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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 THE FACULTY OF (Department of in GRADUATE STUDIES 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 Brit ish 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 Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ABSTRACT Amino acids and other ninhydrin-positive compounds were measured in 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 significantly reduced in choreic thalami, in accord with deficiencies found i n other brain regions choreics (Perry et a l . , 1973a,b). GABA was also significantly reduced in schizophrenic thalami, suggesting a biochemical link between these two diseases, and supporting the hypothesis of a defect in the GABA system in schizophrenia (Roberts, 1972). Homocarnosine, a GABA-containing dipeptide, was also low in choreic and 9 out of 10 schizophrenic thalami. One schizophrenic had extremely high homocarnosine. Glycerophosphoethanolamine was significantly elevated in Huntington's choreics, but not i n schizophrenics. A number of other variables were considered for their potential influence on amino acid concentrations in thalamus. The majority of amino acids were found to rise in a significantly linear fashion in 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 significantly 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 in 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. i i i TABLE OF CONTENTS ABSTRACT •. . - i i LIST OF TABLES v ± LIST OF FIGURES . . v ± ± LIST OF ABBREVIATIONS Y 1 1 1 ACKNOWLEDGEMENTS i x CHAPTER 1 GENERAL INTRODUCTION I HUNTINGTON'S CHOREA A. Historical Background . 1 B. Prevalence and Social Impact.......... 1 C. Pathology 2 D. Cl i n i c a l Features... . . 2 1) Classical HC .2 2) Juvenile Variant .3 3) Westphal Variant 3 E. Age of Onset and Age at Death 4 F. Genetic Studies 4 II SCHIZOPHRENIA A. Historical Background .6 B. Prevalence and Social Impact... 6 C. Symptoms and Classification 6 D. Genetic Studies 7 1) Evidence for Genetic Factors .....8 2) Possible Modes of Transmission..... 8 CHAPTER 2 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 in Schizophrenia..... 13 3) Possible Causes of Dopaminergic Excess 14 iv a) GABA and the Dopamine Hypothesis 14 C. Low P l a t e l e t MAO i n Schizophrenia . 15 D. A l t e r e d Transmitters and Related Enzymes .16 I I HUNTINGTON'S CHOREA A. A l t e r e d Transmitters and Related Enzymes 17 B. Immunological Findings .20 C. Evidence f o r A l t e r e d Membranes 21 D. GABA Receptors. 21 CHAPTER 3 OTHER INFLUENTIAL VARIABLES A. Post-mortem Handling .22 1) GAD 22 2) Amino Acids 22 B. Pre-mortem Factors 23 C. Age 24 D. Drugs .' 25 E. Regional V a r i a t i o n . 26 CHAPTER 4 THE THALAMUS • 27 CHAPTER 5 PURPOSE AND RATIONALE OF THE PRESENT INVESTIGATION 28 CHAPTER 6 MATERIALS AND METHODS A. Sources of B r a i n Tissue. 29 B. Handling 29 C. P r e p a r a t i o n of B r a i n Tissue f o r Amino A c i d A n a l y s i s • • 30 D. Amino Ac 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. Data Used i n S t a t i s t i c a l Analyses ...31 G. The Independent V a r i a b l e s 31 H. The Dependent V a r i a b l e s ....32 I . S t a t i s t i c a l P r o t o c o l 33 CHAPTER 7 RESULTS A. The Independent V a r i a b l e s 1) Age 34 2) Post-mortem Delay. 34 a) Estimates of PMD 34 V b) PMD - Group Differences 36 3) Drug H i s t o r i e s a) Controls 37 b) Huntington's Choreics 37 c) Schizophrenics 37 d) Schizophrenic-like Psychotics 38 4) Cause of Death 38 5) Regions of Thalamus 38 B. The Amino Acids 1) GABA a) Data Uncorrected for Age and PMD 40 b) E f f e c t s of Age and PMD on GABA 40 c) GABA - Differences Among Groups, Accounting for Age and PMD 42 2) Glycerophosphoethanolamine (GLYC-PEA) 47 3) Homocarnosine (HCARN) 48 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 Groups 48 5) Amino Acids Showing a S i g n i f i c a n t Linear Change i n Controls (n=21) with PMD (range, 3-49 hours) but not with Age (range, 21-80 years) 52 6) Amino Acids Showing S i g n i f i c a n t Linear Changes i n Controls (n=21) With Both PMD (range, 3-49 hours) and Age (range, 21-80 hours) 52 CHAPTER 8 DISCUSSION , 55 REFERENCES 59 vi LIST OF TABLES I Studies of Neurotransmitters and Related Enzymes i n HC 18 II Detectable MIF activity in Cultured Lymphocytes (Barkley et a l . , 1977a,b, 1978) 20 III GAD activity in 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 ... 36 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 XII Multiple Regression Analysis of GABA vs. Age and ln(PMD) in Controls (n = 21) for whom Age and PMD were Known 42 XIII Tabulated Variables for Each Individual, and Deviations of GABA Values from Expected GABA Values 44-46 XIV Mean GABA Deviations (umoles /gm wet weight) of Controls, Huntington's Choreics and Schizophrenics (Without estimates of PMD) 47 XV Mean GABA Deviations (jimoles/gm wet weight) of Controls, Huntington's Choreics and Schizophrenics (Including estimates of PMD) .47 XVI Mean GLYC-PEA Concentration (jimoles/gm wet weight) i n Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-like Psychotics 48 XVII Mean HCARN Concentration (umoles/gm wet weight) in Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-like Psychotics . 51 v i i XVIII Amino Acids Showing no Significant Linear Change in Controls (n = 21) with Age (range, 21-80 years) or PMD (range 3-49 hours) and no Significant Differences Among Diagnostic Groups ....51 XIX. Amino Acids Showing a Significant Linear Change in Controls (n = 21) with PMD (range, 3-49 hours) but not with Age (range, 21-80 years) 53 XX Amino Acids Showing Significant Linear Changes in Controls (n = 21) with Both PMD (range, 3-49 hours) and Age (range, 21-80 years) 53 LIST OF FIGURES I Age Distribution in Controls, Huntington's choreics and Schizophrenics 35 II Postmortem delay distribution in Controls, Huntington's choreics and Schizophrenics 35 III GABA Concentration (uncorrected data) in Controls, Huntington's choreics and Schizophrenics 41 IV GLYC-PEA Concentration in Controls, Huntington's choreics and Schizophrenics 49 V Homocarnosine (HCARN) Concentration in Controls, Huntington's choreics ;and Schizophrenics .50 VI Threonine vs. Postmortem Delay 54 v i i i LIST OF ABBREVIATIONS ACh a c e t y l choline HIS h i s t i d i n e AChE ...acetylcholinesterase up . h a l o p e r i d o l ALA alanine HVA homovanillic a c i d ANOVA Analysis of Variance ILE. i s o l e u c i n e ARG arginine LEU leucine ASN asparagine l n n a t u r a l logarithm ASP a s p a r t i c acid LYS. . . . l y s i n e BP bronchopneumonia MAO. . . . monoamine oxidase C.... controls ( i n tables) MET methionine CAT choline a c e t y l transferase Ml myocardial i n f a r c t i o n C P Z chldrpromazine MIF. . . . migration i n h i b i t i o n f actor CSF cerebrospinal f l u i d MS m u l t i p l e s c l e r o s i s ( C Y S ) 2 cystine M Z ..monozygotic CYSTA cystathionine NE . . . .norepinephrine D A : dopamine ORN . . . o r n i t h i n e D ° C dopamine decarboxylase PEA. . . . phosphoethanolamine d f degrees of freedom PHE.. . .phenylalanine DOPAC dihydroxyphenylacetic a c i d PMD . . . post-mortem delay DZ d i z y g o t i c PRO. . . . p r o l i n e E A ethanolamine r 2 . . . c o e f f i c i e n t of determination EPS extra-pyramidal system < = Proportion of t o t a l variance accounted by regres-E S R e l e c t r o n spin resonance s i o n w i t h a given v a r i a b l e ) GABA gamma-amino b u t y r i c a c i d S. . . . schizophrenics ( i n tables) GABA' expected value of GABA (from SEM. . . standard e r r o r of the mean regression analysis) SER.... serine GABA-LYS... ..gamma-amino b u t y r y l l y s i n e SL. . . . s c h i z o p h r e n i c - l i k e psychotics GAD glutamic acid decarboxylase SS. . . . sums of squares ( i n tables) GLU glutamic a c i d TAU. . . taurine GLYC-PEA. . . .glycerophosphoethanolamine THR . . . threonine GLY g l y c i n e TRP. . . tryptophan GP globus p a l l i d u s TYR. . . t y r o s i n e GSH; ..... --reduced glutathionine ( i n tables for t h i s study, t h i s V A L • • - v a l i n e abbreviation r e f e r s to t o t a l GSH (GSH + 2GS-SG)) GS-SG. oxidized glutathione HC Huntington's chorea HCARN homocarnosine i x ACKNOWLEDGEMENTS I would l i k e to thank f i r s t the members of my thesis advisory committee, Dr. J.R. M i l l e r (chairperson), Dr. T.L. Perry (supervisor), Dr. D. Applegarth, Dr. P. MacLeod, and Dr. S. Wood for t h e i r contributions to t h i s study. In p a r t i c u l a r I thank Dr. Perry and Dr. M i l l e r f o r the many moments of t h e i r time, given at a moment's notice f o r the endless questions of a student. I would also l i k e to o f f e r a s p e c i a l thanks to other members of Dr. Perry's lab, Mrs. S h i r l e y Hansen, Mrs. Maureen Murphy and Mr. Stephen Kish for both technical assistance and general support throughout t h i s p r o j e c t . We are indebted to Dr. E.D. B i r d , U n i v e r s i t y of Cambridge, England fo r providing the majority of thalamus specimens used i n t h i s study. I am most g r a t e f u l to Dr. C. Wehrhahn f o r h i s time given i n numerous s t a t i s t i c a l consultations, and f o r programming and running a l l of the computer analyses. I thank also Dr. A Tingle f o r a di s c u s s i o n of immunological studies. The Huntington Society of Canada provided me with a pre-doctoral scholarship, without which t h i s study could not have been undertaken. I appreciate the assistance of Ms. Arleen Hardy and Ms. S h e i l a Manning i n the preparation of the f i n a l manuscript. F i n a l l y , I would l i k e to thank my friends i n the Department of Medical Genetics and at home, who have coped with t h i s thesis admirably. •1 CHAPTER 1 GENERAL INTRODUCTION I HUNTINGTON'S CHOREA A. H i s t o r i c a l Background (Discussed by Myrianthopoulos, 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 1872, George Huntington, a young New York p h y s i c i a n , addressed the medical academy i n M i d d l e p o r t , Ohio. Included i n h i s t a l k was a d e s c r i p t i o n of h e r e d i t a r y chorea w i t h dementia, thought at the time to be the f i r s t such r e p o r t . He recognized 3 marked p e c u l i a r i t i e s of the disease: i t s h e r e d i t a r y n a t u r e , a tendency to 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 disease only i n a d u l t l i f e . Although i t i s now known that r e p o r t s of h e r e d i t a r y chorea had appeared i n the e a r l i e r l i t e r a t u r e ( i n p a r t i c u l a r , one by Waters i n the 1840's), Huntington was the f i r s t to des c r i b e dementia as an e s s e n t i a l f e a t u r e (Maltsberger, 1961). His d i s c u s s i o n was so s u c c i n c t and l u c i d t h a t the name a s s o c i a t i o n has been w e l l j u s t i f i e d . V e s sie (1939) t r a c e d most a f f e c t e d New England f a m i l i e s to 3 B r i t i s h immigrants who a r r i v e d i n America about 1632. S e v e r a l of t h e i r descendants were notorious w i t c h e s , burned f o r t h e i r curse. Nova S c o t i a f a m i l i e s have been traced to the Huguenots who f l e d France a f t e r 1685, and Quebec f a m i l i e s to a s i n g l e ancestor who emigrated from France i n 1645. B. Prevalence and S o c i a l Impact There i s no evidence of r a c i a l , e t h n i c or geographic s e l e c t i v i t y f o r Huntington's Chorea (HC). Most prevalence estimates range from 4 to 7 per 100,000 (Myrianthopoulos, 1973), although i t i s r a r e i n Japan (0.4/100,000) and very p r e v a l e n t i n a few i s o l a t e s such as the Moray F i r t h i n Scotland (560/100,000) ( H e a t h f i e l d , 1973; Myrianthopoulos, 1966). In a Canadian study (Shokeir, 1975), prevalence i n Saskatchewan and Manitoba was estimated at 8.4/100,000. At the end of 1977 there were 61 l i v e cases of HC r e g i s t e r e d 2 with the British 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 Hall , 1978) HC has been the most frequent cause for referral, 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 hospita-liz a t i o n which is 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 proliferation. The caudate is more severely affected than the putamen. Generalized shrinking of the brain is accompanied by dilatation of the lateral ventricles especially in the anterior horns, with flattening of the caudate. There is similar, but less marked involvement of cerebral cortex (especially frontal 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 classical, juvenile, and Westphal variants. 1) Classical HC (Discussed by Heathfield, 1973; Brackenridge, 1971; Maltsberger, 1961) The classical form of the disease may be summarized as a disorder of movement, personality, and cognition. Movement i s described as choreo-athetotic with hyperkinesia. 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. Depression i s common, and apathy and neglect characterize l a t e r stages. Psychotic episodes are not uncommon; frequently schizophrenia i s the i n i t i a l diagnosis. Cognitive impairment often presents as the f i r s t recognized symptom. 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 ; rather, d i f f i c u l t y with tasks requiring 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). Patients eventually become profoundly demented. 2) Juvenile Variant (Discussed by Byers, 1967: Barbeau, 1970; Myrianthopoulos, 1966, 1973; Heathfield, 1973) About 8% of Huntington's choreics have onset before age 20, and 1-2% before age 10. 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), hypokinesia, parkinsonian tremor, convulsions, i n t e l l e c t u a l deterioration, cerebellar signs, and a rapidly progressive course. Pathology i s t y p i c a l of HC but there may be cerebellar disease also (Byers, 1967). Diagnosis of juvenile HC i s generally based on a clear 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 d i s t i n c t variant. The s p e c i f i c modifying e f f e c t of an alternate 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 with juvenile HC. A repeated, but as yet unexplained finding has been the disproportionately high number of males among affected parents of juvenile cases. 3) Westphal Variant (Discussed by Myrianthopoulos, 1966; Heathfield, 1973) The Westphal variant may represent a c l i n i c a l intermediate between the c l a s s i c a l and juvenile forms. I t i s characterized by prominent r i g i d i t y , possibly seizures, and adolescent or early adult onset. The pathology i s again t y p i c a l . 4 E. Age of Onset and Age at Death (Reviewed in Brackenridge, 1971a; Myrianthopoulos, 1966; Heathfield, 1973) Estimates in 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 light of potential confounding variables. Onset in an individual is 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 yield 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 earlier onset is associated with reduced genetic fitness. The artifact of 'anticipation' may arise from the failure to account for living individuals who could, at a later age, begin to manifest symptoms. This bias was avoided in 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 just discussed. Duration of illness can be calculated by difference, and is usually claimed to be about 15 years. F. Genetic Studies HC is a classical example of a dominantly inherited disease with complete penetrance. There are, nonetheless, some genetic peculiarities. Most anomalous observations, such as 'anticipation' result from ascertainment bias. One that seems to be real, however, is p a t r i l i n e a l transmission , a phenomenon that could result from differential mortality.and/or dif 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 in age of onset could favour the male line of descent. 5 Could genetic mechanisms account for the observed phenotypic heterogeneity? Is variation 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 locus, the modifying effect of the alternate 'normal' a l l e l e , or the influence 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 clustering with respect to c l i n i c a l subtypes, age of onset, and the pattern of neurological 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 parent-offspring corr e l a t i o n i n quantitative 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 subjective impression of c l i n i c a l consistency within f a m i l i e s , has persisted i n the l i t e r a t u r e (e.g. Wallace, 1972), and may apply to some families (Myrianthopoulos, 1966). The phenotypic pattern i s related to age of onset (Brackenridge, 1971b; Myrianthopoulos, 1973). The s p e c i f i c influence of the alternate a l l e l e i n determining age of onset i s u n l i k e l y , since correlation c o e f f i c i e n t s for t h i s t r a i t i n sibs and h a l f - s i b s are both close to .5 (Brackenridge, 1972a). As previously discussed, the alternate 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) cited evidence to support the theory that genetic heterogeneity underlies phenotypic v a r i a b i l i t y . Analysis of variance for 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 within. Correlation c o e f f i c i e n t s for these t r a i t s i n choreics did not decrease markedly from s i b - s i b through parent-offspring to cousin-cousin. 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 kindred than within s i b s h i p s , supporting the theory that s i m i l a r i t y i n sibs i s due to common background genes. Wallace proposed that there are at least 2 major (genetically d i s t i n c t ) groups of choreics, distinguished by r e l a t i v e l y early 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 alternatives of a genetic continuum or genocopies, and a single main gene with modifiers cannot be distinguished with present data. The d i s t i n c t i o n might be made through biochemical studies. I I SCHIZOPHRENIA A. H i s t o r i c a l Background (Discussed by Stabenau, 1977; Ban and Lehmann, 1977; Baldessarini, 1977; Kety and Mathysse, 1972) Kraepelin i n 1896 lumped several previously discrete conditions under the name of 'dementia praecox' - impaired cognitive function with onset i n early adulthood - and emphasized i t s inevitable malignant, prognosis. In 1911, Bleuler r e c l a s s i f i e d 'schizophrenia' - fragmentation of mental function - as a group of disorders, enlarging the scope of the e n t i t y . His description of primary (core) and secondary (accessory) symptoms has retained i t s diagnostic value to the present day. B. Prevalence and Social Impact (Discussed by Ban and Lehmann, 1977; Baldessarini, 1977) Schizophrenia i s probably the world's most important p s y c h i a t r i c disorder, and i s one of the greatest public health 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 population, with some v a r i a t i o n associated pri m a r i l y with c r i t e r i a for diagnosis. 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 for the occupancy of up to ha 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 society, 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: ( 1 ) phenomenology, (2) response to therapeutic intervention and (3) cause (Falek and Moser, 1975). These correspond to the l e v e l of understanding of the disease. For 7 s c h i z o p h r e n i a , c l a s s i f i c a t i o n i s s t i l l phenomenological, although drug response c h a r a c t e r i s t i c s are beginning t o be l i s t e d among c r i t e r i a f o r d i a g n o s i s . 'Schizophrenia' 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 should hot be assumed or even expected ( B l a s s , 1977; M e l t z e r , 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 p r o t o c o l s . His core d e f i n i n g symptoms are: (1) a c h a r a c t e r i s t i c thought d i s o r d e r or d i s t u r b a n c e of a s s o c i a t i o n s , (2) i n a p p r o p r i a t e a f f e c t , (3) w i t h d rawal from normal s o c i a l i n t e r a c t i o n s and (4) l a c k of contact w i t h and i n t e r e s t i n e x t e r n a l r e a l i t y . Accessory symptoms i n c l u d e (continuous or i n t e r m i t t e n t ) p e r s e c u t o r y , grandiose or somatic d e l u s i o n s , h a l l u c i n a t i o n s , and c a t a t o n i c symptoms (Kety and Matthysse, 1972). No one symptom i s pathognomonic, and a l l are n o n - s p e c i f i c ; thus i t i s not s u r p r i s i n g t h a t r o u t i n e d i a g n o s t i c methods, even those employing s o p h i s t i c a t e d computer analyses, are unacceptable ( M e l t z e r , 1976; Falek and Moser, 1975). For research purposes, d i a g n o s t i c u n i f o r m i t y i s a minimum requirement that has not yet been achieved. Two aspects 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 i s s u e s . One i s the r e t e n t i o n of B l e u l e r ' s sub-types ( i . e . c a t a t o n i c , paranoid, hebephrenic and simple) 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 ( f o r d i s c u s s i o n see B a l d e s s a r i n i , 1977; Falek and Moser, 1975). The other i s Kety's 'spectrum concept' used t o account f o r the v a r i o u s types of psychopathology (other than f u l l y - d e v e l o p e d s c h i z o p h r e n i a ) which may be present w i t h g r e a t e r frequency i n f i r s t degree r e l a t i v e s of s c h i z o p h r e n i c s ( M e l t z e r , 1976; B a l d e s s a r i n i , 1977). D. Genetic Studies Genetic 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 problems, ascertainment b i a s e s , and probably a complex and heterogeneous e t i o l o g y . The most that can be s a i d w i t h c e r t a i n t y i s that recent s t u d i e s leave l i t t l e doubt • 8 as t o the involvement 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 of s c h i z o p h r e n i a . 1) Evidence f o r Genetic F a c t o r s (Reviewed by Gottesman and S h i e l d s , 1973; DeFries and Plomin, 1978; B a l d e s s a r i n i , .1977; Kety, 1972; Kety and Matthysse, 1972; Tsuang, 1976) Evidence f o r a ge 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 runs along 3 major l i n e s , which w i l l simply 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 of sc h i z o p h r e n i c s than i n the general p o p u l a t i o n . There i s a l s o a c o r r e l a t i o n between the prevalence and degree of r e l a t i o n to the index case. E m p i r i c r i s k to f u l l s i b s ( i n c l u d i n g d i z y g o t i c (DZ) twins) and c h i l d r e n i s i n the order of 10-15%. I f two parents are s c h i z o p h r e n i c the r i s k may be as high as 50%. Second, monozygotic (MZ) twins have a much higher concordance r a t e (more than 50%) than DZ t w i n s , even i f they are reared apart. T h i r d , adoption s t u d i e s (using v a r i o u s methodologies) i n d i c a t e a higher prevalence of 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 of index cases than among adoptive 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 (with the c o r o l l a r y that common parenting 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 environmental f a c t o r has been demonstrated which w i l l , even w i t h moderate p r o b a b i l i t y , induce s c h i z o p h r e n i a . I t i s present i n a l l c o u n t r i e s that have been s t u d i e d , covering a wide range of c u l t u r a l i n f l u e n c e s . This does not imply, of course, t h a t environmental f a c t o r s are not r e l e v a n t to the expression of s c h i z o p h r e n i a . I f so, the concordance r a t e f o r MZ twins would be 100%. 2) P o s s i b l e Modes of Transmission (Reviewed by Kety and Matthysse, 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 simple Mendelian model can account f o r p o p u l a t i o n data on s c h i z o p h r e n i a . Any m o d i f i c a t i o n of 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 are at no higher e m p i r i c r i s k than o f f s p r i n g . Genetic 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 f o r the high frequency of s c h i z o p h r e n i a . There could be a s i n g l e major dominant gene w i t h reduced penetrance, but once the modifying e f f e c t of genes at the same or other l o c i 9 i s allowed, such a model i s d i f f i c u l t to distinguish from polygenic models. If several genes are involved, v a r i a t i o n with respect to a schizophrenic diathesis could be either continuous or quasi-continuous. There seems to be general agreement among reviewers that either mono- or polygenic models w i l l f i t e x i s t i n g data. Cavalli-Sforza and Kidd have suggested that only with the aide of biochemistry i s the genetic basis of schizophrenia l i k e l y to be elucidated (Kety and Matthysse, 1972). CHAPTER 2 10 •INTRODUCTION TO.BIOCHEMICAL THEORIES AND FINDINGS I SCHIZOPHRENIA A. The Transmethylation Hypothesis (Reviewed by Nestoros et a l . , 1977; Smythies, 1976; Matthysse and L i p i n s k i , 1975; Kety, 1972; Kety and Matthysse, 1972; B r o d i e , 1977; Ban and Lehmann, 1977) Proposed by Osmond, Smythies and Harley-Mason over 25 years ago, the t r a n s m y e t h y l a t i o n hypothesis s t i l l has h e u r i s t i c v a l u e . I t s b a s i s was 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 the catecholamines and mescaline (a psychotomimetic dr u g ) . The s u g g e s t i o n was t h a t a d i s t u r b a n c e i n m e t h y l a t i o n might l e a d t o an excess of endogenous, h a l l u c i n o g e n i c , methylated 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 i n d o l a m i n e s ) . Attempts 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 ' (methylated amines) present i n 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 that by P o l i n and co-workers i n 1961 (and confirmed i n a number of l a b o r a t o r i e s ) that methionine^" l o a d i n g caused e x a c e r b a t i o n of s c h i z o p h r e n i c psychoses i n some p a t i e n t s . I t i s not c l e a r , however, whether the psychotogenic e f f e c t was an i n t e n s i f i c a t i o n of the s c h i z o p h r e n i c p r o c e s s , or a superimposed t o x i c p s y c h o s i s . F u r t h e r , the e f f e c t could have r e s u l t e d from other m e t a b o l i c or pharmacological a c t i o n s of methionine and/or i t s 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 . Attempts t o ameliorate symptoms w i t h a methyl acceptor ( n i c o t i n a m i d e or n i c o t i n i c a c i d ) have f a i l e d (e.g. Nestoros, 1977), however S-adenosyl methionine i n b r a i n i s not e f f e c t i v e l y lowered by n i c o t i n a m i d e . B. The Dopamine Hypothesis C u r r e n t l y , the most popular hypothesis f o r a b i o c h e m i c a l d e f e c t i n 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 ' . Very s i m p l y , i t s t a t e s t h a t 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 the 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 , the mesolimbic dopamine t r a c t ) . Support f o r the theory 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 Methionine i s u l t i m a t e l y the source of methyl groups 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-adenosyl-methionine (SAM).  1) Evidence for Dopamine Involvement a) Neuroleptic Drugs (Reviewed in 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 in relieving the fundamental manifestations of schizophrenia; they are truly anti-psychoti The neuroleptics inhibit dopamine (DA)-mediated synaptic transmission in a dose-dependent manner, probably through blackade of DA receptors. Evidence for this i s indirect, but compelling in combination. F i r s t , DA stimulates a specific adenylate cyclase in 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 3 to competitively inhibit binding of H-DA and/or H-HP correlates closely with i t s anti-psychotic potency. The neuroleptic drugs cause an increase in 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 is 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 in feedback inhibition of f i r i n g ('short-loop'). This inhibition can be blocked by neuroleptics. Motor side effects reminiscent of the symptoms of Parkinson's disease (for which a DA deficiency has been well 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 Lipinski, 1975) Alpha-methyl-tyrosine is a potent inhibitor of tyrosine hydroxylase (T-OH), the rate limiting enzyme i n DA biosynthesis. Although nephro-toxicity precludes i t s use in 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 1976; Matthysse and Lipinski, 1975; Kety, 1972) Amphetamine has long been known to possess psychotomimetic properties, capable of e l i c i t i n g in nnn-schizophrenic individuals a c l i n i c a l picture often indistinguishable from acute paranoid schizophrenia. The major pharmacological effect is believed to be induction of catecholamine release. It would seem, therefore, that schizophrenia might also involve an excess activity 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 in NE metabolites. measured following probenecid administration, to prevent loss of the metabolite to venous blood 13 No such increase, however, has been noted in 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 frontal and medial temporal lobes lying above the brainstem, including the amygdala, hippocampus, septum, olfactory tubercle, and hippocampal gyri. It 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 is 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 in the interpeduncular nucleus of the ventral tegmental area. b) The Limbic System in Schizophrenia Support for the involvement of the limbic system in 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 in 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 inter-position at the outflow of limbic structures subserving the primary adaptive processes disturbed in schizophrenia" (Stevens, 1974). There are some reports of EEG abnormalities in schizophrenics, particularly the hypothalamus, and anterior thalamic nuclei, 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 in limbic structures (Stevens, 1973; Meltzer and Stahl, 1976; Torrey and Peterson, 1974). It is generally assumed that the antipsychotic activity of the neuroleptic drugs reflects interaction with DA synapses in the limbic striatum, while the EPS effects result from DA receptor blackade in the striatum 1 (Carlsson, 1978; Meltzer and Stahl, 1976; Snyder, 1972; Baldessarini, 1977). 3) Possible Causes of Dopaminergic Excess (Discussed by Meltzer, 1976; Meltzer and Stahl, 1976; Carlsson, 1978; Stevens, 1973; Matthysse and Lipinski, 1975; Kety, 1972) The real cornerstone of the DA hypothesis i s the evidence for DA blocking activity of the neuroleptics, but whether or not this i s the action required for the antipsychotic effect remains to be demonstrated (Matthysse and Lipinski, 1975; Baldessarini, 1977). A relative increase in dopaminergic activity might come about by any of a number of means. Theoretically at least, there could be (1) an increase in absolute levels of DA due to increased amounts of precursor, increased activity of T-OH, decreased inactivation by degradative enzymes, or failure of control mechanisms for storage and release. Attempts to find alterations in 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 cleft to pre-or post-synaptic c e l l s , resulting in an impaired feedback system, (3) an increase in 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 in the etiology the nigrostriatal DA tract is the site of reduced dopaminergic activity 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 in check by the tonic action of inhibitory (such as GABAergic) neurons, and are released for discharge on demand, through disinhibition. 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 act i v i t y , secondary to deficient inhibitory control (which in turn could be a primary or secondary phenomenon). In light of the focus on limbic system involvement in schizophrenia, some pharmacological evidence for GABA influence on mesolimbic DA neurons supports the general hypothesis. 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 activity of the animals following systemic DA potentiation. The findings supported the hypothesis that behavior characteristic of DA stereotypy resulted from blockade of GABA inhibition on DA neurons (although other explanations were possible). C. Low Platelet MAO in 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)^ " in 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 in the metabolic degradation of a variety of monoamines 16 depression also show reduced p l a t e l e t MAO), and a l l attempts to reveal a deficiency i n post-mortem brain have f a i l e d . Data from MZ twins discordant for schizophrenia suggest that the t r a i t i s genetically determined and thus a possible marker for s u s c e p t i b i l i t y rather than a function of the disease state. Despite the complexities involved, studies of MAO may contribute to the understanding of the schizophrenic phenomenon. D. Altered Transmitters and Related Enzymes Recently, Bird et a l . (1977) published a preliminary report on levels of DA, glutamic acid decarboxylase (GAD), and choline acetyl transferase (CAT), from patients dying with schizophrenia, schizophrenic-like psychoses, and controls"'". They examined 3 parts of the limbic system (nucleus accumbens, amygdala, and hippocampus) as w e l l as putamen. DA was found to be s i g n i f i -cantly increased i n the combined psychotic group i n nucleus accumbens but not putamen, and was not measurable i n amygdala or hippocampus. GAD a c t i v i t y was s i g n i f i c a n t l y decreased i n psychotics i n a l l 4 regions. CAT was s i g n i f i c a n t l y lower i n schizophrenics but not schizophrenic-like psychotics, 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, acetylcholinesterase (AChE), T-OH, dopamine decarboxylase (DDC), and GAD a c t i v i t i e s i n numerous brain regions from 11 schizophrenics and 18 controls. A l l brains had been removed within 2 24 hours of death and cases involving pre-mortem coma had been excluded . The main finding was increased CAT a c t i v i t y i n caudate, putamen, nucleus accumbens and hippocampus of schizophrenic brains. GAD a c t i v i t y was 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 any of these areas. 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 this study and that of Bird et a l , (1977) seems to have resulted from s t r i k i n g differences i n the mean a c t i v i t i e s of the 1 Synthesis of GABA from glutamic acid i s catalysed by GAD. CAT i s responsible for the synthesis of acetylcholine (ACh), another neurotransmitter 2 See following sections, 'pre-mortem factors' and 'post mortem handling' for more detailed discussion of these D o i n t s of concern , 1 7 control 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) following the report of Bird et a l . (1977), the data were re-examined (Bird et a l . , 1978a,b). When only cases for which there was evidence of sudden death by natural causes were included, differences 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 et a l . , 1978), including only sudden death cases, demonstrated no s i g n i f i c a n t difference between controls and schizophrenics for mean GAD a c t i v i t y i n caudate, putamen or nucleus accumbens. (Significant reductions remained for Huntington's choreics). Farley and co-workers (1978) have measured NE i n 4 paranoid schizophrenics and 12 controls. E a r l i e r studies indicated that NE levels did not vary with either age or post-mortem delay. S i g n i f i c a n t elevations of NE were found i n 4 areas of limbic forebrain: nucleus accumbens, mammillary body, s t r i a terminalis and v e n t r a l septum. Drug treatment with neuroleptics and/or cause of death were demonstrated to be unl i k e l y factors to account for the differences. Several other limbic areas showed no s i g n i f i c a n t difference between the 2 groups. I I HUNTINGTON'S CHOREA A. Altered 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 in certain areas of the brain, studies were undertaken to measure GAD, because of the implication that a metabolic error i n biosynthesis might be involved, and because the enzyme was thought to be more stable than GABA immediately after death. Most studies confirmed a decrease i n GAD i n HC brains, i n areas which p a r a l l e l e d the GABA 1 See following sections, 'pre-mortem factors' and 'post-mortem handling' for more detailed discussion of these points of concern TABLE I: STUDIES OF NEUROTRANSMITTERS AND RELATED ENZYMES IN HC AUTHOR GABA GAD CAT OTHER Perry et a l . (1973a) -sig * i n SN -also'V in caudate, putamen-GP - tGLYC-PEA/PEA ratio Perry et a l . (1973b) -striking ^ i n SN, caudate, putamen-GP -sig+ in OC, TC -no sig ^ i n FC, amygdala, thalamus, hypothalamus - t GLYC-PEA - <V HCARN Bird et a l . (1973) - ( 8 5 % ) in caudate, putamen, GP - n o 4 f in FC —|in caudate McGeer et a l . (1973a) - ^ i n caudate, putamen relative to that in thalamus., hippocampus, -patchy in caudate, putamen -T-OH normal -AChE normal amygdala McGeer et a l . (1973b) -uniform "V (50-60%) in caudate, putamen, GP, SN -patchy >|r Bird & Iversen (1974) - i|r in caudate , putamen - -V in caudate, putamen -no-V i n FC -bimodality among ptsT 1 h had normal levels caudate, putamen; *s had striking V -DA normal in most 1 i n 6 ri g i d cases -T-OH normal Stahl & Swanson (1974) - >K88-93%) in advanced HC, no in early HC (1 Pt.) -:.^(73-99%) in striatum -no^in CC -no { in early HC (1 pt. ) -MAO* (50%) in striatum of early HC Urquhart et a l . (1975) -confirmed earlier studies (above) -low i n HC but also low in 2/5 control samples -* HCARN in GP, putamen, CC McGeer et a l . (1976a) - f . \ i n extrapyramidal structures -patchy <Jr in neostriatum Abbreviations: -• SN = substantia nigra PEA = phosphoethanolamine TC = temporal cortex AChE = acetylcholinesterase sig = significant pt. = patient GLYC-PEA = glycerophosphoethanolamine OC = occipital 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 19 decrease. One study from Perry's laboratory, however, (Urquhart et a l , 1975) demonstrated that GAD was also low in 2 of 5 control samples, a finding which could not be accounted for by pre-mortem factors^. Overall, the reduction in GABA has been most striking in the extrapyramidal system (caudate, putamen, GP and SN), a finding in keeping with the motor disturbance of HC. Decreased CAT in HC has been described by McGeer as "patchy", since repeated sampling from caudate or putamen of a given individual may yield 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 in order to answer questions concerning the relevance of decreased GAD activity. The finding of a parellel deficiency of the activity of an enzyme and it s metabolic end product does not necessarily imply that a defective enzyme is primarily responsible for the loss of product. GAD i s localized in inhibitory neurons that u t i l i z e GABA (Bird and Iversen, 1974) and destruction of such neurons would result in concomitant loss of enzyme and product. This is probably the case in HC (Perry et a l . , 1977). The observed neuronal degeneration, most marked in basal ganglia, may represent specific 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 in 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 for causes of sel e c t i v e c e l l death. McGeer and McGeer (1976c) have suggested, for example, that HC might be an exci t o t o x i c phenomenon, r e s u l t i n g from chronic overstimulation 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 migration i n h i b i t i o n factor (MIF) a c t i v i t y (a correlate of delayed hype r s e n s i t i v i t y reaction) i n cultured lymphocyes confronted with brain tissue preparations from various i n d i v i d u a l s . Results are summarized i n Table I I : TABLE I I : Detectable MIF a c t i v i t y i n cultured lymphocytes (Barkley et a l . , 1977a,b, 1978) SOURCE OF LYMPHOCYTES SOURCE OF BRAIN TISSUE (ANTIGEN) Control HC MS Control - - -HC - + -MS - + -AD -Park. -HC = Huntington's Chorea MS = Multiple Sclerosis 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 active 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 virus that has been proposed for the etiology of multiple s c l e r o s i s (MS). Because of the late 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 infectious agent. MS lymphocytes might not respond s i m i l a r l y due to a di f f e r e n t set of immune response genes. They did not comment on the possible relationship of these observations to pathogenesis i n HC. They added that preliminary evidence demonstrated s i m i l a r a n t i g e n i c i ty of HC f i b r o b l a s t s . 2.1 C. Evidence for Altered Membranes B u t t e r f i e l d et a l . (1977) studied electron spin resonance (ESR) ch a r a c t e r i s t i c s of HC erythrocyte membranes. Differences from controls could be interpreted as due to (1) an altered membrane protein, (2) d i f f e r e n t amounts of a membrane component, (3) an altered consituent, organization or (A) a membrane-bound protein i n HC, not found i n controls. I f a s i m i l a r 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 linked to the pathogenesis of HC. Several studies have examined the behavior of HC f i b r o b l a s t s i n c e l l culture. 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 i n v i t r o . Gray and Dana (1977) found no uniform difference i n growth cha r a c t e r i s t i c s between HC and non-HC c e l l s . In contrast, Goetz et a l . (1975), Kirk et a l . (1977) and Leonardi et a l . (1978) found an o v e r a l l superiority 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 saturation density, avoiding an i n i t i a l lag phase and spending longer than normal i n exponential growth. The abnormal behavior may be the r e s u l t of an altered c e l l membrane. D. GABA Receptors 3 H-GABA binding to synaptic membrane preparations can be studied i n post-mortem brain. Findings with respect to altered 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 basal ganglia of Huntington's choreics, whereas Lloyd et a l . (1977) and Iversen et a l . (1978) found a substantial decrease i n binding s i t e density i n caudate and putamen. Iversen et a l . (1978) reported, however, that results were quite 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, investigations should be undertaken to elucidate factors that might be associated with normal or d e f i c i e n t GABA receptors, because of the implications for therapeutic intervention. CHAPTER 3 OTHER INFLUENTIAL VARIABLES A. Post-mortem Handling In order to interpret post-mortem measurements of amino acids and related enzymes, i t is 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 in mouse brain, with cooling conditions programmed to mimic those in 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. Following this, a stable plateau was maintained up to 72 hours. 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 significantly reduced GAD in material obtained more than 48 hours post-mortem. 2) Amino Acids Perry and co-workers (1971 a,b) compared amino acid levels in 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 gamma-aminobutyryl lysine (GABA-LYS)) were also significantly elevated in the 5 23 post-mortem specimens. Concentrations of reduced and oxidized glutathione (GSH 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 biopsied and autopsied specimens, either because they were r e a l l y not altered by post-mortem factors, or because the influence of th i s one variable was obscured by other large sources of v a r i a t i o n . Tews et a l . (1963) demonstrated that GABA i n dog cerebral cortex was elevated 51% by 20 to 23 minutes post-mortem. Minard and Mushahwar (1966) showed that GABA i n rat brain rose to a plateau within 1-2 minutes post-mortem and then remained constant to the end of the test period (30 minutes). Glutamic acid (GLU) was s i g n i f i c a n t l y lower i n brains not immediately frozen, but there were no comparable changes i n the levels of aspartic acid (ASP) or the neutral amino acid f r a c t i o n . B. Pre-mortem Factors There has recently been considerable discussion 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 possible influence of cause of death on post-mortem GAD levels i n the brain. In p a r t i c u l a r , there has been concern that conditions which lead to cerebral hypoxia cause a reduction i n GAD a c t i v i t y . Since causes of death tend to be related to disease conditions, there i s reason for legitimate concern when studying disease group differences. One example may serve to i l l u s t r a t e the magnitude of t h i s influence. McGeer et a l . (1973a) measured GAD i n several b r a i n regions, from a few ind i v i d u a l s . 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) TABLE I I I : GAD A c t i v i t y i n CONTROL HC no coma 7.30 2.90 Thalamus - Effect 4.43 of Coma (McGeer et 4.01 a l . , 1973a) coma 0.40 0.74 enumerated i n 'Discussion' 24 In larger studies (McGeer and McGeer, 1976a; Bowen et a l . , 1976) i t was c l e a r l y demonstrated that GAD a c t i v i t y decreased following coma from either head injury or i l l n e s s , but that there was some regional v a r i a t i o n i n the e f f e c t . The finding has been confirmed by Iversen et a l . (1978) who compared GAD a c t i v i t y i n 3 areas of brain from patients dying with bronchopneumonia to that i n patients dying sudden deaths, and found lower mean GAD a c t i v i t y i n the former group. L i t t l e i s known about the relevance of cause of death to brain amino acids. Tews et a l . (1963) examined effects of pre-mortem anoxia i n dog cerebral cortex. The increase of alanine was the most s t r i k i n g , but increases i n GABA (28%) and several other amino acids were also s i g n i f i c a n t . C. Age Numerous studies (McGeer et a l . , 1973; Bird and Iversen, 1974; Bowen, 1974, 1975; Bowen et a l . , 1976; McGeer and McGeer, 1976a,b; Perry et a l . , 1978a) have attempted to examine the effects of aging on neurotransmitter-related enzymes, p a r t i c u l a r l y , GAD, CAT, T-OH, AChE, and DDC. Results are d i f f i c u l t to interpret since they are probably dependent on the following factors: (1) whether or not post-mortem changes are accounted f o r , (2) the age range examined, (3) whether or not terminally demented and non-demented patients are separated, (4) the region (s) of brain examined and (5) sample s i z e . Decreases have been reported, i n various brain regions, for each of the enzymes l i s t e d above with increasing age. Senile dementia seems to correlate with more s t r i k i n g deficiencies of CAT (Perry et a l . , 1978). I t i s important to note that regional v a r i a t i o n i n these findings i s considerable. A report by TtfcGeer and McGeer (1976b) i s of p a r t i c u l a r i n t e r est for the present study. GAD a c t i v i t y was measured i n 56 brain regions ( w h e r e n i s ^ 5 ) from patients dying without pre-mortem coma or unconsciousness, and on whom autopsies were performed between 2 and 24 hours after death. In general, the thalamic areas showed the greatest decline, i n age ranges 5-20 years, and 20-50 years. C o r t i c a l and rhinencephalic areas followed, and basal ganglia showed r e l a t i v e l y less decline with age. The findings of decreased CAT with age i n some regions, and more s t r i k i n g deficiencies with senile 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) for cholinergic involvement i n memory storage. This function 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 anti-psychotic drugs. Not only i s this a variable that i s almost inex t r i c a b l y linked to diagnosis, but the drugs act on the very neurochemical systems that are being investigated for p o t e n t i a l differences. One means of investigating drug response as a possible source of v a r i a t i o n i s to test the effect i n a controlled animal experiment. Lloyd and Hornykiewicz (1977) treated rats both acutely and chronically with clozapine (chronic, 100 days) or HP (chronic, 167), and measured GABA and GAD i n substantia nigra. Acute treatment with either drug caused a s i g n i f i c a n t reduction i n GABA (but not GAD) compared to salin e - i n j e c t e d controls. Chronically 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 controls. 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 carried out 1. Thirty rats were injected for 100 days; 10 with CPZ (20 mg/kg/day s . c ) , 10 with HP (3 mg/kg/day s.c.) and 10 with ( 0.9% saline (equivalent volume). Following c e r v i c a l d i s l o c a t i o n brains were removed, grossly dissected and frozen i n l i q u i d nitrogen within 25-35 seconds. 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 1 To be presented at NINCDS Huntington's Disease Symposium, San Diego, November 16-18, 1978 limbic forebrain among the three groups. E. Regional Variation There i s considerable regional 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 within the brain (Perry et a l . , 1971a). S i m i l a r l y , there i s regional 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 for amino acid d i s t r i b u t i o n within the thalamus, however because of the presumed association between GAD and GABA, i t i s of interest to note the d i s t r i b u t i o n of the enzyme withi n the thalamus. McGeer and McGeer (1976a) measured GAD a c t i v i t y i n 7 thalamic areas, expressing results as a percentage of a c t i v i t y found i n the caudate. Overall thalamus had 68% a c t i v i t y , with a range from 101% i n anterior thalamus to 32% i n ventral posterior 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', in that stimulation w i l l evoke localized potentials in definite cortical areas. They receive specific 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 in 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 via the mammillothalamic tract and projects to the cingulate gyri. It is therefore included in the limbic system. The ventral lateral nucleus i s important for distribution of impulses from basal ganglia to motor areas of frontal lobe, thereby controlling voluntary movement. The dorso-medial nucleus contributes to mood and related motor responses, and appears to play a role in memory. Thalamic lesions may yield elevated sensory thresholds, with abnormal responses beyond the threshold. There may be spontaneous pain as well as emotional i n s t a b i l i t y . CHAPTER 5 PURPOSE AND RATIONALE OF THE PRESENT INVESTIGATION In t h i s study, amino acids and other ninhydrin-positive compounds were measured i n autopsied brain from patients dying with Huntington's Chorea, with schizophrenia or schizophrenic-like psychoses, and from controls dying without evidence of neurological i l l n e s s . A deficiency of GABA had been noted i n some (but not a l l ) regions of brain i n patients 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 brain was examined as w e l l for two reasons. F i r s t , Huntington's chorea and schizophrenia share certain c l i n i c a l features. It seemed possible, therefore, that they might share a biochemical a l t e r a t i o n . Second, i t had been proposed (Roberts, 19.72) that a deficiency 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. It seemed appropriate, therefore, to look for differences i n GABA levels amongh these groups. St r i k i n g differences i n any of the other amino acids being measured concommitantly would of course be of interest as w e l l . A number of other independent variables, besides disease status, are l i k e l y to influence amino acid levels i n the b r a i n . These have been examined and accounted f o r , as much as possible within the confines of the available material. CHAPTER 6  MATERIALS AND METHODS A. Sources of B r a i n 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. B i r d , MRC Neurochemical Pharmacology U n i t , U n i v e r s i t y of Cambridge, England. Data f o r thalamus of 5 c o n t r o l s and 6 Huntington's c h o r e i c s had been obtained i n Dr. Perry's l a b o r a t o r y , during a p e r i o d of time when thalamus was r o u t i n e l y analysed. These data were i n c l u d e d i n the study. Thalamus from a f u r t h e r 4 choreics had been i n storage here and a v a i l a b l e f o r a n a l y s i s , and that from 2 more HC p a t i e n t s and 5 c o n t r o l s was made a v a i l a b l e during the course of the study. Most of the Vancouver m a t e r i a l was from p a t i e n t s who had d i e d at Riverview H o s p i t a l or the Vancouver General H o s p i t a l , but some had been provided through donations from other North American c e n t r e s . B r a i n d i s s e c t i o n s were performed i n England ( f o r Dr. B i r d ' s m a t e r i a l ) or i n Vancouver. Data from a t o t a l of 70 samples were used f o r the study. B. Handling ( B i r d and I v e r s e n , 1974; B i r d et a l . , 1977) M a t e r i a l from England was handled i n the f o l l o w i n g manner: Whole b r a i n s obtained at necropsy were f r o z e n at -20°C. Frozen b r a i n s were tran s p o r t e d on dry i c e , stored at -20°C and then at -10°C f o r 12 hours before d i s s e c t i o n . D i s s e c t i o n s were c a r r i e d out at -5°C. D i s s e c t e d m a t e r i a l was then chopped and mixed, r e f r o z e n f o r s t o r a g e , then t r a n s p o r t e d to Vancouver on dry i c e where i t was s t o r e d at -80°C. In a l l cases but one, Vancouver m a t e r i a l was handled i n the f o l l o w i n g manner: Whole or h a l f b r a i n s were obtained at necropsy, immediately d i s s e c t e d and p a r t s f r o z e n on dry i c e . They were t r a n s p o r t e d f r o z e n t o storage at 80°C. In one case, the whole b r a i n was s t o r e d at -80°C u n t i l d i s s e c t i o n could be performed, and then p a r t s were r e - f r o z e n and s t o r e d . 30 C. Preparation of Tissue for Amino Acid Analysis A portion of frozen brain, usually approximating 200 mg was weighed, suspended i n cold 0.4 M perchloric acid (0.5 ml per 100 mg), then homogenized i n a tissue grinder with 100 strokes of a motor-driven Teflon pestle. The homogenate was centrifuged at 21,000 x g f o r 10 minutes, the supernatant removed and retained. The p e l l e t was resuspended i n 0.4 M perchloric acid (0.3 ml per 100 mg of o r i g i n a l t i s s u e ) , and rehomogenized (40 strokes). This was recentrifuged for 10 minutes at 21,000 x g, the supernatant 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 for 10 minutes at 21,000 x g to precipitate the potassium perchlorate. The supernatant was removed, volume measured, and concentration calculated using i n i t i a l wet weight. Samples were stored frozen at -80°C u n t i l amino acid analysis could be carried out. D. Amino Acid Analysis Amino acids, small peptides and other ninhydrin-positive compounds were separated on a Technicon automatic amino acid analyser (Perry et a l . , 1968), adjusted for simultaneous analysis of 2 samples.' Molar concentrations of the amino acids were calculated from the chromatograms using a Technicon integrator-calculator. E. S t a t i s t i c a l Methods (Sokal and Rholf, 1969 ) Standard methods were used for the following s t a t i s t i c a l t e s t s : Paired t Test, One-way Analysis of Variance (ANOVA), Linear Regression Analysis, Multiple Linear Regression Analysis, Regression on l n of one v a r i a b l e , and Multiple Regression using l n of one variable. If ANOVA yielded s i g n i f i c a n t r e s u l t s , Scheffe's Test for apo s t e r i o r i comparisons was used to l o c a l i z e differences. 31 F. Data Used i n S t a t i s t i c a l Analyses The majority of analyses were carried out using 63 of the o r i g i n a l 70 samples. Exclusions were made for the following reasons: 1) Three samples from one i n d i v i d u a l and two from another were analysed to see whether there were s t r i k i n g regional differences within the thalamus" for any amino acids. (No marked differences were noted.) Only one sample (anterior) for each i n d i v i d u a l was included i n calculations. 2) Two 'controls' had been treated with neuroleptics. Their control status was therefore questionable. 3) The diagnosis for one choreic was i n question. 4) One Huntington's choreic was an extreme outlyer on a computer analysis that involved grouping individuals on the basis of a l l amino acids. Examination of notes revealed that t h i s brain had been l o s t i n t r a n s i t and arrived i n Vancouver completely thawed. Its exclusion therefore seemed j u s t i f i e d . In a l l , there were maxima of 23 controls (C), 25 Huntington's choreics (HC), 10 schizophrenics (S), and 5 patients with schizophrenic-like psychosis (SL) for any given analysis. The l a t t e r two groups were not pooled since "(those) who were placed i n the schizophrenia-like group ranged from those who narrowly f a i l e d to meet the c r i t e r i a for schizophrenia, to those i n whom the diagnosis of schizophrenia seemed inappropriate" (Bird et a l . , 1977). The SL group was a poor one to deal with because i t was. small, and there were several unknowns among the independent variables. 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 Variables Information concerning the following independent variables was a v a i l a b l e , for most in 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 freezing of brain tissue (post-mortem delay (PMD)), region of the thalamus sampled, immediate cause of death, diagnostic category, and some drug history. Age and PMD are continuously distributed v a r i a b l e s , and therefore readily examinable for differences among groups, and for t h e i r p o t e n t i a l influence on the dependent variables ( i . e . amino acids). Diagnostic categories were, of course, part of the experimental design. Drug h i s t o r y was a d i f f i c u l t variable to deal with. F i r s t , i t was highly confounded with diagnosis, since most schizophrenics and choreics had been treated with neuroleptics, and controls had not. Second, each individual's treatment pattern was l i k e l y to 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 neuroleptics and those who had not. No rigorous analysis of pot e n t i a l drug e f f e c t could be carried out on t h i s sample. S i m i l a r l y , no rigorous analysis of the other discrete v a r i a b l e s , region of thalamus and cause of death, could be carried out. Details were simply tabulated. H. The Dependent Variables Thirty amino acids and other ninhydrin-positive 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 to be quantitated accurately. I. S t a t i s t i c a l Protocol For each amino acid, the following protocol was carried out: Control data were f i r s t submitted to a Multiple Linear Regression Analysis of amino acid on age and PMD. I f this regression was not s i g n i f i c a n t , i t was assumed that these 2 independent variables did not contribute markedly to the va r i a t i o n , and a straight ANOVA was performed for A groups. I f the ANOVA was s i g n i f i c a n t , group differences were lo c a l i z e d using Scheffe's Test (a highly conservative aposteriori test for differences between means taken from a larger group of means). I f the ANOVA was not s i g n i f i c a n t , data for a l l 4 groups were pooled and submitted to a Mult i p l e Linear and Quadratic Regression Analysis (by computer) as a more powerful test of the effects of age and PMD. If the i n i t i a l Multiple Linear Regression was s i g n i f i c a n t , the r e l a t i v e contributions of age and PMD were quantitated, and significance determined. If group differences for these amino acids were to be analysed, correction would have to be made for the influence 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 carried out, except for GABA. The rationale for the further analysis of GABA i s described under 'Results'. CHAPTER 7 RESULTS A. The Independent Variables 1) Age(See Figure 1) There was no significant difference in 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 C HC S n 23 25 10 mean 56.2 54.6 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 total GSH were known, except #39 - an extreme outlyer for ILE FIGURE /. C HC S Age distribution in controls, Huntington's choreics and schizophrenics FIGURE II. • PMD known o PMD estimated 6 0 + 5 0 -4 0 + 3 0 + 2 0 + 1 0 + I • • o OO o C HC S Postmortem delay 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 0.31 0.33 17 (C) 67 0.19 0.41 22 (HC) 21 0.97 0.19 6 (HC) 22 1.09 0.22 6 (HC) 31 0.55 0.29 12 (HC) 32 0.59 0.37 14 (HC) 44 0.25 0.22 14 (HC) 51 0.18 0.45 24 (HC) 53 0.65 0.51 26 (SL) 13 ? 0.44 35** (SL) 37 0 0.67 46 * pinoles/gm wet ** PMD estimated weight 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 significantly longer mean PMD than controls or Huntington's choreics. The latter 2 groups were not significantly different with respect to this variable. XSee figure II and Tables VI and VII.) TABLE VI: Mean PMD (hours) for Controls, Huntington's Choreics and Schizophrenics. (Without estimates of PMD) C HC S n 21 18 10 mean 18.0 16.4 35.0 SEM +2.6 +3.2 +5.0 ANOVA on 3 means: F_ = 6.48 (p <.005) L , HO S vs. C (P <.025); S vs. HC (p< .01) 37 TABLE VII: Mean PMD (hours) for Controls, Huntington's Choreics and Schizophrenics (Including estimates PMD) C HC S n 23 25 10 mean 18.1 15.9 35.0 SEM +2.4 +2.4 +5.0 ANOVA on 3 means: F 2 5 5 = 8.07 (p<.001) S vs. C (p<.025); S vs. HC (p<.01) 3) Drug Histories (Summarized i n Table VIII) a) Controls A l l 23 controls used for 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 patients had been treated with tetrabenezines (similar to reserpine), l i t h i u m , Valium, or miscellaneous drugs but not neuroleptics. These are placed under '-' i n Table V I I I . Nineteen patients had been treated with phenothiazines and/or HP, and are placed under '+' i n Table V I I I . One patient had been treated with phenothiazLnes for 2 years, but these had been discontinued for the l a s t 6 years of l i f e . This patient i s l i s t e d as ' + /-' i n Table V I I I . For one patient, 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'. This patient i s l i s t e d as '?' i n Table V I I I . c) Schizophrenics Nine patients had been treated with phenothiazines and/or HP for at least 1 year, and are placed under '+' i n Table V I I I . One patient was treated without neuroleptics and i s l i s t e d as '-'. ci) 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 Three p a t i e n t s had been t r e a t e d w i t h phenothiazines and/or HP, and are l i s t e d 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 s e d a t i v e s o n l y , and i s l i s t e d as For one p a t i e n t , drug h i s t o r y was unknown. 4) Causes of Death Causes of death 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 are ta b u l a t e d i n Table IX. Those l i s t e d toward the top are 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 without prolonged hypoxia. Bronchopneumonia and h e p a t i c coma would c e r t a i n l y have i n v o l v e d prolonged hypoxia. 5) Regions of Thalamus Most samples had been taken from a n t e r i o r medial thalamus, but some were from other r e g i o n s . For s e v e r a l i n d i v i d u a l s , the r e g i o n of thalamus sampled was not s p e c i f i e d . The regions 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 group, are tab u l a t e d i n Table X. TABLE V I I I : SUMMARY OF NEUROLEPTIC DRUG HISTORIES C HC S SL Treated w i t h n e u r o l e p t i c s (+) H i s t o r y unknown (?) 23 4 1 1 19 9 3 1 1 1 23 25 10 5 TABLE IX: CAUSES OF DEATH C HC S SL Myocardial Infarction (MI) 6 2 1 1 5 1 1 Coronary Thrombosis 1 Suicide (hanging) 1 1 Pulmonary Embolism 1 1 Asphyxia . . . 1 1 Accident 1 Peritonitis 1 Asthma 1 Congestive Heart Failure 1 Cancer (caecum) . 1 Bronchopneumonia (BP) 17 3 1 3 "Others" 3 1 Unknown * 1 4 1 23 25 10 5 TABLE X: REGIONS OF THALAMUS SAMPLED C HC S SL Anterior Medial (AM) Anterior (A) Medial (M) Lateral (Lat) Posterior Lateral (PL) Posterior (Post) Unknown (?) 10 12 9 4 2 5 1 1 2 .1 1 6 9 23 25 10 5 The Amino Acids 1) GABA a) Data Uncorrected f o r Age and PMD The mean GABA concentration i n the thalamus of schizophrenics and of Huntington's choreics was s i g n i f i c a n t l y lower than that of controls, when no co r r e c t i o n was made f o r the e f f e c t s of age and PMD. Schizophreni and choreics did not d i f f e r s i g n i f i c a n t l y from each other. (See Table XI and Figure III.) TABLE XI: Mean GABA Concentration (pmoles/gm wet weight) of Controls, Huntington's Choreics and Schizophrenics C HC S n 23 25 10 mean 2.16 1.64 1.59 SEM +. 13 + .10 +.11 ANOVA on 3 means: F = 2,55 6.41 (p<.005) C vs. HC (p<.01); C vs. S (p<.001); HC vs. S (NS) b) E f f e c t s of Age and PMD on GABA I n i t i a l l y , a multiple l i n e a r regression of GABA on age and PMD was carr i e d out, and f i t to this model was s i g n i f i c a n t . It was decided, however, to do a log transformation of PMD, even though f i t of con t r o l data to t h i s model was not quite as good. The reasons were the following: (1) Analyses of pooled data f o r ILE and GSH had indicated that f i t of data f o r these compounds to a n a t u r a l log curve was better than to a l i n e a r curve. (2) Observations have suggested that GABA increases very r a p i d l y i n the f i r s t hour post-mortem, and very l i t t l e a f t e r that (T.L. Perry, personal communication). I t would make l i t t l e b i o l o g i c a l sense f o r GABA to continue i n c r e a s i n g at a constant rate post-mortem. (3) Linear and n a t u r a l log curves are not very d i f f e r e n t FIGURE III. 3-5 30 + 2 5A-20 1-5 + 10 A-0-5 + C HC S GABA concentration (uncorrected data) in controls, Huntington's choreics and 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 in the extremes of the PMD distribution, and the linear curve is higher in 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 is shown in Table XII. TABLE XII: Multiple Regression Analysis of GABA vs. Age and ln(PMD) in Controls (n = 21) for whom Age and PMD were Known Source of Variation df SS MS F 2 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 significantly to the variation (linear decrease with age, logarithmic increase with PMD). The variables together accounted for 38% of the variation in GABA values in controls. c) GABA - Differences Among Groups, Accounting for Age and PMD Using the equation generated from the multiple regression analysis 43 for controls, an expected (control) value for GABA (GABA') was calculated for 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 for an age- and PMD-matched control. Deviations of observed GABA values from GABA' were calculated. A negative deviation means that the observed value i s less than the mean for an age- and PMD-matched control; that the observed value l i e s below the plane. The converse i s true for a posi t i v e deviation. Table XIII has tabulated individuals for each diagnostic group, i n order of magnitude of deviations from the plane. Details of a l l the independent variables are tabulated as w e l l , for a v i s u a l inspection of 'potential relationships with high or low GABA. Two types of analyses were carried out, each with and without individuals for whom PMD had been estimated. The f i r s t was an analysis of variance on deviations from the control mean (GABA-GABA') among 3 groups (C, HC, S). The second was a paired t t e s t , between GABA and GABA', as i f GABA' represented a matched control for each i n d i v i d u a l . Results are summarized i n Tables XIV and XV. With or without individuals for 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 differences i n mean deviation from expected, among the 3 groups. The differences were lo c a l i z e d to between controls and choreics, and between controls and schizophrenics. Accounting for 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 controls. 44 TABLE XIII: Tabulated Variables f o r Each I n d i v i d u a l , and Deviations of GABA Values from Expec ted GABA V a l u e s GABA GABA' GABA-GABA' PART AGE PMD umoles umoles umoles OF (yrs) (hrs) gm w.w. gm w.w. gm w.w. CAUSE OF DEATH THAL Rx 8 75 26 3.12 1.96 1.16 MI AM -60 48 3 2.63 1.86 0.77 ? 1 -63 40 27 3.29 2.77 0.52 MI • '•!.:' ? -61 21 5 3.12 2.64 0.48 suicide (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 a o r t i c aneurism MED -•J o eH H g 7 70 26 2.21 2.08 0.13 heart attack AM -5 53 24 2.56 2.46 0.10 MI AM -o 10 55 49 2.62 2.63 -0.01 heart attack AM -4 74 20 1.87 1.90 -0.03 asthma AM -69 44 3 1.81 1.95 -0.14 hepatic coma 1 -59 59 13 . . 1.93 2.10 -0.17 cong. heart 1 -62 66 8 1.61 1.78 -0.17 heart attack ? -1 60 19 2.03 2.20 -0.17 coronary thromb. MED -67* 80 22* 1.49 1.80* -0.31* MI AM -l l * 77 17* 1.47 1.78* -0.31* MI AM -70 58 8 1.61 1.96 -0.35 hepatic 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 (caecum) MED -9 73 27 1.48 2.01 -0.54 pulmonary embol. AM -64 31 8 1.45 2.57 -1.12 hepatic coma MED -* PMI ) estimc i t e d Abbreviations • w.w. = wet weight Rx = treatment with neuroleptics GABA' = expected GABA concentration cong. heart = congestive heart for c o n t r o l with given age and PMD Ca = cancer = 2.582 - .023(age) + .330 (ln(PMD)) , . . . pulmonary embol. = pulmonary THAL = Thalamus embolism AM = a n t e r i o r medial , P > p o s t e r i o r , coronary thronib. = coronary thrombosis MED = medial, A; = an t e r i o r , PL = p o s t e r i o r l a t e r a l 45 TABLE XIII: continued GABA GABA' GABA-GABA' PART # AGE PMD umoles umoles pmoles OF (yrs) (hrs) gm w.w. gm w.w. gm w.w. CAUSE OF DEATH THAL Rx 33 64 1 2.30 1.14 1.16 BP 1 + 45 57 1 2.19 1.29 0.90 BP 1 + 21* 72 6* 2.39 1.55* 0.84* ? AM -54 68 5 2.13 1.58 0.55 BP 1 1 31* 60 12* 2.05 2.05* 0.00* BP AM -25 68 . 20 2.00 2.04 -0.04 peritonitis AM -22* 55 6* 1.89 1.93* -0.04* BP AM + 27 72 28 1.85 2.06 -0.21 MI AM + 52 26 24 2.67 3.04 -0.37 1 f + ICS 53* 55 26* 2.01 2.42* -0.41* asphyxia + w g 28 57 22 1.81 2.32 -0.51 BP AM + S3 CJ 57 44 2 1.24 1.82 -0.58 BP 9 + CO 49 35 7 1.85 2.43 -0.58 BP A + riNGTOl 46 39 2 1.24 1.93 -0.69 BP 1 + riNGTOl 44* 62 14* 1.25 2.05* -0.80* MI AM -S3 58 61 2 0.63 1.43 -0.80 ? ? + 55 51 54 1.89 2.75 -0.86 BP 1 + 24 62 18 1.26 2.14 -0.88 ? AM + 32* 54 14* 1.34 2.23* -0.89* BP AM + 42 58 10 1.11 2.03 -0.92 BP AM + 51* 53 24* 1.40 2.43* -1.03* BP AM + 29 58 27 1.30 2.36 -1.06 BP A + 30 53 29 1.18 2.50 -1.32 BP AM + 26 43 24 0.99 2.66 -1.67 BP PL + 47 38 20 1.01 2.71 -1.70 BP ? +/-* PMD estimated 46 TABLE XIII: continued t GABA GABA* GABA-GABA' PART AGE PMD umoles umoles umoles OF # (yrs) (hrs) gm w.w. gm w.w. gm w.w. CAUSE OF DEATH 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 M + 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 47 TABLE XIV: Mean GABA Deviations (umoles/gm wet weight) of Controls, Huntington's Choreics and Schizophrenics (Without estimates of PMD) C HC S n 21 18 10 mean 0.0 -0.53 -0.66 SEM + . 11 +.18 +.17 ANOVA on 3 means: F„ ,r = 5.16 (p < .01) 2,46 C vs. HC (p <.05); C vs. S (p < .025); HC vs. S (NS) Paired t Test (GABA vs. GABA') C: HC: St TABLE XV: Mean GABA Deviations (jamoles/gm wet weight) of : Controls, Huntington's Choreics and Schizophrenics (Including Estimates of PMD) C HC S n 23 25 10 mean -0.03 -0.48 -0.66 SEM +.10 +.15 +.17 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') C HC S 2) Glycerophosphoethanolamine (GLYC-PEA) A multiple 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 for controls, 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 controls. The mean for schizophrenics was intermediate between controls and choreics, not '20 = = -0. 01 (NS) C17 = = -2. 87 (P < .01) fc9 = = -3. 63 (P < .01) t 2 2 = -.269 (NS) t 2 4 = -3.20 (p < .01) t 1 Q = -3.63 (p < .01) 48 s i g n i f i c a n t l y different from either. (See Table XVI and Figure IV ) TABLE XVI: Mean GLYC-PEA Concentration (umoles/gm wet weight) i n Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-Like Psychotics C HC S SL n 23 25 10 5 mean 0.79 1.25 0.92 0.70 SEM + .12 + .08 + .07 + .03 ANOVA on 4 means: F ^ = = 7.51 (p <.001) C vs. HC (p < .001) ; a l l other pairwise comparisons NS . 3) Homocarnosine (HCARN) A multiple l i n e a r regression of HCARN on age and PMD for controls was non-significant, therefore comparisons were made among groups with uncorrected data. One schizophrenic patient had a value for HCARN more than 3 standard deviations beyond the mean for controls (1.58 umoles/ gm wet weight; see Figure V). Analyses were carried out with and without t h i s value. The l a t t e r i s summarized i n Table XVII. With the outlyer, the schizophrenic mean was elevated to 0.49 (+ .12) which was not s i g n i f i c a n t l y different from controls. The o v e r a l l ANOVA on 4 means was correspondingly less s i g n i f i c a n t ( F = 3.68 (p < .01)). J , D O The mean HCARN concentration for Huntington's choreics and for 9 out of 10 schizophrenics was s i g n i f i c a n t l y lower than that for.controls. 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 multiple l i n e a r regression i n controls was non-significant for several other amino acids, therefore comparisons among groups were made with uncorrected data. There were no s i g n i f i c a n t differences among the 4 diagnostic groups for TAU, GLU, CYSTA, (CYS) 0, PHE, TRP, or 49 FIGURE IV. \ 3 i CD 0-5+ 0-2 C HC S SL GLYP~PEA concentration in controls, Huntington's choreics,, schizophrenics and psychotics. 50 FIGURE V. C HC S SL Homocarnosine (HCARN) concentration in controls, Huntington choreics, schizophrenics and psychotics. 51 TABLE XVII: Mean HCARN Concentration (umoles/gm wet weight) in Controls, Huntington's Choreics, Schizophrenics and Schizophrenic-like Psychotics C HC S SL n 22 25 9 5 mean 0.70 0.41 0.37 0.59 SEM +.06 +.05 +.04 + .09 ANOVA on 4 means: F 3 5 ? =5.939 (p < .005) C vs. HC (p <.01); C vs. S (p <025); a l l other pairw.ise comparisons NS GABA-LYS. Means are summarized in 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)2 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 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 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) Amino Acid C HC S SL TAU 0.83 (+.06) 1.03 (+.09) 0.88 (+.07) 0.93 (+.09) GLU 7.94 (+.21) 8.38 (+.53) 8.10 (+.29) 8.80 (+.19) CYSTA 0.89*(+.07) 1.01 (+.12) 1.09 (+.14) 1.35 (+.19) (CYS)2 0.23 (+.02) 0.19 (+.02) 0.25 (+.02) 0.32 (+.01) PHE 0.34 (+.02) 0.28 (+.02) 0.34 (+.02) 0.38 (+.03) 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) * excluding 3 individuals who died i n hepatic coma 52 5) Amino Acids Showing a S i g n i f i c a n t Linear Change i n Controls (n = 21) with PMD (range, 3-49 hours) but not with Age (range,  21-80 years) The majority of amino acids showed a s i g n i f i c a n t l i n e a r increase i n controls between 3 and 49 hours post-mortem. These are l i s t e d i n 2 Table XIX, i n decreaseing order 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 proportion of the t o t a l variance accounted f o r by a l i n e a r regression with PMD). Total GSH and PEA showed s i g n i f i c a n t l i n e a r decreases i n t h i s range. I f group di f f e r e n c e s were to be examined f o r these amino acids, c o r r e c t i o n would have to be made for the in f l u e n c e of PMD on amino acid concentrations. Figure VI i l l u s t r a t e s the change of THR with PMD. 6) Amino Acids Showing S i g n i f i c a n t Linear Changes i n Controls (n =21)  With Both PMD (range, 3-49 hours) and Age (range, 21-80 years) Both age and PMD contributed s i g n i f i c n a t l y to the c o n t r o l v a r i a t i o n i n ORN, HIS, and TYR concentrations (as w e l l as GABA). C o e f f i c i e n t s of determination are l i s t e d f o r each v a r i a b l e , f o r each amino ac i d , i n Table XX. I 53 TABLE XIX : Amino Acids Showing a Significant Linear Change in Controls (n = 21) with PMD (range, 3-49 hours) but not with Age (range, 21-80 years) Amino Acid Coefficient of ^ Determination (r ) Null Probability (P) EA .737 < .001 VAL .571 < .001 PRO .550 < .001 SER .506 < .001 THR .505 < .001 LEU .502 < .001 ILE .486 < .001 Linear MET .461 < .001 Increase LYS .449 < .001 GLY .398 < .005 ASP .375 < .005 ALA .268 < .025 ASN* .192 > .05 GLN* .190 > .05 ARG* .166 > .05 Linear GSH (TOTAL) .432 < .005 Decrease PEA .254 < .025 TABLE XX Amino Acids Showing Significant Linear Changes in Controls (n = 21) with Both PMD (range, 3-49 hours) and Age. (range, 21-80 years) Amino Acid Linear Decrease With Age Linear Increase With PMD Multiple Linear Regression 2 r P < r 2 P< r2 P< ORN HIS TYR .229 .005 .222 .005 .153 .05 .416 .001 .222 .005 .235 .025 .645 .001 .445 .005 .388 .025 FIGURE VI. Threonine vs. postmortem delay 1-2 + 0 0 - 8 + 0 6 + 0 - 4 4-0-.2 + n a a Linear regression through controls Log regression .through controls • Control CD Control PMD estimated • HC CD HC, PMD estimated A Schizophrenia + -I— 2 0 25 3 0 35 Postmortem delay (hours) 10 40 45 50 55 Ln CHAPTER 8 55 ; DISCUSSION The key findings of this study were (1) a deficiency of GABA in the thalamus of Huntington's choreics and schizophrenics, (2) a deficiency of the GABA-containing dipeptide, homocarnosine, in choreics and 9 out of 10 schizophrenics (1 schizophrenic had excessively high HCARN) and (3) an elevated concentration of GLYC-PEA in Huntington's choreics. Compounds which were examined, but showed no differences among groups were: TAU, GLU, CYSTA, (CYS)2, PHE, TRP, and GABA-LYS. There were indications that GABA-LYS might correlate with GABA and HCARN, and thus be reduced in 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 in 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 striking deficiency of GABA in schizophrenics and choreics*. Unfortunately, human material i n general, and autopsied brain in 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, in 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, in 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 light 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 in frontal cortex, and not in occipital cortex, caudate, putamen-GP, or SN. It i s interesting to compare these findings to those of McGeer and McGeer (1976b) who found the most striking decrease of GAD with age to be in the thalamus. The second variable was post-mortem delay. Significant linear increases with PMD in 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 significantly. Significant decreases with PMD were observed for total GSH and PEA. There were no significant linear changes in 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 in 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 significant 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 strikingly higher. The differences between these studies may stem from from the different delay periods being examined, and the nature of the change in any given amino acid. The delay in 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 in 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 li k e l y be more appropriate, but a linear model i s probably not a bad approximation in most cases. It 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 in 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 in 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 in 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, particularly following the results of Bird et a l . (1977) and Iversen et a l . (1978) which suggested that decreased GAD in 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 is quite possible that this variable i s in 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 in 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 restrict 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 medial thalamus. Those that were not may have contributed to the v a r i a t i o n i n amino acids, but were s u f f i c i e n t l y few that they would not l i k e l y have been the cause of group d i f f e r e n c e s . In conclusion, GABA concentration was s i g n i f i c a n t l y decreased i n the thalamus of schizophrenics and Huntington's choreics, whether or not ~ v a r i a t i o n with age and post-mortem delay were accounted f o r . This decrease could be a r e s u l t of the disease process, but could also appear because of the high frequency of bronchopneumonia (and therefore pre-mortem hypoxia) as cause of death i n these groups. The possible e f f e c t of n e u r o l e p t i c drugs can also not be e n t i r e l y ruled out. Homocarnosine was also lower i n these 2 groups, although one schizophrenic had an extremely elevated concentration. Glycerophosphoethanolamine was elevated i n choreics. I t i s hoped that these findings w i l l contribute to an understanding of the biochemical nature of these two horrendous diseases, and of the po s s i b l e r e l a t i o n s h i p between them. That may i n turn contribute to an understanding of e t i o l o g i c a l and pathogenetic f a c t o r s , and suggest appropriate i n t e r v e n t i o n . REFERENCES 59 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. Syst. 38: 665-671 (1977). Barbeau, A., Parental ascent in the juvenile form of Huntington's chorea. Lancet i i : 937 (1970). Barkley, D.S. , Hardiwidjaja, S.I., and Menkes, J.H., Huntington's disease: Delayed hypersensitivity in vitro to human central nervous system antigens. Science 195: 314-316 (1977a). Barkley, D.S., Hardiwidjaja, S.I., Menkes, J.H., Elliso n , G.W., and Myers, L.W., Cellular immune responses in Huntingtron's disease(H.D.). Detection- of H..D. and Multiple Sclerosis (M. S.) .brain antigenicity by H.D. but not M.S. Lymphocytes. Cell. Immunol . 32: 385-390 (1977b). Barkley, D.S., Hardiwidjaja, S.I., Tourtellotte, W.W., and Menkes, J.H., Cellular immune responses in Huntington disease. Specificity of brain antigenicity detected with Huntington disease lymphocytes. Neurology 28: 32-35 (1978). Barr, M.L., The Human Nervous System, an Anotomical Viewpoint. Hagerstown, Maryland: Harper and Row, 1974. Berger, P.A., Ginsberg, R.A., Barchas, J.D., Murphy, D.L., and Wyatt, R.J., Platelet -monoamine oxidase in chronic schizophrenic patients. Am. J. Psychiat. 135 : 95-99 (1978). Bird, E.D., MacKay, A.V.P., Rayner, C.N., and Iversen, L.L., Reduced glutamic acid decarboxylase activity of post-mortem brain in Huntington's chorea. Lancet i : 1090-1092 (1973). Bird, E.D., and Iversen, L.L., Huntington's chorea. Post-mortem measurement of glutamic acid decarboxylase, choline acetylase, and dopamine in basal ganglia. Brain 97: 457-472 (1974). Bird, E.D., Barnes, J. , Iversen, L.L., Spokes, E.G., MacKay, A.V., and Shepherd, M. , Increased brain dopamine and reduced glutamic acid decarboxylase and choline acetyl transferase activity in schizophrenia and related psychoses. Lancet i i _ : 1157-1159 (1977). Bird, E.D., Spokes, E.G., Barnes, J., MacKay, A.V.P., Iversen, L.L., and Shepherd, M. , Brain biochemistry i n Schizophrenia. Lancet jL: 35 (1978a). Bird, E.D., Spokes, E.G., Barnes, J., MacKay, A.V.P., Iversen, L.L., and Shepherd, M., Glutamic-acid decarboxylase in schizophrenia. Lancet 1: 156 (1978b). Bird, T.D. , and Hall, J.G. , Cl i n i c a l neurogenetics. Neurology 2J7_: 1057-1060 (1977). Blass, J.P., and Milne, J.F., Newer concepts of psychiatric diagnosis and biochemical research on mental i l l n e s s . Lancet i _ : 738-740 (1977). Bowen, D.M. , White, P., Flack, R.H.A., Smith, C.B. , and Davidson, A.N., Brain decarboxylase activities as indices of pathological change in senile dementia. Lancet ±: 1247-1249 (1974). Bowen,, D.M. , and Davidson, A.N., Extrapyramidal diseases and dementia. Lancet I: 1199 (1975). Bowen, D.M., Smith, C.B., White, P., and Davidson, A.N. , Senile dementia and related abiotrophies: Biochenical studies on histologically evaluated human post-mortem specimens. In Neurobiology of Aging, edited by R.D. Terry and S. Gershon. New York: Raven Press, 19 76. Brackenridge, C.J., A genetic and s t a t i s t i c a l study of some sex-related factors i n Huntington's disease. Clin. Genet. 1} 267-286 (1971a). Brackenridge, C-J-, The relationship of type of i n i t i a l symptoms and line of transmission to ages at onset and death i n Huntington's disease. Clin. Genet. 2: 287-297 (1971b). Brackenridge, C.J., A s t a t i s t i c a l study of half-sibships born to parents affected with. Huntington's chorea. J. Med. Genet. 9_: 23-27 (1972a). Brackenridge, C.J., Familial correlations for age at onset and age at death in Huntington's disease. J. Med. Genet. 9_: 23-27 (1972b). Brodie, H.K.H., Biology of psychoses: Past research and new frontiers. Southern Med. J. 70: 1064-1066 (1977). Butterfield, D.A., Oeswein, J.Q., and Markesbery, W.R. , Electron spin resonance study of membrane protein alterations in erythrocytes in Huntington's disease. Nature 267: 453-455 (1977). Byers, R.K., and Dodge, J.A., Huntington's chorea in children. Report of four cases. Neurology J J : 587-596 (1967). Caine, E.D., Ebert, M.H., and Weingartner, H., An outline for the analysis of dementia. The memory disorder of Huntington's disease. Neurology 27: 1087-1092 (1977). Carlsson, A., Antipsychotic drugs, neurotransmitters and schizophrenia. Am. J. Psychiat. 135: 164-173 (1978). Critchley, M. , Great Britain and the early history of Huntington's chorea. Adv. Neurol. _1: 13-17 (1973). Crow, T.J., Owen, F., Cross, A.J., Lofthouse, R. , and Longden, A., Brain biochemistry in schizophrenia. Lancet ±_: 1403 (1976). Davies, P. and Maloney, A.F.J., Selective loss of central cholinergic neurons i n Alzheimer's disease. Laneet i i ^ : 1403 (19 76). Davis, K.L. Mohs, R.C., TInklenberg, J.R., Pfefferbaum, A., Holl i s t e r , L.E., and Kopell, B.S., Physostigmine: Improvement of long-term memory processes in normal humans. Science 201: 272-274 (1978). DeFries, J.C, and Plomin, R. , Behavioral Genetics. Ann. Rev. Psychol. 29: 473-515 (1978). DeJong, R.N., The history of Huntington's chorea in the United States of America. Adv. Neurol. U 19-27 (19 73). Drachman, D.A., and Leavitt, J., Human -memory and the cholinergic system. A relationship to aging? Arch. Neurol. 30: 113-121 (1974). Enna, S.J., Bennett, J.P., Bylund, D.B., Snyder, S.H. , Bird, E.D. and Iversen, L.L., Alterations of brain neurotransmitter receptor binding in Huntington's chorea. Brain Res. 116: 531-537 (1976a). Enna, S.J., Bird, E.D., Bennett, J.P., Bylund, D.B., Yamamura, H.I., Iversen, L.L., and Snyder, S.H., Huntington's chorea. Changes i n i neurotransmitter receptors in the brain. N. Engl. J. Med. 294: 1305-1309 (1976b). Falek, A., and Moser, H.M. , Classification in schizophrenia. Arch. Gen. Psych. 32: 59-67 (1975). Farley, I.J., Price, K.S., McCullough, E., Deck, J.H.N. , Hordynski, W. , and Homykiewicz, 0., Norepinephrine in chronic paranoid schizophrenia: Above normal levels in limbic forebrain. Science 200: 456-457 (19 78). Gray, P.N., and Dana, S.L., Analysis of Huntington's chorea induced alterations i n c e l l culture. (Abstrl) F i f t h International Conference on Birth Defects, 1977. Exerpta Medica, International Congress Series no. 426, pp. 93, 1977. Goetz, I., Roberts, E., Comings, D.E., Fibroblasts i n Huntington's disease. New Engl. J. Med. 293: 1225-1227 (1975). Gottesman, I . I . , and Shields, J., Genetic theorizing and schizophrenia. B r i t . J. Psychiat. 122: 15-30 (1973). Heathfield, K.W.G., Huntington's chorea: A centenary review. Postgrad. Med. F. 49: 32-45 (1973). Ingram, W.R. , A Review of Anatomical Neurology. Baltimore: University Park Press, 1976. Iversen, L.L., Bird, E., Spokea,.E., Nicholson, S.H., aid Suckling, C.J., Agonist spe c i f i c i t y of GABA binding sites i n human brain and GABA i n Huntington's disease and schizophrenia. Alfred Benzon symposium 12, GABA -^Neurotransmitters. Copenhagen: Munksgaard (in press). Kety, S., Toward hypotheses.for a biochemical component i n the vulnerability to schizophrenia. Seminars i n Psychiat. 4_: 233-238 (1972). Kety, S. , and Matthysse, S., Prospects for research on schizophrenia. A report based on an N.R.P. work session. Neurosci. Res. Prog. B u l l . 10: 375-507 (19 72). Kirk, D., Parrington, J.M., Corney, G. and Bolt, J.M.W., Anomalous cellular proliferation in vitro associated with Huntington's disease. Hum. Genet. 36t 143-154 (1977). Klintworth, G.K., Huntington's chorea - The morphological contribution of a century. Adv. Neurol. 1_: 353-368 (1973). 62 L e o n a r d i , A., M a r t i n i , I.S., P e r d e l l i , F., Mancardi, G.L., S a l v a r a n i , S., and B u g i a n i , 0. , Skin f i b r o b l a s t s i n Huntington's disease. N. Engl. J . Med. 298: 632 (1978). L l o y d , K.G., D r e k s l e r , S., and B i r d , E.D., A l t e r a t i o n s i n H-GABA b i n d i n g i n Huntington's chorea. L i f e S c i . 21: 747-754 (1977). Ma l t s b e r g e r , J.T., Even unto the t w e l f t h generation - Huntington's chorea. J o u r n a l of the H i s t o r y of Medicine, pp. 1-17 (January, 1961). McGeer, P.L., McGeer, E.G., F i b i g e r , H.C, Choline a c e t y l a s e and glutamic a c i d decarboxylase i n Huntington's chorea. Neurology 23_: 912-916 (1973a). McGeer, P.L. , McGeer, E.G., and F i b i g e r , H.C, Glutamic a c i d decarboxylase and c h o l i n e a c e t y l a s e i n Hunt i n g t o n ' s chorea and Parkinson's d i s e a s e . Lancet i i : 623-623 (1973b). McGeer, P.L., and McGeer, E.G.,, Enzymes a s s o c i a t e d w i t h the metabolism of catecholamines, a c e t y l c h o l i n e and GABA i n p a t i e n t s w i t h Parkinson's disease and Huntington's chorea. J . Neurochem. 26_: 65-76 (1976a). McGeer, E., and McGeer, P.L., Neurotransmitter metabolixm i n the aging b r a i n . In Neurobiology of Aging, e d i t e d by R.D. Terry and S. Gershon. New York: Raven P r e s s , 1976b>, McGeer, E . G . and McGeer, P.L., D u p l i c a t i o n of b i o c h e m i c a l changes of Huntington's chorea by i n t r a s t r i a t a l i n j e c t i o n s of glutamic and k a i n i c a c i d s . Nature 26_3: 517-519 (1976c). McGeer, P.L., and McGeer, E.G., P o s s i b l e changes i n s t r i a t a l and l i m b i c c h o l i n e r g i c systems i n s c h i z o p h r e n i a . Arch. Gen. P s y c h i a t . 34: 1319-1323 (1977). M e l t z e r , H.Y., Sch i z c B u l l . 2: 11-18 (1976). M e l t z e r , H.Y. , and S t a h l , S.M. , The dopamine hypothesis o f s c h i z o p h r e n i a : A review. S c h i z . B u l l . 2: 19-76 (1976). Menkes, J.H., and S t e i n , N., F i b r o b l a s t c u l t u r e s i n Huntington's disease. N. Engl. J . Med. 288: 856-857 (1973). Minard, F.N. and Mushahwar, I.K., Synthesis of ^ -aminobutyric a c i d from a pool of glutamic a c i d i n b r a i n a f t e r d e c a p i t a t i o n . L i f e S c i . .5: 1409-1413 (1966). Myrianthopoulos, M.C, Huntington's chorea. J . Med. Genet. _3: 298-314 (1966). Myrianthopoulos, N.C, Huntington's chorea: The ge n e t i c problem f i v e years l a t e r . Adv. Neu r o l . 1: 149-159 (1973). Nestoros, J.N., Ban, T.A., and Lehmann, H.E. , Transmethylation hypothesis of s c h i z o p h r e n i a : Methionine and n i c o t i n i c a c i d . I n t . Pharmacopsychiat. 12: 215-246 (1977). P e r r y , E.K., P e r r y , R.H., Gibson, P.H., B l e s s e d , G., and Tomlinson, B.E. , A c h o l i n e r g i c connection between normal aging and s e n i l e dementia i n the human hippocampus. N e u r o s c i . L e t t . 6_: 85-89 (1978a). Perry, E.K., Blessed, G., Perry, R.H., and Tomlinson, B.E., Brain biochemistry i n Schizophrenia. Lancet jL: 35-36 (1978b). 63 Perry, T.L., Stedman, D., and Hansen, S., A versatile lithium buffer elution system for single column automatic amino acid chromatography. J. Chromatography 38: 460-466 (1968). Perry, T.L., Berry, K., Hansen, S., Diamond, S., and Mok, C., Regional distribution of amino acids in human brain obtained at autopsy. J. Neurochem. 18: 513-519 (1971a). Perry, T.L., Hansen, S., Berry, K., Mok, C. and Lesk, D., Free amino acids and related compounds in biopsies of human brain. J. Neurochem. 18: 521-528 (1971b). Perry, T.L.,Hansen, S., Lesk, D. and Kloster, M., Amino acids in plasma, cerebrospinal f l u i d , and brain of patients with Huntington's chorea. Adv. Neurol. 1_: 609-618 (1973a). Perry, T.L., Hansen, S., and Kloster, M., Huntington's chorea. Deficiency of <i -aminobutyric acid i n man. N. Engl. J. Med 288: 337-342 (1973b). Perry, T.L., MacLeod, P.M., and Hansen, S., Treatment of Huntington's chorea with isoniazid. N. Engl. J. Med. 297: 840 (1977). Potkin, S.G., Cannon, H.L., Murphy, D.L., and Wyatt, R.J., Are paranoid schizophrenics biologically different.from other schizophrenics? N. Engl. J. Med. 298: 61-66 (1978). Reed, T.E., and Chandler, J.H. , Huntington's chorea i n Michigan. 1^  Demography and genetics. Am. J. Hum. Genet. \Q_\ 201 (1958) (cited In Myrianthopoulos, 1966). Roberts, E. , An hypothesis suggesting that there i s a defect in the GABA system in schizophrenia. Neurosci. Res. Prog. Bul l . ^LO: 468-482 (1972). Shokeir, M.H.K., Investigations on Huntington's disease i n the Canadian prairies. I. Prenalence. Clin. Gaaet. 7_: 345-348 (1975). Sitaram, N., Weingartner, H., and G i l l i n , J.C, Human s e r i a l learning: Enhancement with arecholine and choline and impairment with scopolamine. Science 201: 272-276 (1978). Smythies, J.R., Recent progress in schizophrenia research. Lancet i i : 136-139 (1976). Sokal, R.R. and Rohlf, F.J., Biometry. The principles and practice of statistics in biological research. San Francisco: W.H. Freeman and Co., 1969. Stabenau, J.R., Genetic and other factors in schizophrenic, manic-depressive, and schizo-affective psychoses. J. Nerv. Ment. Dis. 164: 149-168 (1977). Stahl, W.1. ,. and Swanson, ?-.D. , Biochemical- abnormalities i n Huntington's chorea brains. Neurology 24: 813-819 (1974). Stevens, J., An anatomy of schizophrenia? Arch. Gen Psychiat. 29: 177-189 (1973). 64 Stevens, J., Wilson, K. , Foote, W., GABA blockade, dopamine and schizophrenia: Experimental studies i n the cat. Psychopharmacologica (Berl.) 39: 105-119 (1974). Tews, J.K., Free amino acids and related compounds i n dog brain: Post-mortem effects and anoxic changes, effects of ammonium chloride infusion, and levels during seizures induced by p.icrotoxin and by pentylenetetrazol. J. Neurochem. _10_: 641-653 (1963). . Torrey, E.F. and Peterson, M.R., Schizophrenia and the limbic system. Lancet i i : 942-946 (1974). Tsuang, M.T., Genetic Factors i n schizophrenia. In Biological Foundations  of Psychiatry, edited by R.G. Grenell and S. Gabay. New York: Raven Press, 1976. Urquhart, N., Perry, T.L., Hansen, S., Kennedy, J., GABA content and glutamic acid decarboxylase activity i n brain of Huntington's chorea patients and control subjects. J . Neurochem. 24_: 1071-1075 (1975). -Vessie, P.R., Hereditary chorea: St. Anthony's dance and witchcraft i n . colonial Connecticut. J. Conn. State Med. Soc. 3_: 596-600 (1939). Wallace, D.C. and Hall, A.C, Evidence of genetic heterogeneity in Huntington's chorea. J. Neurol. Neurosurg. Psychiat. 35: 789-799 (1972). Wendt, G.G. et a l . , Das erkrankungsalter bei der Huntingtonschen Chorea. Acta Genet. (Basel) 9_: 18 (1959) (Cited i n Brackenridge, 1971a) Wyatt, R.J. and Murphy, D.L., Low platelet monoamine oxidase ac t i v i t y and schizophrenia. Schiz. Bull. 2: 77-89 (1976). 

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