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Huntington’s chorea and schizophrenia : amino acids in thalamus Buchanan, Janet Ann 1978

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HUNTINGTON'S CHOREA AND SCHIZOPHRENIA: AMINO ACIDS IN THALAMUS  by  JANET ANN BUCHANAN B.Sc, McGi 11 University, 1976  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department of Medical Genetics)  We accept this thesis as conforming to the required standard  September, 1978 Janet Ann Buchanan, 1978  In presenting this thesis in partial  fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Depa rtment The University of B r i t i s h Columbia  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date  ABSTRACT Amino acids and other ninhydrin-positive compounds were measured i n post-mortem thalamus from 25 Huntington's choreics, 10 schizophrenics, 5 schizophrenic-like psychotics, and 23 controls dying without neurological disease.  Gamma-aminobutyric acid (GABA) was s i g n i f i c a n t l y reduced i n  choreic thalami, i n accord  with deficiencies found i n other brain regions  choreics (Perry et a l . , 1973a,b).  GABA was also s i g n i f i c a n t l y reduced i n  schizophrenic thalami, suggesting a biochemical l i n k between these two diseases, and supporting the hypothesis of a defect i n the GABA system i n schizophrenia (Roberts, 1972). Homocarnosine, a GABA-containing dipeptide, was also low i n choreic and 9 out of 10 schizophrenic thalami. schizophrenic had extremely high homocarnosine.  One  Glycerophosphoethanolamine  was s i g n i f i c a n t l y elevated i n Huntington's choreics, but not i n schizophrenics. A number of other variables were considered for their potential influence on amino acid concentrations i n thalamus.  The majority of amino  acids were found to rise i n a s i g n i f i c a n t l y linear fashion i n the interval 3 to 49 hours post-mortem, although other models might have described the change better.  GABA, ornithine, histidine and tyrosine were found to  decrease s i g n i f i c a n t l y with increasing age between 21 and 80 years, i n controls.  The effects of pre-mortem hypoxia, regional variation  within the thalamus, and neuroleptic drug treatment could not be rigorously tested with these data.  Neuroleptics were unlikely to have been  the cause of group differences i n GABA concentration, since they failed to deplete GABA i n brain of chronically treated rats.  On the other hand, ..  bronchopneumonia and other causes of pre-mortem hypoxia could not be ruled out as potential contributers to reduced GABA i n thalamus.  iii TABLE OF CONTENTS ABSTRACT  •. . - i i  LIST OF TABLES  v ±  LIST OF FIGURES  ..  LIST OF ABBREVIATIONS  Y  ACKNOWLEDGEMENTS CHAPTER 1  1 1 1  i x  GENERAL INTRODUCTION I HUNTINGTON'S CHOREA A. H i s t o r i c a l Background  .  B. Prevalence and Social Impact.......... C. Pathology D. C l i n i c a l Features...  1  ..  1) Classical HC 2) Juvenile Variant 3) Westphal Variant E. Age of Onset and Age at Death F. Genetic Studies II  1 2 2 .2 .3 3 4 4  SCHIZOPHRENIA A. H i s t o r i c a l Background  .6  B. Prevalence and Social Impact...  6  C. Symptoms and Classification  6  D. Genetic Studies  7  1) Evidence for Genetic Factors 2) Possible Modes of Transmission..... CHAPTER 2  v ± ±  .....8 8  INTRODUCTION TO BIOCHEMICAL THEORIES AND FINDINGS I  SCHIZOPHRENIA A. The Transmethylation Hypothesis  10  B. The Dopamine Hypothesis,  10  1) Evidence for Dopamine Involvement a) Neuroleptic Drugs  11  b) Alpha-methyl Tyrosine  12  c) Amphetamine Psychosis  12  2) The Limbic System a) Anatomy  13  b) The Limbic System i n Schizophrenia.....  13  3) Possible Causes of Dopaminergic Excess  14  iv a) GABA and t h e Dopamine H y p o t h e s i s C. Low P l a t e l e t MAO i n S c h i z o p h r e n i a D. A l t e r e d T r a n s m i t t e r s II  and R e l a t e d  14 .  15  Enzymes  .16  and R e l a t e d Enzymes  17  HUNTINGTON'S CHOREA A. A l t e r e d T r a n s m i t t e r s  B. I m m u n o l o g i c a l F i n d i n g s  CHAPTER 3  .20  C. E v i d e n c e f o r A l t e r e d Membranes  21  D. GABA R e c e p t o r s .  21  OTHER INFLUENTIAL VARIABLES A. Post-mortem H a n d l i n g  .22  1) GAD  22  2) Amino A c i d s  22  B. Pre-mortem F a c t o r s  23  C. Age  24  D. Drugs E. R e g i o n a l  .'  25  Variation.  26  CHAPTER 4  THE THALAMUS  •  CHAPTER 5  PURPOSE AND RATIONALE OF THE PRESENT INVESTIGATION  CHAPTER 6  MATERIALS AND METHODS  28  A. Sources o f B r a i n T i s s u e .  29  B. H a n d l i n g  29  C. P r e p a r a t i o n  o f B r a i n Tissue  f o r Amino A c i d  Analysis  ••  30  D. Amino A c i d A n a l y s i s  30  E. S t a t i s t i c a l Methods  30  F. D a t a Used i n S t a t i s t i c a l A n a l y s e s G. The Independent V a r i a b l e s H. The Dependent V a r i a b l e s I. S t a t i s t i c a l Protocol CHAPTER 7  27  ...31 31 ....32 33  RESULTS A. The Independent V a r i a b l e s 1) Age  34  2) Post-mortem D e l a y .  34  a) E s t i m a t e s o f PMD  34  V  b) PMD - Group D i f f e r e n c e s 3) Drug  B.  36  Histories  a) C o n t r o l s  37  b) H u n t i n g t o n ' s C h o r e i c s  37  c) S c h i z o p h r e n i c s  37  d) S c h i z o p h r e n i c - l i k e P s y c h o t i c s  38  4) Cause o f Death  38  5) Regions o f Thalamus  38  The Amino A c i d s 1) GABA a) Data U n c o r r e c t e d b) E f f e c t s  f o r Age and PMD  o f Age and PMD on GABA  c) GABA - D i f f e r e n c e s Among Groups, A c c o u n t i n g f o r Age and PMD 2) Glycerophosphoethanolamine 3) Homocarnosine  (GLYC-PEA)  (HCARN)  40 40 42 47 48  4) Amino A c i d s Showing no S i g n i f i c a n t L i n e a r Change i n C o n t r o l s (n = 21 ) w i t h Age (range, 21-80 y e a r s ) o r PMD (range, 3-49 h o u r s ) and no S i g n i f i c a n t D i f f e r e n c e s Among Groups 48  CHAPTER 8 REFERENCES  5) Amino A c i d s Showing a S i g n i f i c a n t L i n e a r Change i n C o n t r o l s (n=21) w i t h PMD (range, 3-49 h o u r s ) b u t n o t w i t h Age (range, 21-80 years)  52  6) Amino A c i d s Showing S i g n i f i c a n t L i n e a r Changes i n C o n t r o l s (n=21) With Both PMD (range, 3-49 h o u r s ) and Age (range, 21-80 hours)  52  DISCUSSION  ,  55 59  vi  LIST OF TABLES I II III  Studies of Neurotransmitters and Related Enzymes i n HC  18  Detectable MIF a c t i v i t y i n Cultured Lymphocytes (Barkley et a l . , 1977a,b, 1978)  20  GAD a c t i v i t y i n Thalamus - Effect of Coma (McGeer et a l . , 1973a) *.  23  IV  Mean Age (years) of Controls, Huntington's Choreics and Schizophrenics  34  V  Estimates of PMD for 11 Individuals  VI  Mean PMD (hours) for Controls, Huntington's Choreics and Schizophrenics (Without estimates of PMD)  36  VII  Mean PMD (hours) for Controls, Huntington's Choreics and Schizophrenics (Including estimates of PMD)  37  VIII  Summary of Neuroleptic Drug Histories  38  IX  Causes of Death  39  X  Regions of Thalamus Sampled  39  XI  Mean GABA Concentration (pmoles/gm wet weight) of Controls, Huntington's Choreics and Schizophrenics  40  Multiple Regression Analysis of GABA vs. Age and ln(PMD) i n Controls (n = 21) for whom Age and PMD were Known  42  XII XIII XIV  XV  XVI  XVII  ...  Tabulated Variables for Each Individual, and Deviations of GABA Values from Expected GABA Values Mean GABA Deviations (umoles /gm wet weight) of Controls, Huntington's Choreics and Schizophrenics (Without estimates of PMD) Mean GABA Deviations (jimoles/gm wet weight) of Controls, Huntington's Choreics and Schizophrenics (Including estimates of PMD)  36  44-46  47  .47  Mean GLYC-PEA Concentration (jimoles/gm wet weight) i n Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-like Psychotics  48  Mean HCARN Concentration (umoles/gm wet weight) i n Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-like Psychotics .  51  vii XVIII  XIX.  XX  Amino Acids Showing no Significant Linear Change i n Controls (n = 21) with Age (range, 21-80 years) or PMD (range 3-49 hours) and no Significant Differences Among Diagnostic Groups  ....51  Amino Acids Showing a Significant Linear Change i n Controls (n = 21) with PMD (range, 3-49 hours) but not with Age (range, 21-80 years)  53  Amino Acids Showing Significant Linear Changes i n Controls (n = 21) with Both PMD (range, 3-49 hours) and Age (range, 21-80 years)  53  LIST OF FIGURES I  Age Distribution i n Controls, Huntington's choreics and Schizophrenics  II  Postmortem delay distribution i n Controls, Huntington's choreics and Schizophrenics GABA Concentration (uncorrected data) i n Controls, Huntington's choreics and Schizophrenics GLYC-PEA Concentration i n Controls, Huntington's choreics and Schizophrenics Homocarnosine (HCARN) Concentration i n Controls, Huntington's choreics and Schizophrenics  III IV V  ;  VI  Threonine vs. Postmortem Delay  35 35 41 49 .50 54  viii LIST OF ABBREVIATIONS  ACh  acetyl choline  AChE  ...acetylcholinesterase  HIS  histidine  up  .haloperidol  ALA  alanine  HVA  homovanillic  ANOVA  A n a l y s i s of Variance  ILE.  isoleucine  ARG  arginine  LEU  leucine  ASN  asparagine  l  natural  ASP  aspartic acid  LYS. . . . l y s i n e  BP  bronchopneumonia  MAO.  C....  controls  MET  methionine  CAT  choline acetyl transferase  Ml  myocardial  C P Z  chldrpromazine  MIF. . . . m i g r a t i o n  CSF  cerebrospinal  (  C Y S  )  2  CYSTA D  D  d  (in tables)  fluid  cystine  M  cystathionine  °C f  ORN  . . . ornithine  dopamine d e c a r b o x y l a s e  PEA. . . . phosphoethanolamine  degrees o f freedom  PHE..  dizygotic  PRO.  E  ethanolamine  r  acid  EPS  extra-pyramidal  E  e l e c t r o n s p i n resonance  .  .phenylalanine  PMD . . . post-mortem  2  system  delay  . . . proline . . . c o e f f i c i e n t of =  s  o  n  w  t  a  gamma-amino b u t y r i c a c i d  S. . . . s c h i z o p h r e n i c s  GABA'  expected v a l u e of GABA (from regression analysis)  SEM.  glutamic  acid decarboxylase  GLU  glutamic  acid  GP  globus p a l l i d u s  GSH;  .....  --reduced g l u t a t h i o n i n e ( i n tables f o r t h i s study, t h i s a b b r e v i a t i o n r e f e r s to t o t a l  GSH  (GSH + 2GS-SG))  GS-SG.  oxidized  glutathione  HC  H u n t i n g t o n ' s chorea  HCARN  homocarnosine  e r r o r of the mean  SL. . . . s c h i z o p h r e n i c - l i k e SS. . . . sums o f squares TAU.  GLYC-PEA. . . .glycerophosphoethanolamine glycine  . . standard  (in tables)  SER.... s e r i n e  GABA-LYS... ..gamma-amino b u t y r y l l y s i n e GAD  determination  < P r o p o r t i o n of t o t a l v a r i a n c e accounted by r e g r e s i i h given v a r i a b l e )  GABA  GLY  factor  NE . . . . n o r e p i n e p h r i n e  DZ  R  inhibition  ..monozygotic  dihydroxyphenylacetic  S  infarction  multiple sclerosis  Z  DOPAC  A  logarithm  . . . monoamine o x i d a s e  MS  : dopamine  A  n  acid  . . taurine  THR . . . t h r e o n i n e TRP. TYR. V  A  L  . . tryptophan . . tyrosine • • -valine  psychotics  (in tables)  ix  ACKNOWLEDGEMENTS  I would l i k e  t o thank f i r s t  committee, Dr. J.R. M i l l e r Dr. D. A p p l e g a r t h , to  t h i s study.  ( c h a i r p e r s o n ) , Dr. T.L. P e r r y ( s u p e r v i s o r ) ,  Dr. P. MacLeod, and Dr. S. Wood f o r t h e i r c o n t r i b u t i o n s  In p a r t i c u l a r I thank Dr. P e r r y and Dr. M i l l e r  many moments o f t h e i r questions  the members o f my t h e s i s a d v i s o r y  f o r the  time, g i v e n a t a moment's n o t i c e f o r the e n d l e s s  o f a student.  I would a l s o l i k e Dr. P e r r y ' s  t o o f f e r a s p e c i a l thanks t o o t h e r members o f  l a b , Mrs. S h i r l e y Hansen, Mrs. Maureen Murphy and Mr. Stephen  Kish f o r both  t e c h n i c a l a s s i s t a n c e and g e n e r a l s u p p o r t  throughout  this  project. We are i n d e b t e d for  t o Dr. E.D. B i r d , U n i v e r s i t y o f Cambridge, E n g l a n d  p r o v i d i n g the m a j o r i t y o f thalamus specimens used i n t h i s  study.  I am most g r a t e f u l t o Dr. C. Wehrhahn f o r h i s time g i v e n i n numerous statistical  c o n s u l t a t i o n s , and f o r programming and r u n n i n g  computer a n a l y s e s .  a l l o f the  I thank a l s o Dr. A T i n g l e f o r a d i s c u s s i o n o f  immunological s t u d i e s . The Huntington s c h o l a r s h i p , without  S o c i e t y o f Canada p r o v i d e d me w i t h which t h i s study  a pre-doctoral  c o u l d n o t have been u n d e r t a k e n .  I a p p r e c i a t e the a s s i s t a n c e o f Ms. A r l e e n Hardy and Ms. S h e i l a Manning in  the p r e p a r a t i o n o f the f i n a l F i n a l l y , I would l i k e  Genetics  manuscript.  t o thank my f r i e n d s i n the Department o f M e d i c a l  and a t home, who have coped w i t h  this  thesis  admirably.  •1  CHAPTER 1 GENERAL INTRODUCTION I  HUNTINGTON'S CHOREA  A.  H i s t o r i c a l Background ( D i s c u s s e d by M y r i a n t h o p o u l o s , 1966; H e a t h f i e l d , 1973; V e s s i e , 1939; M a l t s g e r g e r , 1961; C r i t c h l e y , 1973; De Jong, 1973) In  the  1872, George H u n t i n g t o n , a young New Y o r k p h y s i c i a n , a d d r e s s e d  m e d i c a l academy i n M i d d l e p o r t , Ohio.  I n c l u d e d i n h i s t a l k was a  d e s c r i p t i o n o f h e r e d i t a r y c h o r e a w i t h d e m e n t i a , thought a t t h e t i m e t o be the  first  such r e p o r t .  He r e c o g n i z e d 3 marked p e c u l i a r i t i e s o f t h e d i s e a s e :  i t s h e r e d i t a r y n a t u r e , a tendency t o i n s a n i t y and s u i c i d e , and i t s m a n i f e s t a t i o n as a grave d i s e a s e o n l y i n a d u l t l i f e .  A l t h o u g h i t i s now  known t h a t r e p o r t s o f h e r e d i t a r y c h o r e a h a d appeared i n t h e e a r l i e r literature  ( i n p a r t i c u l a r , one by Waters i n t h e 1 8 4 0 ' s ) , H u n t i n g t o n was  the  f i r s t t o d e s c r i b e dementia as an e s s e n t i a l f e a t u r e ( M a l t s b e r g e r , 1961).  His  d i s c u s s i o n was s o s u c c i n c t and l u c i d t h a t t h e name a s s o c i a t i o n has been  well  justified. V e s s i e (1939) t r a c e d most a f f e c t e d New E n g l a n d f a m i l i e s t o 3 B r i t i s h  i m m i g r a n t s who a r r i v e d i n A m e r i c a about 1632. S e v e r a l o f t h e i r were n o t o r i o u s w i t c h e s , burned f o r t h e i r c u r s e .  descendants  Nova S c o t i a f a m i l i e s have  been t r a c e d t o t h e Huguenots who f l e d France a f t e r 1685, and Quebec f a m i l i e s to  B.  a s i n g l e a n c e s t o r who e m i g r a t e d from F r a n c e i n 1645.  P r e v a l e n c e and S o c i a l  Impact  There i s no e v i d e n c e o f r a c i a l , e t h n i c o r g e o g r a p h i c s e l e c t i v i t y f o r H u n t i n g t o n ' s Chorea  (HC).  Most p r e v a l e n c e e s t i m a t e s range from 4 t o 7 p e r  100,000 ( M y r i a n t h o p o u l o s , 1973), a l t h o u g h i t i s r a r e i n Japan (0.4/100,000) and v e r y p r e v a l e n t i n a few i s o l a t e s s u c h as t h e Moray F i r t h i n S c o t l a n d (560/100,000) ( H e a t h f i e l d , 1973; M y r i a n t h o p o u l o s , 1966). s t u d y ( S h o k e i r , 1975), p r e v a l e n c e i n Saskatchewan at  8.4/100,000.  I n a Canadian  and M a n i t o b a was e s t i m a t e d  A t t h e end o f 1977 t h e r e were 61 l i v e cases o f HC r e g i s t e r e d  2  with the B r i t i s h Columbia Health Surveillance Registry, however sources of ascertainment have been limited (Guy Renwick, personal communication) . In one medical genetics c l i n i c (Bird and H a l l , 1978) HC has been the most frequent cause for r e f e r r a l , accounting for 11.4% of a l l i n i t i a l v i s i t s . Clearly the impact weighing on families of a f f l i c t e d individuals i s tremendous, due to the chronic and insidious nature of the disease.  Society  too must share the financial burden of unemployment and the chronic hospital i z a t i o n which i s so frequently inevitable (Myrianthopoulos, 1966). C.  Pathology (Discussed by Heathfield, 1973; Myrianthopoulos, 1966; Klintworth, 1973) The most conspicuous pathological feature i s severe atrophy of the  corpus striatum with (microscopically) loss of small neurons and accompanying astrocytic p r o l i f e r a t i o n .  The caudate i s more severely affected than the  putamen. Generalized shrinking of the brain i s accompanied by d i l a t a t i o n of the l a t e r a l ventricles especially i n the anterior horns, with flattening of the caudate.  There i s s i m i l a r , but less marked involvement of cerebral cortex  (especially f r o n t a l lobes), globus-pallidus (GP), substantia nigra (SN), thalamus, sub-thalamic nuclei, and dentate. D.  C l i n i c a l Features There i s considerable heterogeneity  i n the c l i n i c a l expression of HC.  Three recognizable forms are the c l a s s i c a l , juvenile, and Westphal variants. 1)  C l a s s i c a l HC (Discussed by Heathfield, 1973; Brackenridge, 1971; Maltsberger, 1961)  The c l a s s i c a l form of the disease may be summarized as a disorder of movement, personality, and cognition. athetotic with hyperkinesia.  Movement i s described as choreo-  Involuntary movements, i n i t i a l l y d i f f i c u l t to  distinguish from restlessness, become generalized so that they interfere with voluntary movement. Personality changes may begin with i r r i t a b i l i t y  3 and i r r a t i o n a l impulsive behavior which may progress to unexplained v i o l e n t outbursts. stages.  Depression i s common, and apathy and neglect c h a r a c t e r i z e l a t e r  Psychotic episodes are not uncommon; f r e q u e n t l y schizophrenia i s the  i n i t i a l diagnosis. recognized symptom.  Cognitive impairment often presents as the f i r s t The i n a b i l i t y to remember, organize, and  concentrate,  and the loss of judgement may be the most d e b i l i t a t i n g symptoms.  Generally  there i s no impairment of immediate r e c a l l ; r a t h e r , d i f f i c u l t y w i t h tasks r e q u i r i n g delayed r e c a l l and r e t r i e v a l , s i m i l a r to problems encountered i n normal aging (Caine, 1978). 2)  P a t i e n t s e v e n t u a l l y become profoundly demented.  J u v e n i l e V a r i a n t (Discussed by Byers, 1967: Barbeau, 1970; Myrianthopoulos, 1966, 1973; H e a t h f i e l d , 1973)  About 8% of Huntington's before age 10.  choreics have onset before age 20, and  1-2%  This group has an almost d i s t i n c t c l i n i c a l syndrome,  characterized by r i g i d i t y (rather than chorea), h y p o k i n e s i a , parkinsonian tremor, convulsions, i n t e l l e c t u a l d e t e r i o r a t i o n , c e r e b e l l a r s i g n s , and a r a p i d l y progressive course.  Pathology i s t y p i c a l of HC but there may  c e r e b e l l a r disease a l s o (Byers, 1967).  be  Diagnosis of j u v e n i l e HC i s g e n e r a l l y  based on a c l e a r family h i s t o r y of c l a s s i c a l HC, suggesting that t h i s i s not a genetically distinct variant.  The s p e c i f i c modifying e f f e c t of an a l t e r n a t e  a l l e l e seems u n l i k e l y , since Byers (1967) found a p a i r of h a l f - s i b s , both w i t h j u v e n i l e HC.  A repeated, but as yet unexplained f i n d i n g has been the  d i s p r o p o r t i o n a t e l y high number of males among a f f e c t e d parents of j u v e n i l e cases. 3)  Westphal V a r i a n t (Discussed by Myrianthopoulos, 1973)  1966; H e a t h f i e l d ,  The Westphal v a r i a n t may represent a c l i n i c a l intermediate between the c l a s s i c a l and j u v e n i l e forms.  I t i s c h a r a c t e r i z e d by prominent r i g i d i t y ,  p o s s i b l y s e i z u r e s , and adolescent or e a r l y adult onset. again t y p i c a l .  The pathology i s  E.  Age of Onset and Age at Death (Reviewed in Brackenridge, 1971a; Myrianthopoulos, 1966; Heathfield, 1973)  4  Estimates i n the literature of the mean age of onset, or disease manifestation range from 33.8 years (Brackenridge, 1971a) to 44.0 years (Wendt, 1959).  A l l such estimates must be interpreted i n l i g h t of potential  confounding variables.  Onset i n an individual i s d i f f i c u l t to pin-point since  early signs may not be distinguishable from a 'normal' behavior spectrum. Literature surveys (e.g. Brackenridge, 1971a) may y i e l d an over-abundance of juvenile cases, due to their exceptional interest, which lower the calculated mean age of onset.  Ascertainment of cases through affected offspring w i l l  bias the estimated mean toward later onset i f e a r l i e r onset i s associated with reduced genetic fitness.  The a r t i f a c t of 'anticipation' may arise from  the f a i l u r e to account for l i v i n g individuals who could, at a later age, begin to manifest symptoms. This bias was avoided i n the studies by Brackenridge (1971a) and Wendt (1959) by the exclusion of recent generations. Age at death can be precisely determined, but calculated means are affected by similar factors of ascertainment as those j u s t discussed. Duration of i l l n e s s can be calculated by difference, and i s usually claimed to be about 15 years. F.  Genetic Studies HC i s a c l a s s i c a l example of a dominantly inherited disease with complete  penetrance.  There are, nonetheless, some genetic p e c u l i a r i t i e s .  Most  anomalous observations, such as 'anticipation' result from ascertainment bias. One that seems to be r e a l , however, i s p a t r i l i n e a l transmission , a phenomenon that could result from d i f f e r e n t i a l mortality.and/or d i f f e r e n t i a l genetic fitness (Brackenridge, 1971a). Brackenridge (1971a) reported a significant correlation between age of onset and number of offspring, accounted for primarily by affected mothers; an observation that could lend support to this hypothesis.  Also, a sex difference i n age of onset could favour the male  line of descent.  5  Could genetic mechanisms account f o r the observed phenotypic heterogeneity?  Is v a r i a t i o n the r e s u l t of d i f f e r e n t a l l e l e s at the HC  l o c u s , the modifying e f f e c t of the a l t e r n a t e 'normal' a l l e l e , or the i n f l u e n c e of genetic background? The f i r s t approach to answering these questions i s to determine whether there i s f a m i l i a l c l u s t e r i n g w i t h respect to c l i n i c a l subtypes, age of onset, and the pattern of n e u r o l o g i c a l and p s y c h i a t r i c symptoms.  Most  studies have reported a s i g n i f i c a n t s i b - s i b and p a r e n t - o f f s p r i n g c o r r e l a t i o n i n q u a n t i t a t i v e t r a i t s such as age of onset and age at death (Brackenridge, 1072b; 'Myrianthopoulos, 1966).  The concept of 'biotype', r e f e r r i n g to the  s u b j e c t i v e impression of c l i n i c a l consistency w i t h i n f a m i l i e s , has p e r s i s t e d i n the l i t e r a t u r e (e.g. Wallace, 1972), and may apply to some f a m i l i e s (Myrianthopoulos, 1966).  The phenotypic p a t t e r n i s r e l a t e d to age  of onset (Brackenridge, 1971b; Myrianthopoulos, 1973). The s p e c i f i c i n f l u e n c e of the a l t e r n a t e a l l e l e i n determining age of onset i s u n l i k e l y , since c o r r e l a t i o n c o e f f i c i e n t s f o r t h i s t r a i t i n s i b s and h a l f - s i b s are both close to .5 (Brackenridge, 1972a).  As p r e v i o u s l y  discussed, the a l t e r n a t e a l l e l e i s also u n l i k e l y to s p e c i f i c a l l y cause the juvenile variant. Wallace (1972) c i t e d evidence to support the theory t h a t genetic heterogeneity u n d e r l i e s phenotypic v a r i a b i l i t y .  A n a l y s i s of variance f o r  age of onset and age at death showed s i g n i f i c a n t l y more v a r i a t i o n between kindreds than w i t h i n .  C o r r e l a t i o n c o e f f i c i e n t s f o r these t r a i t s i n choreics  d i d not decrease markedly from s i b - s i b through p a r e n t - o f f s p r i n g to cousincousin.  On the other hand, Reed and Chandler (1958) found s i g n i f i c a n t l y  more v a r i a t i o n between sibships of a k i n d r e d than w i t h i n s i b s h i p s , supporting the theory that s i m i l a r i t y i n s i b s i s due to common background  genes.  Wallace proposed that there are at l e a s t 2 major ( g e n e t i c a l l y d i s t i n c t ) groups of c h o r e i c s , d i s t i n g u i s h e d by r e l a t i v e l y e a r l y or r e l a t i v e l y l a t e onset.  6 Brackenridge ,(1972b.).. and Myrianthopoulos  (1966, 1973) suggested that the  a l t e r n a t i v e s of a genetic continuum or genocopies, and a s i n g l e main gene w i t h m o d i f i e r s cannot be d i s t i n g u i s h e d with present data. The  distinction  might be made through biochemical s t u d i e s . II  SCHIZOPHRENIA  A.  H i s t o r i c a l Background (Discussed by Stabenau, 1977; Ban and Lehmann, 1977; B a l d e s s a r i n i , 1977; Kety and Mathysse, 1972) K r a e p e l i n i n 1896 lumped s e v e r a l p r e v i o u s l y d i s c r e t e c o n d i t i o n s under  the name of 'dementia praecox' - impaired c o g n i t i v e f u n c t i o n with onset i n e a r l y adulthood - and emphasized i t s i n e v i t a b l e malignant, prognosis.  In 1911,  B l e u l e r r e c l a s s i f i e d 'schizophrenia' - fragmentation of mental f u n c t i o n - as a group of d i s o r d e r s , enlarging the scope of the e n t i t y . primary (core) and secondary value to the present B.  His d e s c r i p t i o n of  (accessory) symptoms has r e t a i n e d i t s d i a g n o s t i c  day.  Prevalence and S o c i a l Impact (Discussed by Ban and Lehmann, 1977; B a l d e s s a r i n i , 1977) Schizophrenia i s probably the world's most important p s y c h i a t r i c  d i s o r d e r , and i s one of the greatest p u b l i c h e a l t h problems i n developed countries;  The associated s o c i a l stigma i s of course profound.  Prevalence  i s estimated at about 1% of the world p o p u l a t i o n , w i t h some v a r i a t i o n associated p r i m a r i l y with c r i t e r i a f o r d i a g n o s i s . As of .1977, approximately 20% of a l l schizophrenics i n the United States were h o s p i t a l i z e d  (Baldessarini,  1977), accounting f o r the occupancy of up to h a l f of a l l p s y c h i a t r i c beds (Ban and Lehmann, 1977) at tremendous cost.  Those not h o s p i t a l i z e d are also  expensive to s o c i e t y , even considering unemployment alone. C.  Symptoms and C l a s s i f i c a t i o n Three approaches to disease c l a s s i f i c a t i o n have been:  (2)  ( 1 ) phenomenology,  response to therapeutic i n t e r v e n t i o n and (3) cause (Falek and Moser, 1975).  These correspond to the l e v e l of understanding of the disease.  For  7 schizophrenia,  c l a s s i f i c a t i o n i s s t i l l p h e n o m e n o l o g i c a l , a l t h o u g h drug  r e s p o n s e c h a r a c t e r i s t i c s are b e g i n n i n g diagnosis.  'Schizophrenia'  t o be l i s t e d among c r i t e r i a f o r  i s an o p e r a t i o n a l d e f i n i t i o n , and  i t cannot be  over-emphasized t h a t b i o l o g i c a l homogeneity s h o u l d h o t be assumed or even e x p e c t e d ( B l a s s , 1977;  Meltzer,  1976).  B l e u l e r ' s d e s c r i p t i o n of symptoms has been the b a s i s f o r most d i a g n o s t i c protocols.  H i s core d e f i n i n g symptoms a r e :  d i s o r d e r or d i s t u r b a n c e  (1)  of a s s o c i a t i o n s , (2)  w i t h d r a w a l f r o m n o r m a l s o c i a l i n t e r a c t i o n s and interest i n external reality. intermittent) persecutory, and  i n a p p r o p r i a t e a f f e c t , (3) (4) l a c k of c o n t a c t w i t h  A c c e s s o r y symptoms i n c l u d e ( c o n t i n u o u s  and  or  g r a n d i o s e or s o m a t i c d e l u s i o n s , h a l l u c i n a t i o n s ,  c a t a t o n i c symptoms (Kety and M a t t h y s s e , 1972).  pathognomonic, and  a c h a r a c t e r i s t i c thought  No  a l l are n o n - s p e c i f i c ; thus i t i s not  one  symptom i s  s u r p r i s i n g that  r o u t i n e d i a g n o s t i c methods, even those e m p l o y i n g s o p h i s t i c a t e d computer analyses, research  are u n a c c e p t a b l e ( M e l t z e r , 1976;  issues.  has  achieved.  a s p e c t s of c l a s s i f i c a t i o n have been p a r t i c u l a r l y c o n t r o v e r s i a l One  i s the r e t e n t i o n of B l e u l e r ' s s u b - t y p e s ( i . e . c a t a t o n i c ,  p a r a n o i d , h e b e p h r e n i c and s i m p l e ) as d i s c r e t e d i a g n o s t i c e n t i t i e s d i s c u s s i o n see B a l d e s s a r i n i , 1977;  F a l e k and Moser, 1975).  K e t y ' s 'spectrum c o n c e p t ' used t o account f o r the v a r i o u s  The  p r e s e n t w i t h g r e a t e r f r e q u e n c y i n f i r s t degree r e l a t i v e s of ( M e l t z e r , 1976;  Genetic Genetic  (for  other i s  t y p e s of  psychopathology (other than f u l l y - d e v e l o p e d schizophrenia)  D.  For  p u r p o s e s , d i a g n o s t i c u n i f o r m i t y i s a minimum r e q u i r e m e n t t h a t  n o t y e t been Two  F a l e k and M o s e r , 1975).  w h i c h may  be  schizophrenics  B a l d e s s a r i n i , 1977).  Studies s t u d i e s of s c h i z o p h r e n i a  are confounded by d i a g n o s t i c p r o b l e m s ,  a s c e r t a i n m e n t b i a s e s , and p r o b a b l y a complex and h e t e r o g e n e o u s e t i o l o g y . most t h a t can be s a i d w i t h c e r t a i n t y i s t h a t r e c e n t  studies leave l i t t l e  The doubt  as t o t h e i n v o l v e m e n t 1)  • 8 of h e r e d i t a r y f a c t o r s i n the e t i o l o g y o f s c h i z o p h r e n i a .  E v i d e n c e f o r G e n e t i c F a c t o r s (Reviewed by Gottesman and S h i e l d s , 1973; D e F r i e s and P l o m i n , 1978; B a l d e s s a r i n i , .1977; K e t y , 1972; K e t y and M a t t h y s s e , 1972; Tsuang, 1976)  E v i d e n c e f o r a g e n e t i c d i a t h e s i s i n s c h i z o p h r e n i a r u n s a l o n g 3 major l i n e s , which w i l l s i m p l y be summarized.  F i r s t , there i s a higher prevalence  of s c h i z o p h r e n i a w i t h i n f a m i l i e s o f s c h i z o p h r e n i c s t h a n i n the g e n e r a l population.  There i s a l s o a c o r r e l a t i o n between t h e p r e v a l e n c e and degree of  r e l a t i o n t o the index case.  Empiric r i s k to f u l l sibs (including  (DZ) t w i n s ) and c h i l d r e n i s i n t h e o r d e r o f 10-15%. s c h i z o p h r e n i c t h e r i s k may be as h i g h as 50%.  dizygotic  I f two p a r e n t s a r e  Second, m o n o z y g o t i c (MZ) t w i n s  have a much h i g h e r concordance r a t e (more t h a n 50%) t h a n DZ t w i n s , even i f they a r e r e a r e d a p a r t .  T h i r d , adoption studies (using various  methodologies)  i n d i c a t e a h i g h e r p r e v a l e n c e o f s c h i z o p h r e n i a among b i o l o g i c a l r e l a t i v e s o f i n d e x cases t h a n among a d o p t i v e r e l a t i v e s .  In f a c t , the environmental  i n f l u e n c e of being reared w i t h a s c h i z o p h r e n i c person i s probably not r e l e v a n t ( w i t h t h e c o r o l l a r y t h a t common p a r e n t i n g p r a c t i c e s may be i r r e l e v a n t ) . No p a r t i c u l a r e n v i r o n m e n t a l f a c t o r has been demonstrated w i t h moderate p r o b a b i l i t y , i n d u c e s c h i z o p h r e n i a .  which w i l l ,  even  I t i s present i n a l l  c o u n t r i e s t h a t have been s t u d i e d , c o v e r i n g a wide range o f c u l t u r a l influences.  T h i s does not i m p l y , o f c o u r s e , t h a t e n v i r o n m e n t a l f a c t o r s a r e  not r e l e v a n t t o t h e e x p r e s s i o n o f s c h i z o p h r e n i a .  I f s o , t h e concordance  r a t e f o r MZ t w i n s would be 100%. 2)  P o s s i b l e Modes o f T r a n s m i s s i o n (Reviewed by K e t y and M a t t h y s s e , 1972; Gottesman and S h i e l d s , 1973; Tsuang, 1976; B a l d e s s a r i n i , 1977)  No s i m p l e M e n d e l i a n model can account f o r p o p u l a t i o n d a t a on schizophrenia.  Any m o d i f i c a t i o n o f a s i n g l e r e c e s s i v e gene model can be  r u l e d out s i n c e s i b s a r e a t no h i g h e r e m p i r i c r i s k t h a n o f f s p r i n g . h e t e r o g e n e i t y may be i n v o l v e d , h e l p i n g t o account schizophrenia.  Genetic  f o r the h i g h f r e q u e n c y o f  There c o u l d be a s i n g l e m a j o r dominant gene w i t h  reduced  p e n e t r a n c e , b u t once t h e m o d i f y i n g e f f e c t of genes a t t h e same o r o t h e r  loci  i s allowed, such a model i s d i f f i c u l t to d i s t i n g u i s h from polygenic models. I f s e v e r a l genes are i n v o l v e d , v a r i a t i o n with respect to a schizophrenic d i a t h e s i s could be e i t h e r continuous or quasi-continuous.  There seems t o be  general agreement among reviewers that e i t h e r mono- or polygenic models w i l l f i t e x i s t i n g data.  C a v a l l i - S f o r z a and Kidd have suggested that only  with the aide of biochemistry i s the genetic b a s i s of schizophrenia l i k e l y to be e l u c i d a t e d (Kety and Matthysse, 1972).  9  10  CHAPTER 2 •INTRODUCTION TO.BIOCHEMICAL THEORIES AND  FINDINGS  I  SCHIZOPHRENIA  A.  The T r a n s m e t h y l a t i o n H y p o t h e s i s (Reviewed by N e s t o r o s e t a l . , S m y t h i e s , 1976; M a t t h y s s e and L i p i n s k i , 1975; K e t y , 1972; Kety M a t t h y s s e , 1972; B r o d i e , 1977; Ban and Lehmann, 1977)  1977; and  Proposed by Osmond, Smythies and H a r l e y - M a s o n o v e r 25 y e a r s ago, transmyethylation hypothesis  s t i l l has h e u r i s t i c v a l u e .  Its basis  i n i t i a l l y the s t r u c t u r a l s i m i l a r i t y between t h e c a t e c h o l a m i n e s (a psychotomimetic d r u g ) .  The  s u g g e s t i o n was  and  the  was mescaline  that a disturbance i n  m e t h y l a t i o n might l e a d t o an e x c e s s of endogenous, h a l l u c i n o g e n i c , m e t h y l a t e d d e r i v a t i v e s of the catecholamines  ( l a t e r extended t o i n c l u d e  A t t e m p t s t o f i n d any p o t e n t i a l ' s c h i z o t o x i n ' ( m e t h y l a t e d  indolamines).  amines) p r e s e n t  h i g h e r amounts i n s c h i z o p h r e n i c t i s s u e s have f a i l e d .  The most s u p p o r t i v e  f i n d i n g has been t h a t by P o l i n and  (and c o n f i r m e d  c o - w o r k e r s i n 1961  number of l a b o r a t o r i e s ) t h a t methionine^"  l o a d i n g caused e x a c e r b a t i o n  s c h i z o p h r e n i c p s y c h o s e s i n some p a t i e n t s . the p s y c h o t o g e n i c  e f f e c t was  or p h a r m a c o l o g i c a l  The Dopamine  Attempts t o  ( n i c o t i n a m i d e or n i c o t i n i c  acid)  1977), however S - a d e n o s y l m e t h i o n i n e i n b r a i n  i s not e f f e c t i v e l y l o w e r e d by B.  process,  a c t i o n s of m e t h i o n i n e and/or i t s  a m e l i o r a t e symptoms w i t h a m e t h y l a c c e p t o r (e.g. N e s t o r o s ,  of  F u r t h e r , the e f f e c t c o u l d have r e s u l t e d  d e r i v a t i v e s , not n e c e s s a r i l y i n c r e a s e d t r a n s m e t h y l a t i o n .  have f a i l e d  in a  c l e a r , however, whether  an i n t e n s i f i c a t i o n o f the s c h i z o p h r e n i c  o r a superimposed t o x i c p s y c h o s i s . from other m e t a b o l i c  I t i s not  in  nicotinamide.  Hypothesis  C u r r e n t l y , the most p o p u l a r h y p o t h e s i s s c h i z o p h r e n i a i s the  'dopamine h y p o t h e s i s ' .  f o r a biochemical defect i n Very simply, i t s t a t e s that  s c h i z o p h r e n i a i s r e l a t e d to a r e l a t i v e excess of dopaminergic a c t i v i t y i n t h e b r a i n ' s l i m b i c system ( p a r t i c u l a r l y , t h e m e s o l i m b i c dopamine  tract).  S u p p o r t f o r the t h e o r y i s p r i m a r i l y p h a r m a c o l o g i c a l . 1  M e t h i o n i n e i s u l t i m a t e l y the s o u r c e o f m e t h y l g r o u p s f o r t r a n s m e t h y l a t i o n . r e a c t i o n s , v i a S - a d e n o s y l - m e t h i o n i n e (SAM).  1)  Evidence for Dopamine Involvement a)  Neuroleptic Drugs (Reviewed i n Kety and Matthysse, 1972; Matthysse and Lipinsk'i, 1975; Meltzer and Stahl, 1976; Baldessarini, 1977; Carlsson, 1978; Kety, 1972)  Anti-psychotic drugs which, unlike reserpine, do not deplete monoamine stores, belong to several chemically diverse classes, the major ones being the phenothiazines  (e.g. chlorpromazine (CPZ)  and  fluphenazine), the thioxanthines, the butyrophenones (e.g. haloperidol (HP)) and clozapine.  Not a l l members of these classes have anti-psychoti  action, but those that do are functionally similar, possibly acting on a biological substrate common to many schizophrenics.  Most, but not a l l of  the anti-psychotic drugs cause extrapyramidal (parkinsonian) side effects (EPS effects); thus the term 'neuroleptic'.  They are sometimes called  major tranquilizers, however their action seems to be i n relieving the fundamental manifestations of schizophrenia; they are truly anti-psychoti The neuroleptics i n h i b i t dopamine (DA)-mediated synaptic transmission in a dose-dependent manner, probably through blackade of DA receptors. Evidence for this i s i n d i r e c t , but compelling i n combination.  F i r s t , DA  stimulates a specific adenylate cyclase i n the synaptic c e l l membrane, and the activation can be blocked by administration of CPZ or HP. Second, radio-ligand binding assays indicate that the a b i l i t y of a drug 3  to competitively i n h i b i t binding of  3  H-DA  and/or  H-HP  correlates  closely with i t s anti-psychotic potency. The neuroleptic drugs cause an increase i n DA turnover, with an increased rate of production of DA metabolites  (dihydroxyphenylacetic  acid (DOPAC) and homovanillic acid (HVA)), an increased rate of conversion of tyrosine to DA, and an increased rate of f i r i n g of DA neurons. This i s not true of the non-antipsychotic  phenothiazines.  The effect i s thought to be a compensatory mechanism, secondary to the interruption of synaptic transmission and brought about by a decrease  in the 'long-loop' feedback inhibitory system. Dopaminergic neurons have pre-synaptic autoreceptors that bind DA, resulting i n feedback i n h i b i t i o n of f i r i n g ('short-loop').  This  i n h i b i t i o n can be blocked by neuroleptics. Motor side effects reminiscent of the symptoms of Parkinson's disease (for which a DA deficiency has been w e l l established), are a major complication of antipsychotic drugs.  Conversely, L-DOPA cannot  be used to counteract the EPS effects because i t causes psychotic exacerbation. b)  Alpha-methyl-tyrosine (Discussed by Carlsson, 1978; Meltzer and Stahl, 1976; Matthysse and L i p i n s k i , 1975)  Alpha-methyl-tyrosine i s a potent inhibitor of tyrosine hydroxylase (T-OH), the rate limiting enzyme i n DA biosynthesis. Although nephrot o x i c i t y precludes i t s use i n very high doses, i t can potentiate the antipsychotic effects of neuroleptics, lowering the dose requirement. Presumably i t helps to avoid the almost self-defeating feedback effect of receptor blockade on DA turnover. c)  Amphetamine Psychosis (Discussed by Meltzer and Stahl Matthysse and L i p i n s k i , 1975; Kety, 1972)  1976;  Amphetamine has long been known to possess psychotomimetic properties, capable of e l i c i t i n g i n nnn-schizophrenic individuals a c l i n i c a l picture often indistinguishable from acute paranoid schizophrenia. The major pharmacological effect i s believed to be induction of catecholamine release.  I t would seem, therefore, that schizophrenia  might also involve an excess a c t i v i t y of catecholamines.  Support for  the involvement of DA, rather than norepinephrine (NE) comes from the observation that neuroleptics (which antagonize DA) ameliorate amphetamine psychosis.  Further, amphetamine induces an increase i n  cerebro-spinal f l u i d (CSF) of HVA"'" without an increase i n NE metabolites. measured following probenecid administration, to prevent loss of the metabolite to venous blood  13  No such increase, however, has been noted i n schizophrenics. 2)  The Limbic System a)  Anatomy (Discussed by Stevens, 1973; Meltzer and Stahl, 1976; Torrey and Peterson, 1974; Barr, 1974; Ingram, 1976)  The limbic system comprises f r o n t a l and medial temporal lobes lying above the brainstem, including the amygdala, hippocampus, septum, olfactory tubercle, and hippocampal g y r i .  I t receives afferents from  a l l parts of the brain, and i s functionally associated with emotional aspects of behavior related to the survival of the individual and the species, and with memory. Efferents go to the hypothalamus, basal ganglia, mammillary bodies and thalamic nuclei"'".  Of particular interest  i s the 'limbic striatum' (Stevens, 1973), comprising the bed nucleus of the s t r i a terminalis, the nucleus accumbens, and the olfactory tubercle. The latter structures contain the terminals of the major 'mesolimbic DA tract' which originates i n the interpeduncular nucleus of the ventral tegmental area. b)  The Limbic System i n Schizophrenia  Support for the involvement of the limbic system i n schizophrenia comes from experiments both of nature and of man. Schizophrenic-like psychotic phenomena are frequently associated with psycho-motor (temporal lobe) epilepsy, encephalitis with predominance of temporal lobe involvement, brain tumors i n limbic structures, and stimulation or ablation of the same (Torrey and Peterson, 1974; Stevens, 1973; Baldessarini, 1977). Attention i s drawn to the 'limbic striatum' both because of i t s dopaminergic innervation and i t s "strategic interposition at the outflow of limbic structures subserving the primary adaptive processes disturbed i n schizophrenia" (Stevens, 1974). There are some reports of EEG abnormalities i n schizophrenics, p a r t i c u l a r l y the hypothalamus, and anterior thalamic n u c l e i , being part of the 'limbic c i r c u i t ' are considered by some to be parts of the limbic system  with electrodes implanted i n limbic structures (Stevens, 1973; Meltzer and Stahl, 1976; Torrey and Peterson, 1974). It i s generally assumed that the antipsychotic a c t i v i t y of the neuroleptic drugs reflects interaction with DA synapses i n the limbic striatum, while the EPS effects result from DA receptor blackade i n the striatum (Carlsson, 1978; Meltzer and Stahl, 1976; Snyder, 1972; 1  Baldessarini, 1977). 3)  Possible Causes of Dopaminergic Excess (Discussed by Meltzer, 1976; Meltzer and Stahl, 1976; Carlsson, 1978; Stevens, 1973; Matthysse and L i p i n s k i , 1975; Kety, 1972) The r e a l cornerstone of the DA hypothesis i s the evidence for DA  blocking a c t i v i t y of the neuroleptics, but whether or not this i s the action required for the antipsychotic effect remains to be demonstrated (Matthysse and L i p i n s k i , 1975; Baldessarini, 1977).  A relative increase  i n dopaminergic a c t i v i t y might come about by any of a number of means. Theoretically at least, there could be (1) an increase i n absolute levels of DA due to increased amounts of precursor, increased a c t i v i t y of T-OH, decreased inactivation by degradative enzymes, or f a i l u r e of control mechanisms for storage and release. Attempts to find alterations i n any of these parameters have f a i l e d , although differences might be so narrowly localized as to be undetectable i n large preparations.  There  could be (2) defective transport of DA from the synaptic c l e f t to preor post-synaptic c e l l s , resulting i n an impaired feedback system, (3) an increase i n DA response, (4) an excess of excitatory stimulation on DA neurons, or (5) a deficiency of inhibitory input from an inhibitory transmitter such as gamma-aminobutyric acid (GABA). a)  GABA and the DA Hypothesis  Roberts (1972) proposed that the underlying defect i n the etiology the n i g r o s t r i a t a l DA tract i s the s i t e of reduced dopaminergic a c t i v i t y in Parkinson's disease  . 15  of schizophrenia might be a lower-than-normal inhibitory effect of  GABAergic neurons on other neurons. He suggested that neuronal systems are released, not driven; i n other words, that excitatory neurons are usually held i n check by the tonic action of inhibitory (such as GABAergic) neurons, and are released for discharge on demand, through d i s i n h i b i t i o n . The schizophrenic diathesis might involve a barely adequate inhibitory system which under 'stress' would be incapable of keeping excitatory neurons from excessive f i r i n g , and imbalances would arise.  The problem  could be reflected as a relative increase i n , for example, dopaminergic a c t i v i t y , secondary to deficient inhibitory control (which i n turn could be a primary or secondary phenomenon). In light of the focus on limbic system involvement i n schizophrenia, some pharmacological evidence for GABA influence on mesolimbic neurons supports the general hypothesis.  DA  Stevens et a l . (1974) injected  bicuculline (a putative GABA blocking agent) into the ventral tegmental area of cats. The resulting series of behaviors resembled the stereotypic a c t i v i t y of the animals following systemic DA potentiation. The findings supported the hypothesis that behavior characteristic of DA stereotypy resulted from blockade of GABA i n h i b i t i o n on DA neurons (although other explanations were possible). C.  Low Platelet MAO i n Schizophrenia (Discussed by Wyatt and Murphy, 1976; Potkin et a l . , 1978; Berger et a l . , 1978; Baldessarini, 1978; Brodie, 1977; Meltzer, 1976) A biochemical finding of current interest i s that f i r s t reported by  Murphy and Wyatt i n 1972 of reduced monoamine oxidase (MAO)^" i n platelets of chronic schizophrenics. The finding has not always been confirmed,'but  the  largest decrease may be in 'paraniod schizophrenics' (Potkin et a l . , 1978). The t r a i t i s not specific to these schizophrenics (patients with bipolar ^ an important enzyme i n the metabolic degradation of a variety of monoamines  16  depression a l s o show reduced p l a t e l e t MAO),  and a l l attempts to r e v e a l a  d e f i c i e n c y i n post-mortem b r a i n have f a i l e d .  Data from MZ twins discordant  f o r schizophrenia suggest that the t r a i t i s g e n e t i c a l l y determined and thus a p o s s i b l e marker f o r s u s c e p t i b i l i t y state.  Despite the complexities i n v o l v e d , studies of MAO may  the understanding D.  rather than a f u n c t i o n of the disease contribute to  of the schizophrenic phenomenon.  A l t e r e d Transmitters and Related Enzymes Recently, B i r d et a l . (1977) published a p r e l i m i n a r y report on l e v e l s of  DA, glutamic a c i d decarboxylase  (GAD), and choline a c e t y l t r a n s f e r a s e (CAT),  from p a t i e n t s dying w i t h schizophrenia, s c h i z o p h r e n i c - l i k e psychoses, and controls"'".  They examined 3 p a r t s of the l i m b i c system (nucleus accumbens,  amygdala, and hippocampus) as w e l l as putamen.  DA was found to be  signifi-  c a n t l y increased i n the combined psychotic group i n nucleus accumbens but not putamen, and was not measurable i n amygdala or hippocampus. significantly  decreased i n psychotics i n a l l 4 regions.  GAD  CAT was  activity  was  significantly  lower i n schizophrenics but not s c h i z o p h r e n i c - l i k e p s y c h o t i c s , only i n nucleus accumbens. About the same time, McGeer and McGeer (1977) published r e s u l t s of a s i m i l a r study, i n which they measured CAT, dopamine decarboxylase  (DDC), and GAD  a c e t y l c h o l i n e s t e r a s e (AChE),  T-OH,  a c t i v i t i e s i n numerous b r a i n regions  from 11 schizophrenics and 18 c o n t r o l s .  A l l b r a i n s had been removed w i t h i n 2  24 hours of death and cases i n v o l v i n g pre-mortem coma had been excluded . The main f i n d i n g was increased CAT a c t i v i t y i n caudate, putamen, nucleus accumbens and hippocampus of schizophrenic b r a i n s . significantly  d i f f e r e n t i n any of these areas.  GAD  a c t i v i t y was  not  The discrepancy i n d i r e c t i o n  of change of CAT a c t i v i t y between t h i s study and that of B i r d et a l , (1977) seems to have r e s u l t e d from s t r i k i n g d i f f e r e n c e s i n the mean a c t i v i t i e s of the 1  2  Synthesis of GABA from glutamic a c i d i s catalysed by GAD. CAT i s responsible f o r the synthesis of a c e t y l c h o l i n e (ACh), another neurotransmitter See f o l l o w i n g s e c t i o n s , 'pre-mortem f a c t o r s ' and 'post mortem handling' f o r more d e t a i l e d d i s c u s s i o n of these D o i n t s of concern  ,  1  7  c o n t r o l groups, not the schizophrenics. In l i g h t of considerable c r i t i c i s m i n the Lancet* (Perry et a l . , 1978; Crow et a l . , 1978) f o l l o w i n g the report of B i r d et a l . (1977), the data were re-examined (Bird et a l . , 1978a,b).  When only cases f o r which there was  evidence of sudden death by n a t u r a l causes were i n c l u d e d , d i f f e r e n c e s i n GAD a c t i v i t y between controls and schizophrenics remained s i g n i f i c a n t only i n nucleus accumbens.  A more extensive evaluation (Iversen e t a l . , 1978),  i n c l u d i n g only sudden death cases, demonstrated no s i g n i f i c a n t d i f f e r e n c e between c o n t r o l s and schizophrenics f o r mean GAD a c t i v i t y i n caudate, putamen or nucleus accumbens.  ( S i g n i f i c a n t reductions remained f o r Huntington's  choreics). F a r l e y and co-workers (1978) have measured NE i n 4 paranoid and 12 c o n t r o l s .  schizophrenics  E a r l i e r studies i n d i c a t e d that NE l e v e l s d i d not vary with  e i t h e r age or post-mortem delay. areas of l i m b i c f o r e b r a i n : and v e n t r a l septum.  S i g n i f i c a n t e l e v a t i o n s of NE were found i n 4  nucleus accumbens, mammillary body, s t r i a t e r m i n a l i s  Drug treatment with n e u r o l e p t i c s and/or cause of death  were demonstrated to be u n l i k e l y f a c t o r s t o account f o r the d i f f e r e n c e s .  Several  other l i m b i c areas showed no s i g n i f i c a n t d i f f e r e n c e between the 2 groups.  II  HUNTINGTON'S CHOREA  A.  A l t e r e d Transmitters and Related Enzymes Results of studies on HC, i n which measurements of GABA, GAD, and CAT  were made are summarized i n Table I .  Following the i n i t i a l reports of Perry et  a l . (1973a, 1973b) of a reduction of GABA i n c e r t a i n areas of the b r a i n , studies were undertaken to measure GAD, because of the i m p l i c a t i o n that a metabolic e r r o r i n biosynthesis might be i n v o l v e d , and because the enzyme was thought to be more s t a b l e than GABA immediately a f t e r death. confirmed  Most studies  a decrease i n GAD i n HC b r a i n s , i n areas which p a r a l l e l e d the GABA  1 See f o l l o w i n g s e c t i o n s , 'pre-mortem f a c t o r s ' and 'post-mortem handling' f o r more d e t a i l e d d i s c u s s i o n of these points of concern  TABLE I: STUDIES OF NEUROTRANSMITTERS AND RELATED ENZYMES IN HC AUTHOR Perry et a l . (1973a) Perry et a l . (1973b)  Bird et a l . (1973)  GABA -sig * i n SN -also'V i n caudate, putamen-GP - s t r i k i n g ^ i n SN, caudate, putamen-GP -sig+ i n OC, TC -no s i g ^ i n FC, amygdala, thalamus, hypothalamus  GAD  McGeer et a l . (1973b) Bird & Iversen - i|r in caudate , putamen (1974) Stahl & Swanson (1974) Urquhart et a l .-confirmed e a r l i e r studies (above) (1975) McGeer et a l . (1976a)  - ( 8 5 % ) i n caudate, putamen, GP  —|in  caudate  FC  - ^ i n caudate, putamen -patchy i n caudate, relative to that i n putamen thalamus., hippocampus, amygdala -uniform "V (50-60%) i n -patchy >|r caudate, putamen, GP, SN - -V i n caudate, putamen -bimodality among ptsT h had normal levels -no-V i n FC caudate, putamen; *s had striking V - >K88-93%) i n advanced -:.^(73-99%) i n striatum HC, no i n early HC - n o ^ i n CC (1 Pt.) -no { i n early HC (1 pt. ) -low i n HC but also low in 2/5 control samples - f . \ i n extrapyramidal -patchy <Jr in neostriatum structures 1  -T-OH normal -AChE normal  -DA normal i n most 1 i n 6 r i g i d cases -T-OH normal -MAO* (50%) i n striatum of early HC -* HCARN i n GP, putamen, CC  -•  Abbreviations: SN = substantia nigra PEA = phosphoethanolamine TC = temporal cortex AChE = acetylcholinesterase s i g = significant pt. = patient  OTHER - tGLYC-PEA/PEA r a t i o - t GLYC-PEA - < V HCARN  -no4f in  McGeer et a l . (1973a)  CAT  GLYC-PEA = glycerophosphoethanolamine OC = o c c i p i t a l cortex T-OH = tyrosine hydroxylase CC = cerebellar cortex MAO = monoamine oxidase HC = Huntington's chorea  GP = globus pallidus HCARN = homocarnosine FC = frontal cortex DA = dopamine 4^ = decrease t = increase  decrease.  19 One study from Perry's laboratory, however, (Urquhart et a l , 1975)  demonstrated that GAD was also low i n 2 of 5 control samples, a finding which could not be accounted for by pre-mortem factors^.  Overall, the reduction i n  GABA has been most s t r i k i n g i n the extrapyramidal system (caudate, putamen, GP and SN), a finding i n keeping with the motor disturbance of HC. Decreased CAT i n HC has been described by McGeer as "patchy", since repeated sampling from caudate or putamen of a given individual may y i e l d variable results. GAD deficiency i s not specific to HC. Bowen (1974, 1975) reported a f a i r l y widespread but severe deficiency of GAD in brains of patients dying with senile dementia.  Davies and Maloney (1976), on the other hand, found 2  GAD levels within normal range i n 3 Alzheimer's patients . In Parkinson's disease, GAD deficiency may be secondary to treatment with anticholinergics (McGeer et a l . , 1973b).  Clearly, carefully designed experiments need to be  carried out i n order to answer questions concerning the relevance of decreased GAD a c t i v i t y . The finding of a p a r e l l e l deficiency of the a c t i v i t y of an enzyme and i t s metabolic end product does not necessarily imply that a defective enzyme i s primarily responsible for the loss of product.  GAD i s localized i n  inhibitory neurons that u t i l i z e GABA (Bird and Iversen, 1974) and destruction of such neurons would result i n concomitant loss of enzyme and product. i s probably the case i n HC (Perry et a l . , 1977).  This  The observed neuronal  degeneration, most marked i n basal ganglia, may represent s p e c i f i c loss of GABAergic and possible small cholinergic neurons (Bird and Iversen, 1974). C l i n i c a l manifestations suggest DA hyperactivity, although no evidence of increased DA turnover has been demonstrated (except i n 6 r i g i d cases as reported by Bird and Iversen (1974)). As discussed for schizophrenia, DA hyperactivity could result from a relative deficiency of GABA. 1  See following section on 'pre-mortem factors'  2 See also following section on 'age'  20  There are numerous p o s s i b i l i t i e s f o r causes of s e l e c t i v e c e l l death. McGeer and McGeer  (1976c) have suggested, f o r example, that HC might be an  e x c i t o t o x i c phenomenon, r e s u l t i n g from chronic o v e r s t i m u l a t i o n of glutamate receptors, p a r t i c u l a r l y i n the caudate. B.  Immunological Findings Barkely and co-workers (1977a,b, 1978) measured m i g r a t i o n i n h i b i t i o n  f a c t o r (MIF) a c t i v i t y (a c o r r e l a t e of delayed h y p e r s e n s i t i v i t y reaction) i n c u l t u r e d lymphocyes confronted with b r a i n t i s s u e preparations from various individuals. TABLE I I :  Results are summarized i n Table I I :  Detectable MIF a c t i v i t y i n c u l t u r e d lymphocytes (Barkley et a l . , 1977a,b, 1978) SOURCE OF LYMPHOCYTES Control SOURCE OF BRAIN HC MS TISSUE (ANTIGEN) AD Park.  Control -  HC + +  MS -  HC = Huntington's Chorea MS = M u l t i p l e S c l e r o s i s AD = Alzheimer's Disease Park. = Parkinson's Disease The authors hypothesized that the HC gene could be a p a r t i a l v i r a l genome, becoming a c t i v e i n middle l i f e , and producing a gene product with some s i m i l a r i t i e s to a v i r u s that has been proposed f o r the e t i o l o g y of m u l t i p l e s c l e r o s i s (MS). Because of the l a t e appearance of the gene product, i t could e l i c i t an immune response s i m i l a r to that invoked by an i n f e c t i o u s agent. lymphocytes might not respond s i m i l a r l y due t o a d i f f e r e n t set of immune response genes.  They d i d not comment on the p o s s i b l e r e l a t i o n s h i p of these  observations t o pathogenesis i n HC.  They added that p r e l i m i n a r y evidence  demonstrated s i m i l a r a n t i g e n i c i t y of HC f i b r o b l a s t s .  MS  2.1  C.  Evidence f o r A l t e r e d Membranes B u t t e r f i e l d et a l . (1977) studied e l e c t r o n s p i n resonance  c h a r a c t e r i s t i c s of HC erythrocyte membranes.  (ESR)  D i f f e r e n c e s from c o n t r o l s  could be i n t e r p r e t e d as due to (1) an a l t e r e d membrane p r o t e i n , (2) d i f f e r e n t amounts of a membrane component, (3) an a l t e r e d consituent, o r g a n i z a t i o n or (A)  a membrane-bound p r o t e i n i n HC, not found i n c o n t r o l s .  If a similar  a l t e r a t i o n were to e x i s t i n neuronal c e l l s , i t could be l i n k e d to the pathogenesis of  HC.  Several s t u d i e s have examined the behavior of HC f i b r o b l a s t s i n c e l l culture. in vitro.  Menkes (1973) found that HC f i b r o b l a s t s performed r e l a t i v e l y poorly Gray and Dana (1977) found no uniform d i f f e r e n c e i n growth  c h a r a c t e r i s t i c s between HC and non-HC c e l l s .  In c o n t r a s t , Goetz et a l . (1975),  K i r k et a l . (1977) and Leonardi et a l . (1978) found an o v e r a l l s u p e r i o r i t y of growth of HC f i b r o b l a s t s .  Methodology appears to have been an  important  v a r i a b l e ; nonetheless, i t seems that HC c e l l s can reach higher s a t u r a t i o n d e n s i t y , avoiding an i n i t i a l l a g phase and spending longer than normal i n exponential growth.  The abnormal behavior may  be the r e s u l t of an a l t e r e d  c e l l membrane. D.  GABA Receptors 3 H-GABA binding to synaptic membrane preparations can be studied i n post-  mortem b r a i n .  Findings with respect to a l t e r e d density of GABA binding s i t e s  i n HC are c o n f l i c t i n g .  Enna et a l . (1976a,b) found no change i n receptor  density i n b a s a l g a n g l i a of Huntington's c h o r e i c s , whereas Lloyd et a l . (1977) and Iversen et a l . (1978) found a s u b s t a n t i a l decrease i n binding s i t e density i n caudate and putamen.  Iversen et a l . (1978) reported, however,  that r e s u l t s were q u i t e v a r i a b l e , with some samples showing e s s e n t i a l l y normal binding and others very low values.  I f such heterogeneity  can be  confirmed, i n v e s t i g a t i o n s should be undertaken to e l u c i d a t e f a c t o r s that might be associated with normal or d e f i c i e n t GABA r e c e p t o r s , because of the i m p l i c a t i o n s f o r therapeutic i n t e r v e n t i o n .  CHAPTER 3 OTHER INFLUENTIAL VARIABLES A.  Post-mortem Handling In order to interpret post-mortem measurements of amino acids and  related enzymes, i t i s important to know how closely they represent levels that would have been present during l i f e .  The interval from death to  freezing of brain tissue i s a poorly controlled variable and may sometimes not be accurately estimated. 1) GAD Iversen et a l . (1978) recently carried out a carefully controlled study of GAD levels i n mouse brain, with cooling conditions programmed to mimic those i n human cadavers.  With cooling to 4°C, they observed a rapid i n i t i a l  decline (during the f i r s t few hours) to 80% of i n i t i a l values. t h i s , a stable plateau was maintained up to 72 hours.  Following  This confirms results  of earlier studies (McGeer et a l . , 1973a; Bird and Iversen, 1974: Urquhart et a l . , 1975; McGeer and McGeer, 1976a,b) which demonstrated relative s t a b i l i t y of GAD during storage at 4°C.  Decline was greater, however, at  room temperature (Bird and Iversen, 1974; McGeer and McGeer, 1976) or i f tissue had been frozen and then thawed (McGeer and McGeer, 1976a,b). The one discrepant finding has been that by Crow et a l . (1978) of s i g n i f i c a n t l y reduced GAD i n material obtained more than 48 hours post-mortem. 2) Amino Acids Perry and co-workers (1971 a,b) compared amino acid levels i n biopsied human cerebral cortex (immediately frozen) with those i n tissue obtained at autopsy and frozen 2.5-27 hours post-mortem. Most amino acids which were components of protein rose significantly after death, presumably due to proteolysis, and hydrolysis of N-acetylated amino acids.  Ethanolamine (EA),  GABA, and 2 GABA-containing dipeptides (homocarnosine (HCARN) and gammaaminobutyryl lysine (GABA-LYS)) were also s i g n i f i c a n t l y elevated i n the 5  23 post-mortem specimens. (GSH  Concentrations of reduced and o x i d i z e d  glutathione  and GS-SG), on the other hand were s i g n i f i c a n t l y lower i n autopsied  Levels of a number of compounds* were not s i g n i f i c a n t l y d i f f e r e n t i n b i o p s i e d and autopsied  specimens, e i t h e r because they were r e a l l y not  a l t e r e d by post-mortem f a c t o r s , or because the i n f l u e n c e of t h i s one v a r i a b l e was  obscured by other large sources of v a r i a t i o n . Tews e t a l . (1963) demonstrated that GABA i n dog c e r e b r a l cortex was  elevated 51% by 20 t o 23 minutes post-mortem.  Minard and Mushahwar (1966)  showed that GABA i n r a t b r a i n rose to a plateau w i t h i n 1-2 minutes post-mortem and then remained constant to the end of the t e s t period (30 minutes). Glutamic a c i d (GLU)  was s i g n i f i c a n t l y lower i n b r a i n s not immediately f r o z e n ,  but there were no comparable changes i n the l e v e l s of a s p a r t i c a c i d (ASP) or the n e u t r a l amino a c i d f r a c t i o n . B.  Pre-mortem Factors There has r e c e n t l y been considerable d i s c u s s i o n i n the l i t e r a t u r e  (e.g.  Perry et a l . , 1978b; Crow et a l . , 1978) about the p o s s i b l e i n f l u e n c e of cause of death on post-mortem GAD l e v e l s i n the b r a i n .  I n p a r t i c u l a r , there  has been concern that conditions which lead t o c e r e b r a l hypoxia cause a reduction i n GAD a c t i v i t y .  Since causes of death tend to be r e l a t e d to  disease c o n d i t i o n s , there i s reason f o r l e g i t i m a t e concern when studying disease group d i f f e r e n c e s . One example may serve t o i l l u s t r a t e the magnitude of t h i s i n f l u e n c e . McGeer et a l . (1973a) measured GAD i n s e v e r a l b r a i n regions, from a few individuals.  Results  f o r t h e thalamus are summarized i n Table I I I . ;  GAD a c t i v i t y (nmoles/gm/hour) CONTROL HC no coma  7.30  2.90 4.43 4.01  coma  0.40  0.74  enumerated i n 'Discussion'  TABLE I I I : GAD A c t i v i t y i n Thalamus - E f f e c t of Coma (McGeer et a l . , 1973a)  24 In l a r g e r studies (McGeer and McGeer, 1976a; Bowen et a l . , 1976) c l e a r l y demonstrated that GAD  i t was  a c t i v i t y decreased f o l l o w i n g coma from e i t h e r  head i n j u r y or i l l n e s s , but that there was some r e g i o n a l v a r i a t i o n i n the effect. GAD  The f i n d i n g has been confirmed by Iversen et a l . (1978) who  compared  a c t i v i t y i n 3 areas of b r a i n from p a t i e n t s dying with bronchopneumonia to  that i n p a t i e n t s dying sudden deaths, and found lower mean GAD  activity in  the former group. L i t t l e i s known about the relevance of cause of death to b r a i n amino acids.  Tews et a l . (1963) examined e f f e c t s of pre-mortem anoxia i n dog  c e r e b r a l cortex.  The increase of alanine was  the most s t r i k i n g , but  increases  i n GABA (28%) and s e v e r a l other amino acids were a l s o s i g n i f i c a n t . C.  Age Numerous studies (McGeer et a l . , 1973;  1974,  B i r d and Iversen, 1974; Bowen,  1975; Bowen et a l . , 1976; McGeer and McGeer, 1976a,b; Perry et a l . ,  1978a) have attempted to examine the e f f e c t s of aging on r e l a t e d enzymes, p a r t i c u l a r l y , GAD,  CAT,  T-OH,  neurotransmitter-  AChE, and DDC.  Results are  d i f f i c u l t to i n t e r p r e t since they are probably dependent on the f o l l o w i n g factors:  (1) whether or not post-mortem changes are accounted f o r , (2) the  age range examined, (3) whether or not t e r m i n a l l y demented and non-demented p a t i e n t s are separated,  (4) the region (s) of b r a i n examined and (5) sample  size. Decreases have been reported, i n various b r a i n regions, f o r each of the enzymes l i s t e d above with i n c r e a s i n g age. with more s t r i k i n g d e f i c i e n c i e s of CAT  S e n i l e dementia seems to c o r r e l a t e  (Perry et a l . , 1978).  I t i s important  to note that r e g i o n a l v a r i a t i o n i n these f i n d i n g s i s considerable. A report by TtfcGeer and McGeer the present study.  GAD  (1976b) i s of p a r t i c u l a r i n t e r e s t f o r  a c t i v i t y was measured i n 56 b r a i n regions  (wherenis^5)  from patients dying without pre-mortem coma or unconsciousness, and whom autopsies were performed between 2 and 24 hours a f t e r death.  on  In general,  the thalamic areas showed the greatest d e c l i n e , i n age ranges 5-20 years, and 20-50 years.  C o r t i c a l and rhinencephalic areas followed, and b a s a l  g a n g l i a showed r e l a t i v e l y l e s s d e c l i n e with age. The f i n d i n g s of decreased CAT with age i n some r e g i o n s , and more s t r i k i n g d e f i c i e n c i e s with s e n i l e dementia support the  pharmacological  evidence (Drachman and L e a v i t t , 1974; Davis et a l . , 1978; Sitaram et a l . , 1978) f o r c h o l i n e r g i c involvement i n memory storage.  This f u n c t i o n diminishes  with aging (Drachman and L e a v i t t , 1974) and i s d e f i c i e n t i n HC (Caine, 1978). D.  Drugs The vast majority of diagnosed schizophrenics and Huntington's choreics  are treated with one or a combination of a n t i - p s y c h o t i c drugs.  Not only i s  t h i s a v a r i a b l e that i s almost i n e x t r i c a b l y l i n k e d t o d i a g n o s i s , but the drugs act on the very neurochemical systems that are being i n v e s t i g a t e d f o r potential differences. One means of i n v e s t i g a t i n g drug response as a p o s s i b l e source of v a r i a t i o n i s to t e s t the e f f e c t i n a c o n t r o l l e d animal experiment.  Lloyd  and Hornykiewicz (1977) treated r a t s both acutely and c h r o n i c a l l y with clozapine (chronic, 100 days) or HP (chronic, 167), and measured GABA and GAD i n s u b s t a n t i a n i g r a .  Acute treatment with e i t h e r drug caused a  s i g n i f i c a n t reduction i n GABA (but not GAD) compared t o s a l i n e - i n j e c t e d controls.  C h r o n i c a l l y treated r a t s , on the other hand, had GABA and GAD  values not s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l s . In conjunction with the present i n v e s t i g a t i o n , a s i m i l a r drug experiment was c a r r i e d o u t . T h i r t y r a t s were i n j e c t e d f o r 100 days; 10 1  with CPZ (20 mg/kg/day s . c ) , 10 with HP (3 mg/kg/day s.c.) and 10 with  ( 0.9% s a l i n e (equivalent volume).  Following c e r v i c a l d i s l o c a t i o n b r a i n s  were removed, grossly d i s s e c t e d and frozen i n l i q u i d n i t r o g e n w i t h i n 25-35 seconds. 1  GABA concentrations were not s i g n i f i c a n t l y d i f f e r e n t i n  To be presented at NINCDS Huntington's Disease Symposium, San Diego, November 16-18, 1978  l i m b i c f o r e b r a i n among the three groups. E.  Regional V a r i a t i o n There i s considerable  r e g i o n a l v a r i a t i o n i n the d i s t r i b u t i o n of amino  acids w i t h i n the b r a i n (Perry et a l . , 1971a).  S i m i l a r l y , there i s r e g i o n a l  v a r i a t i o n i n the d i s t r i b u t i o n of enzymes such as GAD and CAT (McGeer and McGeer, 1976a).  No breakdown has been done f o r amino acid d i s t r i b u t i o n  w i t h i n the thalamus, however because of the presumed a s s o c i a t i o n between GAD and GABA, i t i s of i n t e r e s t t o note the d i s t r i b u t i o n of the enzyme w i t h i n the thalamus.  McGeer and McGeer (1976a) measured GAD a c t i v i t y i n  7 thalamic areas, expressing r e s u l t s as a percentage of a c t i v i t y found i n the caudate.  O v e r a l l thalamus had 68% a c t i v i t y , with a range from 101%  i n a n t e r i o r thalamus t o 32% i n v e n t r a l p o s t e r i o r thalamus.  27  CHAPTER 4 THE .THALAMUS. The thalamus (Discussed by Barr, 1974; Ingram, 1976) i s a large mass of grey matter making up most of the diencephalon.  It may be subdivided into  several nuclei on the basis of fibre connections and phylogeny.  Some of  these nuclei are 'specific', i n that stimulation w i l l evoke localized potentials i n definite c o r t i c a l areas.  They receive s p e c i f i c sensory input  and project to sensory areas of cerebral cortex.  Stimulation of 'non-  specific' nuclei, on the other hand, w i l l evoke potentials over wide neocortical areas of both hemispheres.  These are functionally related to  association areas of cortex, and participate i n emotional response to sensory stimuli.  In general, the thalamus i s a relay station, modulating and  controlling contacts between cerebral cortex and the outside world. The anterior nucleus receives afferents v i a the mammillothalamic tract and projects to the cingulate g y r i . limbic system.  I t i s therefore included i n the  The ventral l a t e r a l nucleus i s important for distribution  of impulses from basal ganglia to motor areas of f r o n t a l lobe, thereby controlling voluntary movement. The dorso-medial nucleus contributes to mood and related motor responses, and appears to play a role i n memory. Thalamic lesions may yield elevated sensory thresholds, with abnormal responses beyond the threshold. emotional i n s t a b i l i t y .  There may be spontaneous pain as well as  CHAPTER 5 PURPOSE AND  RATIONALE OF THE PRESENT INVESTIGATION  In t h i s study, amino acids and other n i n h y d r i n - p o s i t i v e compounds were measured i n autopsied b r a i n from p a t i e n t s dying with Huntington's Chorea, with schizophrenia or s c h i z o p h r e n i c - l i k e psychoses, and from c o n t r o l s dying without evidence of n e u r o l o g i c a l i l l n e s s . A d e f i c i e n c y of GABA had been noted i n some (but not a l l ) regions of b r a i n i n p a t i e n t s dying with Huntington's Chorea (Perry et a l . , 1973a,b). The thalamus had not been thoroughly  examined.  Knowledge of the d i s t r i b u t i o n  of the biochemical a l t e r a t i o n could contribute to an understanding of the pathogenesis underlying t h i s disease. Schizophrenic b r a i n was examined as w e l l f o r two reasons.  First,  Huntington's chorea and schizophrenia share c e r t a i n c l i n i c a l features. seemed p o s s i b l e , t h e r e f o r e , that they might share a biochemical  It  alteration.  Second, i t had been proposed (Roberts, 19.72) that a d e f i c i e n c y of GABAergic a c t i v i t y might underly the elevated dopaminergic a c t i v i t y that i s thought by many to be a key feature of schizophrenia.  I t seemed appropriate,  therefore, to look f o r differences i n GABA l e v e l s amongh these groups. S t r i k i n g d i f f e r e n c e s i n any of the other amino acids being measured concommitantly would of course be of i n t e r e s t as w e l l . A number of other independent v a r i a b l e s , besides disease s t a t u s , are l i k e l y to i n f l u e n c e amino a c i d l e v e l s i n the b r a i n .  These have been  examined and accounted f o r , as much as p o s s i b l e w i t h i n the confines of the available material.  CHAPTER 6 MATERIALS AND A.  Sources of B r a i n  METHODS  Tissue  The m a j o r i t y of m a t e r i a l used was  obtained  from Dr. E.D.  Bird,  MRC  N e u r o c h e m i c a l Pharmacology U n i t , U n i v e r s i t y of Cambridge, E n g l a n d . thalamus of 5 c o n t r o l s and 6 H u n t i n g t o n ' s c h o r e i c s had Dr. P e r r y ' s analysed.  been o b t a i n e d  l a b o r a t o r y , d u r i n g a p e r i o d of time when thalamus was These d a t a were i n c l u d e d i n the s t u d y .  4 c h o r e i c s had been i n s t o r a g e h e r e and  in  routinely  Thalamus from a f u r t h e r  a v a i l a b l e f o r a n a l y s i s , and  from 2 more HC p a t i e n t s and 5 c o n t r o l s was of the s t u d y .  Data f o r  that  made a v a i l a b l e d u r i n g the  Most of the Vancouver m a t e r i a l was  from p a t i e n t s who  course had  at R i v e r v i e w H o s p i t a l or the Vancouver G e n e r a l H o s p i t a l , b u t some had provided  t h r o u g h d o n a t i o n s from o t h e r N o r t h A m e r i c a n c e n t r e s .  died  been  Brain  d i s s e c t i o n s were p e r f o r m e d i n E n g l a n d ( f o r Dr. B i r d ' s m a t e r i a l ) or i n Vancouver. B.  D a t a from a t o t a l of 70 samples were used f o r the  Handling  ( B i r d and  I v e r s e n , 1974;  M a t e r i a l from E n g l a n d was brains obtained  B i r d e t a l . , 1977)  h a n d l e d i n the f o l l o w i n g manner:  a t n e c r o p s y were f r o z e n at -20°C.  t r a n s p o r t e d on d r y i c e , s t o r e d a t -20°C and before d i s s e c t i o n . m a t e r i a l was  then a t -10°C  t h e n chopped and m i x e d , r e f r o z e n f o r s t o r a g e ,  Whole or h a l f b r a i n s were o b t a i n e d  d i s s e c t e d and p a r t s f r o z e n on d r y i c e . In one  Dissected then  transported  h a n d l e d i n the f o l l o w i n g  at n e c r o p s y , i m m e d i a t e l y  They were t r a n s p o r t e d f r o z e n  c a s e , the whole b r a i n was  d i s s e c t i o n c o u l d be p e r f o r m e d , and  f o r 12 h o u r s  s t o r e d at -80°C.  I n a l l cases but one, Vancouver m a t e r i a l was  s t o r a g e at 80°C.  Whole  F r o z e n b r a i n s were  D i s s e c t i o n s were c a r r i e d out at -5°C.  t o Vancouver on d r y i c e where i t was  manner:  study.  to  s t o r e d a t -80°C u n t i l  t h e n p a r t s were r e - f r o z e n and  stored.  C.  30  Preparation of Tissue f o r Amino Acid A n a l y s i s A p o r t i o n of frozen b r a i n , u s u a l l y approximating  200 mg was weighed,  suspended i n c o l d 0.4 M p e r c h l o r i c a c i d (0.5 ml per 100 mg), then homogenized i n a t i s s u e grinder with 100 strokes of a motor-driven pestle.  Teflon  The homogenate was centrifuged at 21,000 x g f o r 10 minutes, the  supernatant  removed and r e t a i n e d .  The p e l l e t was resuspended i n 0.4 M  p e r c h l o r i c a c i d (0.3 ml per 100 mg of o r i g i n a l t i s s u e ) , and rehomogenized (40 s t r o k e s ) . supernatant  This was recentrifuged f o r 10 minutes at 21,000 x g, the  removed and combined with the f i r s t supernatant.  The pooled  supernatant was adjusted to pH 2.5-3.0 with KOH and centrifuged f o r 10 minutes at 21,000 x g t o p r e c i p i t a t e the potassium p e r c h l o r a t e .  The  supernatant was removed, volume measured, and concentration c a l c u l a t e d using i n i t i a l wet weight.  Samples were stored frozen at -80°C u n t i l  amino a c i d a n a l y s i s could be c a r r i e d out. D.  Amino Acid A n a l y s i s Amino a c i d s , s m a l l peptides and other n i n h y d r i n - p o s i t i v e compounds  were separated on a Technicon automatic amino a c i d analyser (Perry et a l . , 1968), adjusted f o r simultaneous  a n a l y s i s of 2 samples.' Molar concentrations  of the amino acids were c a l c u l a t e d from the chromatograms using a Technicon integrator-calculator. E.  S t a t i s t i c a l Methods (Sokal and R h o l f , 1969 ) Standard methods were used f o r the f o l l o w i n g s t a t i s t i c a l t e s t s :  Paired  t Test, One-way A n a l y s i s of Variance (ANOVA), Linear Regression A n a l y s i s , M u l t i p l e Linear Regression A n a l y s i s , Regression on l n of one v a r i a b l e , and M u l t i p l e Regression using l n of one v a r i a b l e .  I f ANOVA y i e l d e d s i g n i f i c a n t  r e s u l t s , Scheffe's Test f o r a p o s t e r i o r i comparisons was used to l o c a l i z e differences.  F.  Data Used i n S t a t i s t i c a l  31  Analyses  The m a j o r i t y of analyses were c a r r i e d out using 63 of the o r i g i n a l 70 samples. 1)  Exclusions were made f o r the f o l l o w i n g reasons: Three samples from one i n d i v i d u a l and two from another were analysed t o see whether there were s t r i k i n g r e g i o n a l d i f f e r e n c e s w i t h i n the thalamus" f o r any amino a c i d s . were noted.)  (No marked d i f f e r e n c e s  Only one sample ( a n t e r i o r ) f o r each i n d i v i d u a l was  included i n c a l c u l a t i o n s . 2)  Two  ' c o n t r o l s ' had been t r e a t e d with n e u r o l e p t i c s .  Their c o n t r o l  s t a t u s was therefore questionable. 3)  The diagnosis f o r one choreic was i n question.  4)  One Huntington's choreic was an extreme o u t l y e r on a computer a n a l y s i s that involved grouping i n d i v i d u a l s on the b a s i s of a l l amino a c i d s .  Examination of notes revealed that t h i s b r a i n had  been l o s t i n t r a n s i t and a r r i v e d i n Vancouver completely thawed. I t s e x c l u s i o n therefore seemed j u s t i f i e d . I n a l l , there were maxima of 23 c o n t r o l s (C), 25 Huntington's choreics (HC), 10 schizophrenics (S), and 5 p a t i e n t s w i t h s c h i z o p h r e n i c - l i k e psychosis (SL) f o r any given a n a l y s i s .  The l a t t e r two groups were not pooled since  "(those) who were placed i n the s c h i z o p h r e n i a - l i k e group ranged from those who narrowly f a i l e d to meet the c r i t e r i a f o r schizophrenia, t o those i n whom the diagnosis of schizophrenia seemed inappropriate" ( B i r d et a l . , 1977). The SL group was a poor one to deal with because i t was. s m a l l , and there were s e v e r a l unknowns among the independent v a r i a b l e s . Although i t was included i n some analyses, no conclusions about i t should be drawn from t h i s sample. G.  The Independent V a r i a b l e s Information concerning the f o l l o w i n g independent v a r i a b l e s was a v a i l a b l e ,  f o r most i n d i v i d u a l s included i n the study:  age, i n t e r v a l between death and  f r e e z i n g of b r a i n t i s s u e (post-mortem delay (PMD)), r e g i o n of the thalamus  sampled, immediate cause of death, d i a g n o s t i c category, and some drug history. Age and PMD are continuously d i s t r i b u t e d v a r i a b l e s , and therefore r e a d i l y examinable f o r d i f f e r e n c e s among groups, and f o r t h e i r p o t e n t i a l influence on the dependent v a r i a b l e s ( i . e . amino a c i d s ) . Diagnostic categories were, of course, part of the experimental Drug h i s t o r y was a d i f f i c u l t v a r i a b l e to deal w i t h .  design.  F i r s t , i t was  h i g h l y confounded with d i a g n o s i s , since most schizophrenics and choreics had been treated with n e u r o l e p t i c s , and c o n t r o l s had not.  Second, each  i n d i v i d u a l ' s treatment pattern was l i k e l y t o have been d i f f e r e n t , and the r e l i a b i l i t y of recorded  information was questionable.  A d i v i s i o n was made  somewhat a r b i t r a r i l y , between those who had been treated with n e u r o l e p t i c s and those who had not.  No rigorous a n a l y s i s of p o t e n t i a l drug e f f e c t could  be c a r r i e d out on t h i s sample. S i m i l a r l y , no rigorous a n a l y s i s of the other d i s c r e t e v a r i a b l e s , region of thalamus  and cause of death, could be c a r r i e d out.  D e t a i l s were simply  tabulated. H.  The Dependent V a r i a b l e s T h i r t y amino acids and other n i n h y d r i n - p o s i t i v e compounds were  examined s t a t i s t i c a l l y .  Others had been measured but were present i n  amounts too small t o be quantitated accurately. I.  S t a t i s t i c a l Protocol For each amino a c i d , the f o l l o w i n g p r o t o c o l was c a r r i e d out:  data were f i r s t submitted acid on age and PMD.  Control  t o a M u l t i p l e Linear Regression A n a l y s i s of amino  I f t h i s r e g r e s s i o n was not s i g n i f i c a n t , i t was assumed  that these 2 independent v a r i a b l e s d i d not contribute markedly t o the v a r i a t i o n , and a s t r a i g h t ANOVA was performed f o r A groups. was  I f the ANOVA  s i g n i f i c a n t , group d i f f e r e n c e s were l o c a l i z e d using Scheffe's Test  (a h i g h l y conservative  a p o s t e r i o r i t e s t f o r d i f f e r e n c e s between means  taken from a l a r g e r group of means).  I f the ANOVA was not s i g n i f i c a n t ,  data f o r a l l 4 groups were pooled and submitted t o a M u l t i p l e Linear and Quadratic Regression Analysis  (by computer) as a more powerful t e s t of  the e f f e c t s of age and PMD. I f the i n i t i a l M u l t i p l e Linear Regression was s i g n i f i c a n t , the r e l a t i v e c o n t r i b u t i o n s of age and PMD were q u a n t i t a t e d , and s i g n i f i c a n c e determined.  I f group d i f f e r e n c e s f o r these amino acids were t o be  analysed, c o r r e c t i o n would have t o be made f o r the i n f l u e n c e of the ( s i g n i f i c a n t ) independent v a r i a b l e ( s ) .  Such analyses were not c a r r i e d o u t ,  except f o r GABA. The r a t i o n a l e f o r the f u r t h e r a n a l y s i s of GABA i s described  under  'Results'.  CHAPTER 7 RESULTS A.  The Independent Variables 1) Age(See Figure 1) There was no significant difference i n mean age among controls,  Huntington's choreics, and schizophrenics  (see Table IV).  Schizophrenics,  however, tended to be distributed towards the older end of the age spectrum. TABLE IV: Mean Age (years) of Controls, Huntington's Choreics and Schizophrenics n mean  C 23 56.2  HC 25 54.6  S 10 64.9  SEM  +3.5  +2.3  +3.7  ANOVA on 3 means:  ^ = 1 .95 (NS)  Post-mortem Delay (PMD) a)  Estimates of PMD  PMD was unknown for a number of samples, (2 controls, 7 choreics and 2 schizophrenic-like psychotics).  Since this variable influences the  levels of most amino acids, i t was useful to have an estimate of PMD for those unknowns. Total GSH and ILE were chosen to base predictions on, since a preliminary analysis indicated that they were the compounds most highly correlated with PMD, without being confounded by other variables.  A natural log transformation of PMD allowed the best f i t of 1  2  data to a multiple regression (n = 49 , r = .661, F2 ^ =44.95, p <T.001). From the multiple regression, the equation for prediction of PMD was: ln(PMD)' = 2.4668 - 1.0234(GSH) + 2.0301(ILE).  See Table V  for the estimates of PMD. including a l l individuals for whom both ILE and t o t a l GSH were known, except #39 - an extreme outlyer for ILE  FIGURE  FIGURE  /.  II.  • PMD  o  known PMD estimated  6 0 +  50-  4 0 +  3 0 +  2 0 +  I  HC  S  Age distribution in controls, Huntington's choreics and schizophrenics  OO  o  10+  C  ••o  C  HC  Postmortem delay  S distribution in  w  • Ln  controls, Huntington's choreics and schizophrenics  TABLE V: Estimates of PMD for 11 Individuals ID# •  GSH*  ILE*  PMD' (hrs)  (C) 11 (C) 67 (HC) 21  0.31  0.33 0.41 0.19 0.22  17 22  (HC) 22 (HC) 31 (HC) 32 (HC) 44 (HC) 51 (HC) 53 (SL) 13  0.19 0.97 1.09 0.55 0.59 0.25  6 6 12  0.29 0.37 0.22  0.18 0.65  14 14 24 26  0.45 0.51 0.44  ?  35**  46 (SL) 37 0 0.67 * pinoles/gm wet weight ** PMD estimated from regression of ILE on ln(PMD) b)  PMD - Group Differences  Calculations of mean PMD and comparisons among groups were made both with and without individuals for whom PMD had been estimated. Either way, schizophrenics had a s i g n i f i c a n t l y longer mean PMD than controls or Huntington's choreics.  The l a t t e r 2 groups were not  s i g n i f i c a n t l y different with respect to this variable. XSee figure I I and Tables VI and VII.) TABLE VI: Mean PMD (hours) for Controls, Huntington's Choreics and Schizophrenics. (Without estimates of PMD) C n mean SEM  HC  S  21  18  10  18.0  16.4  35.0  +2.6  +3.2  +5.0  ANOVA on 3 means: F_ L  , HO  = 6.48 (p <.005)  S vs. C (P <.025); S vs. HC (p< .01)  37 TABLE V I I :  Mean PMD (hours) f o r C o n t r o l s , Huntington's Choreics and Schizophrenics (Including estimates PMD) C  n mean SEM  S  HC  23  25  10  18.1  15.9  35.0  +2.4  +2.4  ANOVA on 3 means:  F  2  5 5  +5.0 = 8.07  S vs. C (p<.025); S vs. HC 3)  (p<.001)  (p<.01)  Drug H i s t o r i e s (Summarized i n Table V I I I ) a)  Controls  A l l 23 c o n t r o l s used f o r s t a t i s t i c a l analyses had been treated without drugs, or with d i u r e t i c s or 'others'.  A l l are placed under  '-' i n Table V I I I . b)  Huntington's choreics  Four p a t i e n t s had been treated with tetrabenezines ( s i m i l a r to r e s e r p i n e ) , l i t h i u m , Valium, or miscellaneous These are placed under '-' i n Table V I I I .  drugs but not n e u r o l e p t i c s .  Nineteen p a t i e n t s had been  treated with phenothiazines and/or HP, and are placed under '+' i n Table V I I I .  One p a t i e n t had been t r e a t e d w i t h phenothiazLnes f o r 2 years,  but these had been discontinued f o r the l a s t 6 years of l i f e . p a t i e n t i s l i s t e d as ' + /-' i n Table V I I I .  This  For one p a t i e n t , nothing was  known about the l a s t 2 years of l i f e , but at the time of death drugs were l i s t e d as 'none'. c)  This p a t i e n t i s l i s t e d as '?' i n Table V I I I .  Schizophrenics  Nine p a t i e n t s had been treated with phenothiazines and/or HP f o r at l e a s t 1 year, and are placed under '+' i n Table V I I I . was treated without n e u r o l e p t i c s and i s l i s t e d as  '-'.  One p a t i e n t  ci)  Schizophrenic-like  Psychotics  Three p a t i e n t s had been t r e a t e d w i t h p h e n o t h i a z i n e s are l i s t e d under '+' i n T a b l e V I I I .  and/or HP, and  One p a t i e n t h a d been t r e a t e d  s e d a t i v e s o n l y , and i s l i s t e d as  with  F o r one p a t i e n t , d r u g h i s t o r y  was unknown. 4)  Causes o f Death  Causes o f d e a t h f o r p a t i e n t s i n each d i a g n o s t i c group a r e t a b u l a t e d i n Table IX.  Those l i s t e d toward t h e t o p a r e more l i k e l y t o have i n v o l v e d a  r a p i d death w i t h o u t p r o l o n g e d h y p o x i a . would c e r t a i n l y have i n v o l v e d p r o l o n g e d 5)  Bronchopneumonia and h e p a t i c  coma  hypoxia.  Regions o f Thalamus  Most samples had been t a k e n from a n t e r i o r m e d i a l t h a l a m u s , b u t some were f r o m o t h e r r e g i o n s .  F o r s e v e r a l i n d i v i d u a l s , t h e r e g i o n o f thalamus  sampled was n o t s p e c i f i e d .  The r e g i o n s  sampled, f o r i n d i v i d u a l s i n each  d i a g n o s t i c g r o u p , a r e t a b u l a t e d i n T a b l e X. TABLE V I I I :  SUMMARY OF NEUROLEPTIC DRUG HISTORIES C  HC  S  SL  23  4  1  1  19  9  3  T r e a t e d w i t h n e u r o l e p t i c s (+)  1 H i s t o r y unknown ( ? )  1 23  25  1 10  5  TABLE IX: CAUSES OF DEATH  Myocardial Infarction (MI)  C  HC  S  SL  6  2  1  1  5 1 Coronary Thrombosis  1  Suicide (hanging)  1 1  Pulmonary Embolism Asphyxia . . . Accident  1  1 1 1  1  1  Peritonitis Asthma Congestive Heart Failure Cancer (caecum) Bronchopneumonia (BP)  1  .  1 1 1 17  3  1 1  1  4  3 1  23  25  10  5  C  HC  S  SL  10  12  9  4  3 "Others" Unknown  *  TABLE X: REGIONS OF THALAMUS SAMPLED  Anterior Medial (AM) Anterior (A)  2  Medial (M)  5  Lateral (Lat)  2  1  Posterior Lateral (PL)  .1  Posterior (Post)  1  Unknown (?)  6  9  23  25  10  1  5  The  Amino A c i d s  1)  GABA a)  Data U n c o r r e c t e d  f o r Age and PMD  The mean GABA c o n c e n t r a t i o n i n the thalamus o f s c h i z o p h r e n i c s and of  Huntington's  c h o r e i c s was s i g n i f i c a n t l y  lower  when no c o r r e c t i o n was made f o r the e f f e c t s and  c h o r e i c s d i d not d i f f e r s i g n i f i c a n t l y  than t h a t of c o n t r o l s ,  of age and PMD.  from each o t h e r .  Schizophreni (See  Table XI and F i g u r e I I I . )  TABLE X I :  Mean GABA C o n c e n t r a t i o n (pmoles/gm wet weight) of C o n t r o l s , Huntington's C h o r e i c s and S c h i z o p h r e n i c s C  n  S  HC  23  10  25  mean  2.16  1.64  1.59  SEM  +. 13  + .10  +.11  ANOVA on 3 means:  F  2,55  = 6.41 ( p < . 0 0 5 )  C v s . HC ( p < . 0 1 ) ; C v s . S ( p < . 0 0 1 ) ; HC v s . S (NS)  b)  E f f e c t s of Age and PMD on GABA  Initially, carried  a m u l t i p l e l i n e a r r e g r e s s i o n o f GABA on age and PMD was  o u t , and f i t t o t h i s model was s i g n i f i c a n t .  I t was d e c i d e d ,  however, t o do a l o g t r a n s f o r m a t i o n of PMD, even though f i t of c o n t r o l d a t a t o t h i s model was n o t q u i t e as good. following:  (1)  Analyses  of p o o l e d  The reasons  were the  d a t a f o r I L E and GSH had i n d i c a t e d  t h a t f i t o f d a t a f o r these compounds t o a n a t u r a l l o g curve was b e t t e r than t o a l i n e a r curve.  (2)  Observations  have suggested  t h a t GABA  i n c r e a s e s v e r y r a p i d l y i n the f i r s t hour post-mortem, and v e r y a f t e r t h a t (T.L. P e r r y , p e r s o n a l communication).  little  I t would make  little  b i o l o g i c a l sense f o r GABA t o c o n t i n u e i n c r e a s i n g a t a c o n s t a n t r a t e post-mortem.  (3)  L i n e a r and n a t u r a l l o g c u r v e s  are not very  different  FIGURE  III.  3-5  30 +  2 5A-  20  1-5 +  10 A-  0-5 +  C  HC  GABA  concentration  data)  in controls,  choreics  and  S (uncorrected Huntington's  schizophrenics  in the middle range of PMD values.  See, for example Figure VI, a plot  of THR vs. PMD with the best f i t t i n g linear and natural log curves through control data.  The biggest differences between the two plots  are i n the extremes of the PMD d i s t r i b u t i o n , and the linear curve i s higher i n both regions.  Since a number of choreics had a very short  PMD, and a number of schizophrenics had a very long PMD, their amino acid values might seem excessively low in relation to the linear plot through controls. The natural log transformation was seen as a more conservative estimate of the control mean. The regression analysis i s shown i n Table XII.  TABLE XII: Multiple Regression Analysis of GABA vs. Age and ln(PMD) i n Controls (n = 21) for whom Age and PMD were Known 2 Source of Variation df SS MS F r Total 20 8.476 Multiple Regression 2 3.221 1.610 5.516 (p <.025) .380 Regression with Age 1 1.860 1.860 6.370 (p <.025) .219 Regression with In(PMD) 1 1.361 1.361 4.661 (p <.05) .161 Residual 18 5.255 0.292  The best f i t t i n g plane through GABA values, plotted against age and ln(PMD) was defined by the equation: GABA' = 2.582 - .023(age) + .330(In(PMD)) The f i t of this plane to observed GABA values was significant at the 2.5% level.  Both independant variables (age and ln(PMD)) contributed  s i g n i f i c a n t l y to the variation (linear decrease with age, logarithmic increase with PMD).  The variables together accounted for 38% of the  variation i n GABA values i n controls. c)  GABA - Differences Among Groups, Accounting for Age and PMD  Using the equation generated from the multiple regression analysis  43  f o r c o n t r o l s , an expected (control) value f o r GABA (GABA') was c a l c u l a t e d f o r each i n d i v i d u a l .  This value would l i e on the plane,  and would be the best estimate of GABA f o r an age- and PMD-matched control.  Deviations of observed GABA values from GABA' were  calculated.  A negative d e v i a t i o n means that the observed value i s  l e s s than the mean f o r an age- and PMD-matched c o n t r o l ; that the observed value l i e s below the plane. positive deviation.  The converse i s true f o r a  Table X I I I has tabulated i n d i v i d u a l s f o r each  d i a g n o s t i c group, i n order of magnitude of d e v i a t i o n s from the plane. D e t a i l s of a l l the independent v a r i a b l e s are tabulated as w e l l , f o r a v i s u a l i n s p e c t i o n of 'potential r e l a t i o n s h i p s w i t h high or low GABA. Two types of analyses were c a r r i e d out, each with and without i n d i v i d u a l s f o r whom PMD had been estimated.  The f i r s t was an a n a l y s i s  of variance on d e v i a t i o n s from the c o n t r o l mean (GABA-GABA') among 3 groups (C, HC, S).  The second was a p a i r e d t t e s t , between GABA and  GABA', as i f GABA' represented a matched c o n t r o l f o r each i n d i v i d u a l . Results are summarized i n Tables XIV and XV.  With or without  i n d i v i d u a l s f o r whom PMD was estimated, there were s i g n i f i c a n t negative deviations of HC and schizophrenic GABA values from values expected of controls.  From the ANOVA, there were s i g n i f i c a n t d i f f e r e n c e s i n mean  d e v i a t i o n from expected, among the 3 groups.  The d i f f e r e n c e s were  l o c a l i z e d t o between c o n t r o l s and c h o r e i c s , and between c o n t r o l s and schizophrenics.  Accounting f o r age and PMD, Huntington's choreics and  schizophrenics have s i g n i f i c a n t l y lower mean GABA than c o n t r o l s .  44 TABLE X I I I :  T a b u l a t e d V a r i a b l e s f o r Each I n d i v i d u a l , and D e v i a t i o n s of GABA V a l u e s from Expec ted GABA V a l u e s  AGE (yrs)  PMD (hrs)  GABA umoles gm w.w.  GABA' umoles gm w.w.  GABA-GABA' umoles gm w.w.  3.12  1.96  1.16  PART OF THAL  Rx  MI  AM  -  1  -  ?  -  CAUSE OF DEATH  8  75  26  60  48  3  2.63  1.86  0.77  ?  63  40  27  3.29  2.77  0.52  MI  61  21  5  3.12  2.64  0.48  s u i c i d e (h anging)  ?  -  3  56  10  2.51  2.08  0.43  MI  AM  -  2  21  38  3.59  3.31  0.28  accident  AM  -  65  63  7  2.05  1.80  0.25  aortic  MED  -  •J 7 o  70  26  2.21  2.08  0.13  heart  AM  -  5  53  24  2.56  2.46  0.10  MI  AM  -  10  55  49  2.62  2.63  -0.01  heart  AM  -  4  74  20  1.87  1.90  -0.03  asthma  AM  -  69  44  3  1.81  1.95  -0.14  h e p a t i c coma  1  -  59  59  13 . . 1.93  2.10  -0.17  cong. h e a r t  1  -  62  66  8  1.61  1.78  -0.17  heart  ?  -  1  60  19  2.03  2.20  -0.17  coronary  MED  -  67*  80  22*  1.49  1.80*  -0.31*  MI  AM  -  ll*  77  17*  1.47  1.78*  -0.31*  MI  AM  -  70  58  8  1.61  1.96  -0.35  h e p a t i c coma  MED  -  68  74  23  1.49  1.95  -0.4.6  heart  attack  LAT  -  6  44  27  2.19  2.68  -0.49  heart  attack  LAT  -  66  52  7  1.55  2.05  -0.50  Ca  MED  -  9  73  27  1.48  2.01  -0.54  pulmonary  AM  -  64  31  8  1.45  2.57  -1.12  h e p a t i c coma  MED  -  eH H  g o  • '•!.:'  aneurism attack  attack  attack thromb.  (caecum) embol.  * PMI) estimci t e d  Abbreviations • w.w.  = wet  Rx = treatment  weight  with neuroleptics  cong. h e a r t = c o n g e s t i v e h e a r t GABA' = expected GABA c o n c e n t r a t i o n f o r c o n t r o l w i t h g i v e n age and Ca = c a n c e r PMD , . . . = 2.582 - .023(age) + .330 (ln(PMD)) pulmonary embol. = pulmonary embolism THAL = Thalamus AM = a n t e r i o r m e d i a l , P >  posterior,  MED = m e d i a l , A; = a n t e r i o r , PL = p o s t e r i o r  coronary lateral  thronib. =  coronary thrombosis  45 TABLE XIII:  #  33 45 21* 54 31* 25 22* 27 52  AGE (yrs)  continued PMD (hrs)  GABA GABA' GABA-GABA' umoles umoles pmoles gm w.w. gm w.w. gm w.w.  CAUSE OF DEATH  PART OF THAL  Rx  64  1  2.30  1.14  1.16  BP  1  +  57  1  2.19  1.29  0.90  BP  1  +  72 68  6* 5  2.39 2.13  1.55* 1.58  0.84* 0.55  ?  AM  -  BP  1  1  12* 60 68 . 20 55 6* 72 28 26 24 55 26* 57 22 44 2  2.05 2.00 1.89  2.05* 2.04  BP peritonitis BP  AM AM  -  AM  +  1.85 2.67 2.01 1.81 1.24 1.85 1.24  2.06 3.04 2.42* 2.32 1.82  0.00* -0.04 -0.04* -0.21  MI  AM  +  1  f  +  1.93*  -  1.43 2.75  18  1.26  2.14  -0.88  ?  AM  +  54  14*  1.34  2.23*  BP  AM  +  42  58  BP  AM  +  53  1.11 1.40  2.03  51*  10 24*  -0.89* -0.92  29 30  58  27  1.30  53  29  26 47  43 38  24 20  1.18 0.99 1.01  ICS  54  1.25 0.63 1.89  -0.37 -0.41* -0.51 -0.58 -0.58 -0.69 -0.80* -0.80 -0.86  53* w 28 gS3 CJ 57 CO  35 39 62 61 51 62  32*  riNGTOl  49 46 44* 58 S3 55 24  7 2 14* 2  * PMD estimated  2.43 1.93 2.05*  asphyxia BP BP BP BP MI  +  AM  +  9  +  A  +  1  +  AM  -  ?  ?  +  BP  1  +  BP  AM  +  2.36 2.50  -1.03* -1.06 -1.32  BP BP  A AM  + +  2.66 2.71  -1.67 -1.70  BP BP  PL  +  2.43*  ?  +/-  46  TABLE XIII:  continued  t  #  AGE (yrs)  PMD (hrs)  GABA umoles gm w.w.  GABA* umoles gm w.w.  GABA-GABA' umoles gm w.w.  CAUSE OF DEATH  PART OF THAL  Rx  + -  16  70  12  2.11  1.82  0.29  others  AM  40  88  18  1.39  1.55  -0.16  others  AM  18  80  35  1.67  1.95  -0.28  BP  AM  15  64  13  1.62  1.98  -0.36  pulmonary embol.  AM  19  57  50  1.96  2.57  -0.63  asphyxia  AM  50  52  24  1.67  2.46  -0.79  MI  17  54  48  1.91  2.64  -0.73  others  AM  20  72  56  1.42  2.29  -0.87  BP  AM  12  56  48  1.13  2.60  -1.47  BP  AM  14  56  46  1.02  2.58  -1.56  ?  AM  41  92  8  2.04  1.19  0.85  MI  AM  36  64  10  1.98  1.90  -0.16  heart attack  AM  +  35  44  17  2.36  2.52  -0.16  others  AM  37*  47  45*  1.74  2.78*  -1.04*  suicide  AM  + +  13*  52  35*  1.03  2.58*  -1.55*  BP  M ED  *PMD estimated  M  + + + + + + + +  ?  47 TABLE XIV: Mean GABA Deviations (umoles/gm wet weight) o f Controls, Huntington's Choreics and Schizophrenics (Without estimates of PMD)  n mean  C  HC  S  21  18  10  -0.53  -0.66  +.18  +.17  0.0  SEM  + . 11 F„ ,  ANOVA on 3 means:  = 5.16 (p < .01)  r  2,46  C vs. HC (p <.05);  C vs. S (p < .025); HC vs. S (NS)  P a i r e d t Test (GABA vs. GABA')  C: HC: St  TABLE XV:  '20  =  17  =  C  9  fc  =  = -0.01 (NS) = -2.87 (P < .01) = -3. 63 (P < .01)  Mean GABA Deviations (jamoles/gm wet weight) o f Controls, Huntington's Choreics and Schizophrenics (Including Estimates of PMD) :  C  HC  S  23  25  10  mean  -0.03  -0.48  -0.66  SEM  +.10  +.15  +.17  n  ANOVA on 3 means:  F  2 5 5  = 4.88  (p < .025)  C vs. HC (p < .05) ; C vs. S (p < .05);  HC vs. S (NS)  Paired t Test (GABA vs. GABA')  2)  C  t  2 2  = -.269 (NS)  HC  t  2 4  = -3.20  (p < .01)  S  t  1 Q  = -3.63  (p < .01)  Glycerophosphoethanolamine (GLYC-PEA) A m u l t i p l e l i n e a r regression of GLYC-PEA on age and PMD was non-  s i g n i f i c a n t f o r c o n t r o l s , therefore comparisons were made among groups with uncorrected data.  Huntington's  choreics had a mean GLYC-PEA  concentration s i g n i f i c a n t l y higher than that of c o n t r o l s . The mean f o r schizophrenics was intermediate between c o n t r o l s and c h o r e i c s , not  48 s i g n i f i c a n t l y d i f f e r e n t from e i t h e r . TABLE XVI:  n  Mean GLYC-PEA Concentration (umoles/gm wet weight) i n Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-Like Psychotics C  HC  S  SL  23  25  10  5  mean  0.79  1.25  0.92  0.70  SEM  + .12  + .08  + .07  + .03  ANOVA on 4 means:  F ^  C vs. HC (p < .001) ;  3)  (See Table XVI and Figure IV )  == 7.51  (p <.001)  a l l other p a i r w i s e comparisons NS .  Homocarnosine (HCARN) A m u l t i p l e l i n e a r regression of HCARN on age and PMD  for controls  was n o n - s i g n i f i c a n t , therefore comparisons were made among groups with uncorrected data.  One schizophrenic p a t i e n t had a value f o r HCARN more  than 3 standard deviations beyond the mean f o r c o n t r o l s (1.58 umoles/ gm wet weight; see Figure V). without t h i s value.  Analyses were c a r r i e d out w i t h and  The l a t t e r i s summarized i n Table XVII.  o u t l y e r , the schizophrenic mean was elevated to 0.49 not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s . means was  With the  (+ .12) which was  The o v e r a l l ANOVA on 4  correspondingly l e s s s i g n i f i c a n t ( F = J , D  3.68  (p <  .01)).  O  The mean HCARN concentration f o r Huntington's choreics and f o r 9 out of 10 schizophrenics was s i g n i f i c a n t l y lower than that f o r . c o n t r o l s . 4)  Amino Acids Showing no S i g n i f i c a n t Linear Change i n Controls (n = 21) with Age (range, 21-80 years) or PMD (range, 3-49 hours) and no S i g n i f i c a n t Differences Among Diagnostic Groups The m u l t i p l e l i n e a r regression i n c o n t r o l s was n o n - s i g n i f i c a n t  f o r s e v e r a l other amino a c i d s , therefore comparisons among groups were made w i t h uncorrected data.  There were no s i g n i f i c a n t d i f f e r e n c e s  among the 4 diagnostic groups f o r TAU,  GLU,  CYSTA, (CYS) , PHE, 0  TRP,  or  49  FIGURE  IV.  \  3 i  CD  0-5+  0-2 C GLYP~PEA Huntington's psychotics.  HC concentration choreics,,  S in  SL controls,  schizophrenics  and  50  FIGURE  V.  C  HC  Homocarnosine in controls, and  (HCARN)  S  SL  concentration  Huntington choreics,  psychotics.  schizophrenics  51 TABLE XVII: Mean HCARN Concentration (umoles/gm wet weight) i n Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-like Psychotics  n  C  HC  S  SL  22  25  9  5  mean  0.70  0.41  0.37  0.59  SEM  +.06  +.05  +.04  + .09  ANOVA on 4 means: F C vs. HC (p <.01); comparisons NS  3 5 ?  =5.939 (p < .005)  C vs. S (p <025);  a l l other pairw.ise  GABA-LYS. Means are summarized i n Table XVIII.  Since there were no  no significant differences among groups,.data for these amino acids were pooled and submitted to a linear/quadratic regression analysis.  With  this, (CYS) showed a combined linear increase and quadratic decrease 2 with PMD (r = .26), suggesting that i t increased i n i t i a l l y and then 2 2  plateaued.  PHE showed a straight linear increase with PMD (r = .29).  Other amino acids of this group s t i l l showed no significant change with age (range, 21-92 years) or PMD (range, 1-56 hours). TABLE XVIII:  Amino Acid  Amino Acids Showing no Significant--Linear Change i n Controls (n = 21) with. Age (range, 21-80 years) or PMD G r a n g e 3-49 hours) and no Significant Differences -Among Diagnostic Groups Mean Concentration (+ SEM) (umoles/gm wet weight) C HC S SL  TAU  0.83 (+.06)  1.03 (+.09)  0.88 (+.07)  0.93 (+.09)  GLU CYSTA  7.94 (+.21) 0.89*(+.07)  8.38 (+.53) 1.01 (+.12)  0.23 (+.02)  8.10 (+.29) 1.09 (+.14) 0.25 (+.02)  8.80 (+.19) 1.35 (+.19) 0.32 (+.01)  0.34 (+.02)  0.38 (+.03)  (CYS) PHE  0.34 (+.02)  0.19 (+.02) 0.28 (+.02)  TRP  0.07 (+.01)  0.07 (+.01)  0.09 (+.01)  0.10 (+.01)  GABA-LYS  0.07 (+.01)  0.05 (+.02)  0.02 (+.01)  0.03 (+.01)  2  * excluding 3 individuals who died i n hepatic coma  52 5)  Amino A c i d s Showing a S i g n i f i c a n t L i n e a r Change i n C o n t r o l s (n = 21) w i t h PMD (range, 3 - 4 9 hours) but n o t w i t h Age (range, 21-80 years) The m a j o r i t y of amino a c i d s showed a s i g n i f i c a n t  i n c o n t r o l s between 3 and 4 9 hours post-mortem.  l i n e a r increase  These are l i s t e d  in  2 T a b l e XIX,  i n d e c r e a s e i n g o r d e r of r  (the c o e f f i c i e n t  of  determination,  which i n d i c a t e s the p r o p o r t i o n o f the t o t a l v a r i a n c e accounted a l i n e a r r e g r e s s i o n w i t h PMD). l i n e a r decreases  i n t h i s range.  T o t a l GSH  and PEA  showed  f o r by  significant  I f group d i f f e r e n c e s were to be  examined f o r these amino a c i d s , c o r r e c t i o n would have t o be made f o r the i n f l u e n c e o f PMD change o f THR 6)  on amino a c i d c o n c e n t r a t i o n s .  with  F i g u r e VI i l l u s t r a t e s  the  PMD.  Amino A c i d s Showing S i g n i f i c a n t L i n e a r Changes i n C o n t r o l s (n =21) With Both PMD (range, 3 - 4 9 hours) and Age (range, 21-80 years) Both age and PMD  i n ORN,  HIS,  and TYR  contributed s i g n i f i c n a t l y concentrations  of d e t e r m i n a t i o n are l i s t e d i n Table  to the c o n t r o l  (as w e l l as GABA).  f o r each v a r i a b l e ,  variation  Coefficients  f o r each amino a c i d ,  XX.  I  53 TABLE XIX :  Amino Acids Showing a Significant Linear Change i n Controls (n = 21) with PMD (range, 3-49 hours) but not with Age (range, 21-80 years)  Amino Acid  Coefficient of ^ Determination (r )  EA VAL PRO SER THR  Linear Increase  LEU ILE MET LYS GLY  .737 .571 .550  < < < <  .506 .505 .502  .001 .001 .001 .001  < .001 < .001  .486 .461  < .001  ALA  .375 .268  < < < < <  ASN*  .192  > .05  GLN* ARG*  .190 .166  > .05 > .05  GSH (TOTAL) PEA  .432  < .005 < .025  ASP  Linear Decrease  Null Probability (P)  .449 .398  .254  .001 .001 .005 .005 .025  TABLE XX Amino Acids Showing Significant Linear Changes i n Controls (n = 21) with Both PMD (range, 3-49 hours) and Age. (range, 21-80 years) Amino Acid  ORN HIS TYR  Linear Decrease With Age 2 r P< .229 .005 .222 .005 .153 .05  Linear Increase With PMD r  2  .416 .222 .235  P< .001 .005 .025  Multiple Linear Regression r  2  .645 .445 .388  P<  .001 .005 .025  FIGURE VI.  Threonine vs. postmortem delay  1-2 +  0 a 0-8 +  06 +  0 - 4 4Linear regression through controls Log regression .through controls  n  0-.2 +  a  10  +  -I—  20 25 30 35 Postmortem delay (hours)  40  • Control CD Control PMD estimated • HC CD HC, PMD estimated A Schizophrenia  45  50  55  Ln  55  CHAPTER 8 ;  DISCUSSION  The key findings of this study were (1) a deficiency of GABA i n the thalamus of Huntington's choreics and schizophrenics, (2) a deficiency of the GABA-containing dipeptide, homocarnosine, i n choreics and 9 out of 10 schizophrenics (1 schizophrenic had excessively high HCARN) and (3) an elevated concentration of GLYC-PEA i n Huntington's choreics. Compounds which were examined, but showed no differences among groups were:  TAU, GLU,  CYSTA, (CYS) , PHE, TRP, and GABA-LYS. There were indications that GABA-LYS 2  might correlate with GABA and HCARN, and thus be reduced i n choreics and schizophrenics as well, but i t s mean concentration was too low for group differences to be extracted with these techniques. A l l of the findings with respect to HC are i n accord with studies of amino acids i n other parts of HC brain (Perry et a l . , 1973a,b; Urquhart et  '  a l . , 1975). No other studies of amino acids i n schizophrenic brain have been published to date.  A concurrent study of nucleus accumbens demonstrated  a similar, but more s t r i k i n g deficiency of GABA i n schizophrenics and choreics*. Unfortunately, human material i n general, and autopsied brain i n particular comprises a poor system for experimental design.  I t i s extremely  d i f f i c u l t to control for a number of variables, besides diseases being studied, that may influence the biochemical parameters under investigation.  Statistics  can, i n some cases, mimic the controls of experimental design, but problems are encountered when independent variables are not distributed homogeneously among groups. The f i r s t independent variable considered was age.  Significant linear  decreases with age, i n control thalami, were observed for GABA, ORN, HIS, and TYR. Similar decreases of other amino acids may have been masked by the * to be presented at NINCDS Huntington's Disease Symposium, San Diego, November 16 - 18, 1978  56 over-riding influence of PMD. In l i g h t of the finding of decreased GABA with age, data for a number of other brain regions were examined. Linear regression of GABA with age was significant i n frontal cortex, and not i n o c c i p i t a l cortex, caudate, putamen-GP, or SN. I t i s interesting to compare these findings to those of McGeer and McGeer (1976b) who found the most s t r i k i n g decrease of GAD with age to be i n the thalamus. The second variable was post-mortem delay.  Significant linear increases  with PMD i n controls were observed for the majority of amino acids: EA, VAL, PRO, SER, THR, LEU, ILE, MET, LYS, GLY, ASP, ALA, ORN. HIS, TYR, and GABA. In a pooled sample, (CYS^ and PHE also increased s i g n i f i c a n t l y . decreases with PMD were observed for t o t a l GSH and PEA.  Significant  There were no  significant linear changes i n TAU, GLU, CYSTA, TRP, GABA-LYS, GLYC-PEA or i  HCARN between 1 and 56 hours post-mortem. ASN, GLU, and ARG showed nearly significant linear decreases with PMD i n controls (range, 3-49 hours). These findings with respect to post-mortem changes do not correspond exactly with findings of Perry et a l . (1971b), who compared biopsied and autopsied cortex specimens.  They noted no s i g n i f i c a n t differences for  GLYC-PEA, TAU, PEA, TRP, ORN, and CYSTA, between 8 biopsied and 5 autopsied specimens.  GSH and GLN were lower i n autopsied cortex, while other amino  acids were s t r i k i n g l y higher.  The differences between these studies may stem  from from the different delay periods being examined, and the nature of the change i n any given amino acid. The delay i n the present study (for controls) ranged from 3 to 49 hours.  A rapid and marked change during the f i r s t 3 hours,  followed by a relative plateau, would appear as no significant change with PMD i n this analysis. Further, other models, such as logarithmic transformations or power curve f i t s were not tested for most amino acids. Such other models would l i k e l y be more appropriate, but a linear model i s probably not a bad approximation i n most cases.  I t i s a matter of concern  that the distribution of PMD was not the same i n the 3 diagnostic groups.  Extrapolation of the control curve to account for choreics and schizophrenics i n the extremes of the distribution was not entirely appropriate.  On the  other hand, a log transformation was seen as a conservative means of coping with the variable, for reasons previously discussed,and also because a large change i n PMD at the higher end of the distribution would make l i t t l e difference i n the correction factor for GABA. Clearly an experiment designed to test the change i n amino acid concentrations with PMD, without other confounding variables, needs to be carried out. Differences i n cause of death among the 3 groups are also a matter of concern, p a r t i c u l a r l y following the results of Bird et a l . (1977) and Iversen et a l . (1978) which suggested that decreased GAD i n various parts of schizophrenic brain was no longer significant when cases involving pre-mortem hypoxia were excluded.  No conclusions can be drawn from the present study  about the possible effect of pre-mortem hypoxia on amino acids, but i t i s quite possible that this variable i s i n f l u e n t i a l , and could account for part of the observed group differences.  The effect of neuroleptic drugs was seen as a potential candidate for causing differences i n GABA concentrations among groups.  As discussed  previously, this variable was highly confounded with diagnosis, and could not be separated adequately.  The chronic drug experiments on rats (Lloyd and  Hornykiewicz, 1977; Perry et a l . *) suggested, however, that CPZ and HP are not the cause of decreased GABA i n choreics and schizophrenics. Were this experiment to be done again, i t would be better to r e s t r i c t samplesto one'part of.the thalamus, or to use a homogenate of whole thalamus.  *to be presented at NINCDS Huntington's Disease Symposium, San Diego, November 16-18, 1978.  58 Most samples were from a n t e r i o r m e d i a l thalamus. have c o n t r i b u t e d  Those t h a t were not  to the v a r i a t i o n i n amino a c i d s , but were s u f f i c i e n t l y  t h a t they would not l i k e l y have been the cause o f group  I n c o n c l u s i o n , GABA c o n c e n t r a t i o n was thalamus  may  differences.  s i g n i f i c a n t l y d e c r e a s e d i n the  of s c h i z o p h r e n i c s and H u n t i n g t o n ' s  v a r i a t i o n w i t h age  few  c h o r e i c s , whether or not  and post-mortem d e l a y were a c c o u n t e d  for.  ~  T h i s decrease  c o u l d be a r e s u l t o f the d i s e a s e p r o c e s s , but c o u l d a l s o appear because  of  the h i g h f r e q u e n c y o f bronchopneumonia (and t h e r e f o r e pre-mortem h y p o x i a ) cause of death i n these groups.  The p o s s i b l e e f f e c t o f n e u r o l e p t i c  can a l s o not be e n t i r e l y r u l e d o u t . 2 groups, a l t h o u g h one  Homocarnosine was  as  drugs  a l s o lower i n t h e s e  s c h i z o p h r e n i c had an e x t r e m e l y e l e v a t e d c o n c e n t r a t i o n .  Glycerophosphoethanolamine  was  elevated i n choreics.  I t i s hoped t h a t these f i n d i n g s w i l l  c o n t r i b u t e to an u n d e r s t a n d i n g o f  the b i o c h e m i c a l n a t u r e o f these two horrendous r e l a t i o n s h i p between them.  That may  d i s e a s e s , and o f the p o s s i b l e  i n t u r n c o n t r i b u t e t o an u n d e r s t a n d i n g  o f e t i o l o g i c a l and p a t h o g e n e t i c f a c t o r s , and suggest a p p r o p r i a t e i n t e r v e n t i o n .  59  REFERENCES Baldessarini, R.J., Schizophrenia. N. Engl. J. Med. 297: 988-995 (1977).  Ban, T.A. and Lehman, H.E., Myths, theories and treatment of schizophrenia. Dis. Nerv. 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