. 
Mellstrom  (Visiting  Fellow)  has  searched  for  an  effective  inhibitor  which  is 
capable  of  crossing  the  blood  brain  barrier  to  block  the  brain  dipeptidyl 
carboxypeptidase.  HOE  498,  2-(N-(s)-l-ethoxycarbonyl-3-phenylpropyl-L-alanyl ) 
-  IS,  35,  55)-2-azabicyclo  (3,3,0)  octan-3  carboxylic  acid  was  found  to  be  a 
potent  inhibitor.  This  compound  was  tested  for  its  ability  to  cross  the  blood 
brain  barrier.  The  presence  of  HOE  498  in  rat  CSF  after  i.v.  or  i^p.  injection 
was, demonstrated.  HOE  498  was  found  to  prjDtect  the  injected  met  -enkephalin- 
arg  -phe  .  Furthermore,  the  recovery  of  met  -enkephalin-arg  -phe  released  from 
striatal  tissue  slices  was  increased  in  the  presence  of  HOE  498.  Whether  this 
inhibitor  can  potentiate  the  analgesic  effect  of  met  -enkephalin-arg  phe  still 
remains  to  be  investigated. 

Dr.  M.  ladarola  (Staff  Fellow)  has  investigated  the  role  of  opioid  peptides  in 
chronic  pain.  Chronic  pain  was  induced  by  injecting  one  hind  paw  with  Freund 
adjuvant.  In  the  dorsal  cord  ipsilateral  Ix)  the  injection^,  the  yContent  of 
dynorphin  A  was  found  to  increase  while  met  -enkephalin-arg  -gly  -  remained 
constant.  The  dynorphin  content  of  the  ventral  cord  failed  to  change.  The  data 
suggest  that  chronic  pain  and/or  inflammation  in  a  peripheral  limb  provoke  a 
selective  increase  in  the  dynorphin  content  of  dorsal  spinal  chord.  Whether  the 
increase  in  dynorphin  content  reflects  an  increase  or  decrease  in 
dynorphinergic  neurons  still  remains  to  be  investigated.  In  addition,  Drs. 
ladarola,  Naranjo,  and  Nicoletti  are  investigating  the  changes  in  the  dynamic 
state  of  brain  neuropeptide  stores  during  kindling. 

Section  on  Molecular  Neurobiology  (Chief,  E.  Costa,  M.D) 

A).  Affective  disorders:  biochemistry  and  pharmacology:  Drs.  M.L.  Barbaccia 
(Guest  Researcher)  U.  Ravizza  (Guest  Researcher)  and  0.  Pozzi  (Guest 
Researcher)  have  investigated  the  mechanisms  of  action  of  typical  and  atypical 
antidepressants.  Specific  high  affinity  recognition  sites  for  several 
antidepressants  are  present  in  crude  synaptic  membranes  prepared  from  brain  of 
various  animal  species.  The  imipramine  recognition  sites  are  functionally 
associated  though  not  identical  to  the  serotonin  reuptake  site.  Previous  wgrk 
from  our  laboratory  has  contributed  evidence  suggesting  that  these  H- 
imipramine  recognition  sites  are  pharmacologically  relevant  for  the  expression 
of  the  changes  in  beta-adrenergic  receptor  function  which  have  been  related  to 
the  therapeutic  action  of  this  drug.  We  suggested  that  these  sites  are 
physiologically  regulated  by  an  endogenous  putative  modulator  (endacoid)  which 
regulates  5HT  uptake,  an  important  step  in  serotonergic  transmission.  Hence, 

193 


Dr.  Barbaccia  has  partially  purified  from  brain  a  smal 1,  molecular  weight 
thermostable  nonpeptidic  putative  endacoid  that  displaces  H-imipramine  from 
binding.  Its  biological  activity  is  lost  in  0.1  N  solutions  of  strong  acid  (HCl 
or  HCIO.).  It  is  polar  and  as  such  is  soluble  in  methanol  and  ethanol  but  much 
less  in  propanol  or  acetonitri le.  It  can  be  separated  from  endogenous  5HT  on  a 
reversed  phase  HPLC  column.  This^ndacoid  inhibits  in  a  similar  dose  dependent 
manner  H-imipramine  binding  and  H-5HT  uptake  in  rat  brain.  In  rat,  its  brain 
content  is  not  affected  by  a  pretreatment  with  reserpine  in  doses  that  deplete 
catecholamines  and  serotonin  stores  or  p-chlorophenylalanine  (P-CPA)  in  doses 
that  inhibit  5HT  synthesis.  However,  a  selective  lesion  of  5HT  axon  terminals 
by  i.c.v.  injection  of  5,-7-dihydroxytryptamine  decreases  while  a  preloading 
of  the  rats  with  tryptophan  ethyl  ester  increases  the  whole  brain  content  of  the 
putative  endacoid. 

HPLC  retention  time,  GC/MS  fragmentation  pattern  and  other  chemico-physical 
characteristics  show  that  this  putative  endacoid  differs  from  a  series  of 
indolealkylamine  derivatives  and  from  tryptoline,  5-OH  tryptoline,  5- 
methoxytryptoline.  The  tryptoJine  derivatives  inhibit  in  the  high  nanomolar 
range  H-5HT  uptake  and  H-imipramine  binding.  In  particular  5- 
methoxytryptol ine  has  been  reported  to  be  present  in  high  concentrations  in 
human  pineal.  On  the  contrary,  in  rat  brain  we  could  not  detect  significant 
amounts  of  5-methoxytyptol ine.  Moreover,  the  putative  endacoid  extracted  from 
rat  brain  appears  to  have  a  different  regional  distribution  when  compared  to  5- 
methoxytryptol ine:  the  highest  amount  is  located  in  corpus  striatum  followed  by 
hippocampus,  cerebral  cortex,  diencephalon  brain  stem  cerebellum,  hypothalamus, 
olfactory  bulb.  A  knowledge  of  the  chemical  nature  of  this  putative  endacoid 
that  still  eludes  identification  would  prompt  new  information  on  the 
supramolecular  organization  of  the  5HT  reuptake  system,  on  the  multiplicity  of 
signals  at  the  synaptic  level  and  would  enable  us  to  measure  it  in  biological 
fluids  of  patients  suffering  from  various  kinds  of  affective  disorders. 

Drs.  Ravizza,  Barbaccia  and  Pozzi  have  studied  the  pharmacological  profile  of 
another  antidepressant:  maprotiline  which  is  a  drug  of  clinical  efficacy.  Its 
most  peculiar  pharmacological  property  is  that  it  potently  and  selectively 
inhibits  the  reuptake  of  norepinephrine  (NE)  in  vitro.  Despite  this  in  vitro 
effect,  when  i.p.  maprotiline  was  given  to  rats  daily  for  3  weeks,  the  activity 
of  the  cAMP  generating  system  coupled  to  beta-adrenoceptor  and  the  number  of 
beta-adrenoceptor  recognition  sites  was  not  reduced.  This  was  at  variance  with 
what  one  would  expect  following  continuous  blockade  of  NE  reuptake  and  chronic 
exposure  of  the  postsynaptic  receptors  to  increased  amounts  of  endogenous 
agonist.  Furthermore,  3  weeks  of  treatment  with  maprotiline  failed  to  decrease 
the  number  of  SHT^  recognition  sites  or  of  H-mianserin  recognition  sites, 
although  maprotiline  has  a  reasonably  high  affinity  for  these  two  recognition 
sites  in  vitro.  However,  the  same  treatment  schedule  elicited  a  decrease  in  H- 
f lunitrazepam  and  H-pC  binding  to  the  hypothalamic  and  hippocampal  membranes. 
These  effects  (decreased  Vmax  for  H-flu  and  decreased  affinity  for  H-^C 
binding)  do  not  appear  to  be  related  to  changes  in  endogenous  GABA  levels. 
These  results  raise  several  possibilities,  1)  the  down  regulation  of  the  beta- 
adrenoceptor  linked  cAMP  generating  system  following  repeated  daily  injections 
of  antidepressants  does  not  seem  to  depend  merely  on  the  increase  in  synaptic 
levels  of  endogenous  transmitters;  2)  there  appears  to  be  more  and  more 
exceptions  to  the  rule  that  all  antidepressants  decrease  the  beta  adrenoceptor 
function  upon  chronic  treatment;  and  3)  the  brain  B-pC  recognition  sites  appear 
to  be  involved  in  the  long  term  action  of  antidepressants.    Hence, 


194 


antidepressants  of  clinical  significance  appear  to  be  a  nonhomogeneous  class  of 
drugs  which  relieve  the  symptoms  of  affective  disorders  by  a  varipfv'  of 
mechanisms.  All  these  indirectly  trigger  a  mouification  or  several  syu^^dc 
mechanisms. 

2+ 
B)  GABAp  receptors  and  Ca — channels 

Drs.  W.  Wojcik  (Staff  Fellow),  J.  Xu  (Visiting  Fellow)  and  E.  Carboni  (Guest 
Researcher)  continued  their  studies  on  the  adenosine  Al  and  the  GABA  B  receptors 
which  are  found  to  inhibit  the  activity  of  adenylate  cyclase  coupled  to  the 
guanine  nucleotide  inhibitory  (Ni)  unit  of  adenylate  cyclase  to  prevent  calcium 
uptake  and  block  the  release  of  excitatory  transmitters.  The  activation  of  this 
group  of  receptors  attenuates  cyclic  AMP  formation  and  subsequently  reduces 
cyclic  AMP  dependent  protein  phosphorylation.  Furthermore,  the  Ca  uptake, 
which  is  essential  for  the  release  of  transmitt^^,  was  hypothesized  to  occur 
through  the  dihydropyridine-voltage  sensitive  Ca  channels  (VSCC).  They  have 
postulated  that  VSCC  can  be  modulated  by  cyclic  AMP  dependent  phosphorylation. 
For  example,  phosphorylation  of  the  channel  protein  may  facilitate  Ca  entry 
during  depolarization  while  no  phosphorylation  may  inhibit  Ca  entry.  The 
primary  cultures  of  cerebellar  granule  cells  express  GABA  B  receptors,  the  VSCCs 
and  the  excitatory  transmitters  are  used  as  a  model  for  these  studies. 

C) .  Receptors  for  excitatory  dicarboxylic  acids 

Studies  by  Drs.  J.  Wroblewski  (Guest  Researcher),  F.  Nicoletti  (Guest 
Researcher)  and  A.  Novelli  (Guest  Researcher)  indicate  that  also  receptors  for 
dicarboxylic  excitatory  amino  acids  functioning  as  putative  transmitters  are 
supramolecular  structures  which  includes  at  least  three  subunits:  a  recognition 
site  for  the  transmitter,  a  coupling  mechanism,  and  a  signal  transducing  device. 
There  are  several  types  of  dicarboxylic  amino  acid  receptors  which  are 
diffentiated  by  the  characteristics  of  the  recognition  sites.  Moreover, 
couplers  and  transducers  contribute  to  create  additional  diversity  of  these 
receptors.  Drs^^  Wroblewski  and  NicolettJ^  have  detected  two  couplers,  one 
inhibited  by  Mg  which  is  related  to  a  Ca  channel  whose  activation  couples 
the  recognition  sites  with  guanylate  cyclase  functioning  as  a  signal 
transducer.  In  the  frame  of  reference  suggested  above,  this  receptor  would  have 
the  signal  transducing  subunit  located  intracellularly.  There  are  two  types  of 
Ca  channels  that  participate  in  the  dicarboxylic  amino  acid  signal 
transduction,  one  is  voltage  dependent  and  the  other  voltage  independent,  the 
latter  is  functionally  linked  to  the  recognition  site.  Another  transducer 
system  operative  in  these  receptors  is  a  phosphatidyl  inositide  specific 
phospholipase  A  which  catlyzes  the  format ioru^  of  inositol  phosphates. 
Biochemical ly,  the  receptor  that  is  coupled  with  Ca  channel  can  be  studied  by 
measuring  Ca  uptake  and  or  cyclic  GMP  formation,  the  phospholipase  linked 
receptor  can  be  studied  by  measuring  inositol  I  phosphate  accumulation  in  the 
presence  of  high  concentrations  of  Li.  In  hippocampal  slices  prepared  from  rats 
the  latter  receptorial  system  is  abundant  and  is  inhibited  by  2- 
aminophosphobutyric  acid.  The  activity  of  this  receptorial  system  is  enhanced 
by  denervation  and  is  particularly  high  in  new  born  animals.  The  recognition 
sites  specific  for  aspartate,  NMDA  and  ibotenate  are  active  only  in  the  absence 
of  Mg  ,  kainate  and  glutama^e  recognition  sites,  also  express  receptor 
activity  in  the  presence  of  Mg  . 


195 


D)  Trans-synaptic  control  of  the  Ca^^/phospholipid-dependent  protein  kinase. 

O  J. 

The  Ca  /phospholipid-dependent  protein  kinase  (protein  kinase  C)  is  believed 
to  be  an  intracellular  mediator  of  the  actions  of  the  extracellular  signals  that 
stimulate  the  hydrolysis  of  phosphatidylinositol  to  inositol  phosphate  and 
diacylglycerol  via  activation  of  a  specific  inositide  phospholipase  A. 
Diacylglycerol  is  an  in  vitro  activator  of  protein  kinase  C  and  may  act  as  a 
second  messenger  intracel lularly.  Dr.  B.  Wise  (Staff  Fellow)  found  that  tumor 
promoting  phorbol  esters  mimic  the  action  of  diacylglycerol  on  protein  kinase  C 
activity,  and  in  chromaffin  cells  these  compounds  inhibit  membrane 
translocation  of  soluble  protein  kinase  C  activity.  The  effects  of  phorbol 
esters  are  rapid  and  biologically  specific.  In  chromaffin  cells,  they  produce  a 
dose-dependent  release  of  both  NE  and  epinephrine.  Several  neurotransmitters 
and  peptides  were  studied  for  their  effects  on  protein  kinase  C  translocation 
from  cytosol  to  membranes  of  chromaffin  cells.  No  consistent  effects  have  yet 
been  detected. 

The  presence  of  neurotransmitter  stimulated  activation  of  protein  kinase  C  was 
also  investigated  in  primary  cultures  of  cerebellar  granule  cells.  Drs.  Wise 
and  Nicoletti  have  demonstrated  that  glutamate  which  stimulates 
phosphatidylinositol  hydrolysis  in  these  cells  caused  about  a  20  to  30%  increase 
in  membrane  associated  kinase  activity.  Although  preliminary  in  nature,  these 
results  suggest  that  glutamate  by  stimulating  diacylglycerol  formation  may 
activate  protein  kinase  C  with  a  consequent  translocation  and  specific 
phosphorylation  of  endogenous  membrane  proteins. 

E)  Neurotrophic  factors 

Trophic  factors  have  been  postulated  to  exist  for  central  nervous  system  (CNS) 
neurons.  The  predominantly  cholinergic  septo  hippocampal  pathway  was  chosen  by 
Drs.  Wise  and  Emerit  (Visiting  Fellow)  as  the  model  to  study  CNS  target  derived 
(i.e.  hippocampal)  trophic  factors.  Fimbria  fornix  lesions  of  the  septal 
hippocampal  pathway  are  known  to  decrease  cholinergic  activity  in  the  target 
hippocampus  and,  so,  may  induce  the  synthesis  of  a  neurotrophic  factor.  Primary 
explant  and  dissociated  cell  cultures  of  the  fetal  rat  septal  area  have  been  set 
up  to  study  the  pre-  and  post  lesion  levels  of  hippocampal  neurotrophic  factors 
and  also  as  an  assay  system  for  the  purification  and  characterization  of  the 
factor(s).  In  addition,  a  molecular  biological  approach  to  tlpis  problem  is 
being  carried  out.  Complementary  DNA  to  rat  hippocampal  poly  (A  )  RNA  has  been 
synthesized  by  Dr.  Emerit  and  will  be  used  in  cDNA-mRNA  hybridizations  to 
enrich  those  mRNAs  that  are  induced  as  a  result  of  septo  hippocampal  lesions. 
The  induced  or  novel  mRNA  species  can  be  detected  by  in  vitro  translation  and 
identification  of  the  synthesized  proteins  by  molecular  weight  by  functional 
assays.  The  eventual  goal  is  to  clone  the  cDNA  for  the  neurotrophic  factor  in 
order  to  delineate  its  stucture  and  to  study  the  regulation  of  its  expression. 

Dr.  J.  Byrd  (Guest  Researcher)  studied  the  promoters  in  the  PC12 
pheochromocytoma  cell  line.  The  differentiation  promoter,  butyric  acid,  has 
rapid  marked  effects  on  both  the  morphology  and  biochemical  nature  of  these 
cells.  These  changes  include  the  following:  a  marked  increase  in  cell-cell  and 
cell-substratum  adhesion;  ultrastructural  changes  in  the  cell  nucleus 
suggestive  of  neuronal  differentiation;  cessation  of  cell  proliferation  and  DNA 
synthesis;  stimulation  of  neuron-specific  enolase,  a  marker  for  APUD  cells; 
appearance  of  proenkephal in  mRNA  and  its  gene  products,  and  stimulation  of 


196 


transglutaminase  (TGASE),  an  enzyme  whose  activity  has  been  correlated  with 
both  the  state  of  differentiation  as  well  as  the  state  of  transformation  of  a 
variety  of  tissues.  The  tissue  form  and  the  epidermal  form  of  TGASE  are  present 
in  PC  12  cells.  Sodium  butyrate  not  only  increased  the  activity  of  the 
epidermal  form  of  the  enzyme,  but  also  stimulated  the  synthesis  of  tissue 
transglutaminase  as  well.  Dr.  Byrd  is  currently  exploring  how  the  differential 
expression  of  this  enzyme  following  exposure  to  these  two  different  promotors 
migh  account  for  the  different  morphological  characteristics  of  the  treated 
cells,  the  forms  of  which  are  each  quite  distinct. 

E)  Brain  muscarinic  receptors 

The  occupation  of  brain  muscarinic  receptors  by  specific  agonists  stimulates 
the  phosphatidil inositol  (PI)  turnover  in  vitro.  Dr.  C.  Eva  (Visiting  Fellow) 
investigated  the  effect  of  the  ionic  composition  of  the  incubation  medium  on  the 
efficacy  of  this  signal  transduction.  In  rat  hippocampal  slices  kept  in  Krebs- 
Henseleit  medium,  an  increase  of  K  ions  to  12  mM  potentiates  the  stimulation  of 
PI  turnover  elicited  by  carbachol  and  cis  (+)methyldioxolane.  Oxotremorine  is 
inactive  if  tested  in  Krebs  Henselei"^  medium  but  it  stimulates  by  220%  the  PI 
turnover  in  the  presence  of  high  K  .  The  facilitation  of  the  carbachol 
stimulation  of  PI  turnover  was  blocked  by  pirenzepine,  a  muscarinic  antagonist. 
This  drug  was  equally  potent  in  inhibiting  the  carbachol  stimulation  of  PI 
turnover  both  in  normal  and  12  mM  K  Krebs  medium.  This  facilitatory  effect  of 
K  appears  to  be  preferential  for  muscarinic  receptors  since  it  failed  to 
increase  the  activtion  of  PI  turnover  induced  by  NE  norepinephrine  and 
histamine.  Dr.  Eva  is  now  characterizing  the  two  classes  of  brain  muscarinic 
receptors,  one  linked  to  adenylate  cyclase  and  the  other  to  phospholipase  in 
cholinergically  denervated  tissue;  moreover,  the  role  of  these  receptors  in  the 
behavioral  responses  to  cholinergic  drugs  is  being  studied  using  cloning  in  a 
maze  situation.  The  purpose  is  to  characterize  the  role  of  the  cholinergic 
receptors  biochemically  (denervation)  and  behaviorally. 

F).  Opiate  Receptors: 

Dr.  R.  Rothman  (Guest  Researcher)  and  Ms.  J.  Danks  (Guest  Researcher)  have  been 
studying  the  use  of  site-directed  alkylating  agents  to  define  opiate  receptor 
subtypes.  Previous  work  demonstrated  that  pretreatment  of  m^mbranes2with  t^e 
fentanyl  derivative  FIT  eliminates  the  higher  affinity  H-D-Ala  -0-Leu  - 
enkephalin  binding  site.  Additional  work  has  shown  that  this  delta^selecti^e 
irreversible  ligand^lso  alters  the  properties  of  the  lower  affinity  H-D-Ala  - 
D-Leu  -enkephalin  (  H-DADL)  binding  site.  Additional  experiments  have  shown 
that  pretreatment  of  membranes  with  both  FIT  and  the  mu-receptor  directs  an 
irreversible  ligand,  BIT,  and  produces  a  membrane  preparation  highly  enriched 
with  kappa  opiate  receptors.  Using  this  protocol,  the  distribution  of  kappa 
receptors  in  the  hippocampus  of  several  species  was  determined  using 
autoradiographic  techniques.  Of  some  interest  is  the  finding  that  opiate 
receptors  in  the  rat  pituitary  are  confined  to  the  neural  lobe  and  are 
exclusively  kappa  receptors.  A  second  line  of  investigation  was  to  identify  jn 
vivo  manipulations  of  rats  that  will  produce  selective  alterations  in  opiate 
receptors  assayed  in  vitro.  Thus  using  the  site-directed  alkylating  agents  to 
define  3H-DALA  binding  sites,  we  have  shown  that  whereas  chronic  morphine  causes 
an  up  regulation  of  the  lower  affinity  3H-DADL  binding  site,  chronic 
electroconvulsive  shock  causes  a  down-regulation  of  this  binding  site.  It  is 
likely  that  these  observations  may  lead  to  some  understanding  of  the  receptor 

197 


modifications  underlying  the  development  of  tolerance  and  dependence  to 
opiates. 


198  <.U.S.  GOVERNMENT  PRINTING  OFFICE:  1986-'+91-292!'*1011 


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