REGIONAL DISTRIBUTION OF ENZYMES ASSOCIATED WITH NEUROTRANSMISSION BY MONOAMINES, ACETYLCHOLINE AND GABA IN THE HUMAN BRAIN A. V. P. MACKAY,' P. DAVIES.'. A. J. D E W A Rand ~ C. M. Y a m MRC Brain Metabolism Unit. 1 George Square. Edinburgh, EH8 9JZ, Scotland, U.K. (Receiced 29 April 1977. Recised 31 Augusr 1977. Accepted 16 Srpremher 1977)
Abstract-The activities of tyrosine hydroxylase (T-OH). DOPA decarboxylase (DDC). dopamine-/]hydroxylase (DBH). monoamine oxidase (MAO). choline acetyltransferase (ChAT), acetylcholinesterase (AChE).L-glutamic acid decarboxylase (GAD) and the concentrations of DNA and RNA were measured in I f 2 O areas of post-mortem brain tissue from neurologically and psychiatrically normal individuals. Emphasis has been put on regional distribution rather than establishing normal values and detailed comparisons have been made with previously published work on the normal human brain. Despite expressing all results relative to an internal reference point there was substantial inter-brain variability. There was no apparent relation between age. sex. medication, cause of death or time lag between death and dissection and any of the enzyme activities. Enzyme activities were fairly evenly distributed throughout cerebral cortex whereas clear differences existed along the rostro-caudal axis of the brain. It is hoped that this paper. with its companion paper on amine and metabolite concentrations. will be useful as a reference work for investigators of the chemical pathology of the human brain.
THE CLASSICAL studies of Vogt and her colleagues (FELDBEXG & VOGT. 1948; VOGT. 1954) marked the beginning of extensive investigations into the neuro-
distribution of acetyl cholinesterase (EC 3.1.1.7. AChE) in human brain came from MCGEER & MCGEER in 1971. Catecholamine-related enzymes have been extensively investigated, none probably more than monoamine oxidase (EC 1.4.3.4. MAO) since the original report of GANROT er a/. (1962) on the M A 0 activity of the human mesencephalon. Interest has been focussed upon the catecholamine systems in human brain following the formulation of the catecholamine hypothesis of affective disorder (SCHILDKRAUT, 1965). by the progress made in elucidating the chemopathology of Parkinson's disease (for 1966; FAHN er al.. 1971) reviews see HORNYKIEWICZ, and most recently by the dopamine hypothesis of 1972; MATTHYSSE, 1973). The schizophrenia (SNYDER. activities of tyrosine hydroxylase (EC 1.14.16.2, T-OH) DOPA decarboxylase (EC 4. I . 1.28. DDC) and dopamine-8-hydroxylase (EC 1.14.17.1, DPH) in human brain were first systematically investigated by VOGEL et a/. (1969). although limited mapping of the neurotransmitters dopamine (DA) and noradrenaline (NA) was already established due to interest in the extrapyramidal disorders (EHRINGER & HORNYKIEW-
chemical topography of the mammalian brain. Understanding human neurological and psychiatric disorder requires a detailed knowledge of the arrangement of neurotransmitter systems in the human brain, but within the mammalian species man has received less attention than the rat. The distribution of cholinergic neurons in the human brain was first investigated by ZETTLER& SCHLOSSER (1955) and by HEBB& SILVER (1956) who used the now widely adopted principle of measuring the activity of a biosynthesising enzyme, in this case choline acetyltransferase (EC 2.3.1.6, ChAT) as a marker with which to identify the presence of a particular class of neuron. The first report of the regional ~
~~
Present addresses: MRC Neurochemical Pharma' cology Unit, Department of Pharmacology. Medical School, Hills Road, Cambridge, CB2 2QD, U.K. * Department of Pathology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A. Shell Toxicology Laboratory (Tunstall). Sittingbourne. Kent, England. U.K. Abbreviations used: ChAT. choline acetyltransferase; T-OH. tryosine hydroxylase; DDC. DOPA decarboxylase; DBH, dopamine-S-hydroxylase; GAD. L-glutamic acid decarboxylase; MHPG. 3-methoxy4hydroxyphenylglycol; DA. dopamine ; HVA, homovanillic acid; DOPAC. 3, 4-dihydroxy-phenylaceticacid; 5-HIAA. 5-hydroxyindoleacetic acid; PLP. pyridoxal phosphate; AOAA. aminooxyacetic acid.
'
N.C. M/&K
ICZ,
1960).
The functional importance of ?-amino butyric acid (GABA) has received attention relatively recently and the contemporary view is that GABA is probably the major inhibitory neurotransmitter in the mammalian brain. The regional distribution of the synthesising enzyme L-glutamic acid decarboxylase (EC 4.1.1.15, GAD) in human brain was first reported by MULLER, & LANGEMAN (1962) but interest in this area has been stimulated by the report by BIRD et a!. (1973) that
827
A. V. P. MACKAY. P. DAVIES,A. J. DEWARand C. M. YATES
828
loss of G A D activity from t h e basal ganglia is t h e most striking neurochemical abnormality in Hunting-
cryptogenic viral infection developing in anyone exposed to the brain tissue.
ton's chorea a n d by the provocative hypothesis that G A B A systems may be deficient in t h e schizophrenias
Dissection
(ROBERTS.1972). T h e main purpose of this study was t o investigate the regional distribution and inter-relationship of various neurotransmitter systems within t h e normal h u m a n brain. W e have placed emphasis on t h e relationships rather t h a n t h e absolute values observed a n d t o this e n d we have investigated a large n u m b e r of neurochemical variables in t h e s a m e sample of brain tissue from a limited number of individual brains. W e n o w report a n investigation of T-OH. DDC, DBH, MAO, ChAT, AChE a n d GAD activities in twenty areas of h u m a n brain. T h e s a m e post-mortem brain tissue was t h e subject of assays for NA. 3-methoxy-4-hydroxyphenylglycol ( M H P G ) . DA. homovanillic acid (HVA) 3. 4-dihydroxyphenylacetic acid ( D O P A C ) a n d 5-hydroxyindoleacetic acid (5-HIAA) which are reported in the accompanying rr al.. 1978). paper (MACKAY
MATERIALS AND METHODS Sortrce marrrial
Whole brains from psychiatrically and neurologically normal patients (average age 60.8 years, range 4 6 7 4 ) who had died without protracted ante-mortem coma were obtained by courtesy of Professor CURRYat routine necropsies performed by the University Department of Pathology, Edinburgh Royal Infirmary. The average time lag between death and nenopsy was 30.1 h (range 2>36 h). In all cases the cadaver reached the mortuary cold-room ( 4 C ) within 3 h of death and remained there until postmortem examination was performed. Table 1 summarises the clinic-pathological characteristics of the ten cases from which brains were obtained. Once removed from the skull each whole brain was taken on ice to a cold room ( 4 C ) in which the brain was halved sagittaly; one hemibrain was fixed in formalin for gross pathological assessment (including serial sectioning) the other hemi-brain was immediately dissected into 20 constituent areas. Safery precaurions
Any case in which there was a suggestion of septicaemia hepatitis or massive blood transfusion was avoided. A sample of serum from every cadaver was submitted to the Regional Blood Transfusion Service Laboratory for Australia antigen detection and a negative result was required before the brain tissue was subjected to biochemical assay. Disposable gloves. aprons and face masks were worn at all times when brain tissue was being handled; mouth pipetting was strictly avoided. During homogenisation venti-mask dust respirators (Pneu-Seal. Leyland & Birmingham Rubber Co.. Ltd.) were worn and the homogeniser was used within a fume cupboard. Disposable clothing and apparatus were incinerated and non-disposable apparatus was sterilised in a 30% (v/v) aqueous solution of chloros (ICI). Laboratory personnel were advised to have a sample of blood taken prior to the commencement of the project and samples of serum have been stored to provide baseline antibody profiles against the remote possibility of a
All dissection was performed in a designated area within a cold room with an ambient temperature of 4'C. After careful removal of the meninges. each hemi-brain was dissected into the following twenty areas. usually in the order given. It was found that with the exception of the nucleus accumbens dissection of the areas described was best achieved in three dimensions on a hemi-brain which had not been serially sliced. The instrument best suited t o this purpose was a Swan Major scalpel with a crescent shaped disposable blade (shape U) and this blade was particularly suited to taking cortical shavings. Anatomical definitions were mainly those of ROMANES (1964). 1. Cerebellar cortex: the hemisphere was detached after cutting through the cerebellar peduncles and shavings of cerebellar cortex (2 mm thickness) were taken from the postero-lateral convexity of the cerebellar hemisphere. 2. Floor of fourth ventricle (hereafter referred to as 'Pons'): A block of tissue was excised from the floor of the fourth ventricle whose boundaries were from the midline to 3 mm lateral to the midline. 2 mm deep to the floor and approx 12mm in length. The sample included the locus cwruleus, the melanotic pigmentation of which aided dissection. 3. Pars compacta of the substantia nigra: a cut was made through the mid-brain at the level of the upper border of the superior colliculus to pass anteriorly and emerge on the ventral surface of the brain stem just posterior t o the mammillary body. The mid-brain was also divided transversely below the superior colliculus and the caudal portion of the brainstem removed. Pars compacta was defined by melanotic pigmentation and was exposed after removal of the crus cerebri. 4. Mesencephalic tegmentum (hereafter referred to as 'mid-brain'): this portion consisted of the mesencephalic tegmentum. the substantia higra having been removed and the tectum and crus cerebri discarded. 5. Hypothalamus: a 1.5mm deep block was cut from the medial aspect of the diencephalon. limited anteriorly by the anterior commissure and lamina terminalis, inferiorly by the upper border of the infundibulum. posteriorly by the mammillary body and upper border of the midbrain and superiorly by the hypothalamic sulcus. 6. Olfactory area: the area composed mainly of the olfactory trigone. limited anteriorly by the bifurcation of the olfactory tract into medial and lateral olfactory striae, posteromedially by the optic chiasma and tract and posterlaterally by the medial surface of the amygdala. The inferior surface is easily visible as the anterior perforated substance and a cut of 2 mm depth under this area was taken to include the olfactory tubercle. 7. Parahippocampal gyrus: slices of the total thickness of cortical grey matter were taken from the medial surface of the gyrus extending from the uncus posteriorly as far as the anterior end of the calcarine sulcus. 8. Temporal cortex: slices of the entire thickness of cortical grey were taken from the whole anteroposterior extent of the middle temporal gyrus. 9. Amygdala: a coronal section was taken through the entire body of the temporal lobe at the level of the mammillary body and this was found to transect the anterior tip of the inferior horn of the lateral ventricle. The amygda-
Enzyme distribution in human brain loid body could then be shelled out from the anterior pole of the temporal lobe. 10. Convexity frontal cortex: slices of the entire thickness of cortical grey were taken from a large area anterior to the precentral gyrus and including the superior and lateral surfaces of the superior frontal gyrus and the lateral surfaces of the middle and inferior frontal gyri. 11. Orbital frontal cortex: slices of the entire thickness 01 cortical grey were taken from the orbital surface of the frontal lobe to include the gyrus rectus. 12. Motor cortex: slices were taken of the entire thickness of cortical grey matter from the superolateral surface of the pre-central gyrus. The course of the central sulcus was remarkably variable between brains and thus in orderto define pre-' and post-central gyri with some confidence it was necessary to cut wedges out of adjacent gyri which appeared to occupy roughly the correct position. When a pair of gyri were found of which the anterior gyrus had clearly thicker cortex than the posterior then the former was taken to be the pre-central gyrus and the latter the post-central gyrus. 13. Sensory cortex: slices were taken of the entire thickness of cortical grey matter from the superolateral surface of the post-central gyrus as defined above. 14. Parietal cortex: slices were taken of the entire thick- ' ness of cortical grey matter from the lateral surfaces of the superior parietal lobule (posterior to the superior postcentral sulcus) and the anterior middle and posterior parts of the inferior parietal lobule. IS.Occipital cortex: slices were taken of the entire thickness of cortical grey matter from the lips and apposed surfaces of the calcarine sulcus extending anteriorly as far as the point at which the calcarine sulcus is joined by the parieto-occipital sulcus. 16. Mammillary body: the clearly definable ball containing the mammillary nucleus was excised from the inferior border of the hypothalamic bed. 17. Cingulate cortex: slices were taken of the entire thickness of cortical grey matter from the medial surface of the full length of the cingulate gyrus as defined superiorly by the sulcus cinguli. inferiorly by the callosal sulcus. anteriorly as far as the genu of the corpus callosum and posteriorly as far as the splenium of the corpus callosum. 18. Nucleus accumbens: this proved the most difficult area to identify and dissect with confidence. The most successful approach was found to be a coronal section through the frontal lobe at the level of the anterior border of the head of the caudate nucleus (Fig. I). Serial thin sections cut in the coronal plane moving posteriorly through the level of the optic chiasma revealed a small nucleus situated inferior to the lower margin of the anterior limb of the internal capsule between the caudate nucleus and putamen (METTLER.1948; RILEY, 1943). The antero-posterior dimension of this nucleus was taken to be approx 5 mm. Only those cases were included in which it was possible clearly to differentiate between the head of the caudate and the nucleus accumbens; in this series only five cases out of ten. 19. Caudate: the head and body of the caudate were reflected medially from the internal capsule by blunt dissection. Those portions of the head of the caudate which had been included in the coronal sections described above were excised from the slices and added to the bulk of the head and body. The tail of the caudate was not included in our estimations. 20. Thalamus: a block of tissue 5 mm thick in the antero-
829
posterior dimension was cut through the entire mediolateral breadth of the thalamus at the level of the interthalamic adhesion. Once removed, each of the twenty areas was chopped with a scalpel into very small fragments and then thoroughly mixed in order to make the sample homogeneous. Thereafter each area was divideo into SIX aliquots. each of which was stored in a screw-capped polycarbamate vial (Union Carbide) under liquid nitrogen ( - 196 C). By this means, individual aliquots could be taken from store and used for a small number of estimations at a time. thus avoiding the necessity of thawing and re-freezing. Provided that thawing did not occur between initial freezing and the time of assay the biochemical values to be reported did not change with storage for up to I year. Reproducibility between aliquots within any one area varied between 5 and 10'; for GAD and AChE. A ssa)s
After thawing from liquid nitrogen all tissue extracts were handled at (r-4 C unless otherwise stated. 1. G A D acririry was determined by measuring the rate of formation of I4CO2 from ~-[I-~*C]glutamic acid using a modification of the method of ROBERTS & SIMONSEN (1963). Tissue was weighed immediately after thawing and a 10% homogenate (w/v) prepared in 1 mkc-potassium phosphate buffer (pH 6.5) containing 0.1:; (v/v) Triton X-100 and 0.1 mM-pyridoxal phosphate (PLP) using a manual glass homogeniser. Ten pI homogenate were added to 1Opl of substrate mix which contained the following ingredients, giving final concentrations in the 20 pI incubation mixture as follows: 100 mkc-potassium phosphate buffer (pH 6.5); 0.5 mM-PLP; 0.5 mwdithiothreitol; 0.88 mM ~-[I-'~C]glutamic acid (23 mCi/mmol, Radiochemical Centre, Amersham. U.K.). 0.5 mkc-potassium L-glutamic acid; 0.1'); (v/v) Triton X-100; I mht-sodium arsenite. The samples were incubated at 37'C for 20min and a further 20min were allowed for I4CO2 evolution. Production of i 4 C 0 2 increased linearly with time up to 30min but levelled OR markedly at longer incubation times. There was a linear relationship with tissue concentration between the limits 2.5':" to 25"" (w/v) of homogenate strength. Sodium arsenite ( I mM) was added routinely to the incubation mixture and it was found that addition of 1 mwamino-oxyacetic acid. (AOAA) to samples of human caudate nucleus. amygdala and frontal cortex reduced apparent GAD activity to S",, of control values. 2. TOH activity was assayed by the method of HENDRY & IVERSEN(1971). except that tetrahydrobiopterin (a gift from Dr. R. F. LONG,Roche Products Ltd.). w a s used as cofactor in place of 6. 7-dimethyltetrahydropteridine. 3. DDC activity was measured by determining the rate of formation of ['*C]dopamine from ~-[Z-"C]dihydroxyphenyl alanine (L-DOPA). Homogenates (lo", w/v) of samples of brain regions were made in 25mM-sodium phosphate buffer. pH 7.0. containing 25 mM-EDTA. 0.1",, Triton X-100 and 0.1 mM-PLP. The conditions of incubation, and for extraction of [i4C]dopamine were exactly as described by EMSONer a/. (1974). 4. DBH activity was assayed by the two-stage procedure of MOLINOFFef a/. (1971) using phenylethylamine as substrate, except that 5 mM-EDTA was included in the phenylethanolamine-N-methyl transferase reaction mixture. Because of the possibility that different brain regions con-' lain different concentrations of the so-called 'endogenous
830
A. V. P. MACKAY, P. DAVIES.A. J. DEWAR and C. M. YATES
values were normally distributed. Means, standard errors. rank order and product moment correlations were all calculated using computer programmes compiled by Dr. GRAHAM HILL. RESULTS
Neuroparhological usscssmenf On the ten hemi-brains obtained for neuropathological examination from patients in whom no neurological or psychiatric abnormality was noted prior to death. eight were judged to be normal (Table 1). However minor focal abnormalities were detected in the brains from two 70 year-old males (Table I).Biochemical values observed in the corresponding contralateral halves did not differ significantly from the average and it was concluded that the neuropathological lesions. of an ischaemic nature, were strictly localised. Such changes are probably not unusual in a neurologically 'normal' population in later life. Post morrcm srahility FIG. I. Diagrammatic representation of a coronal section of the human brain passing through the rostra1 tip of the
globus pallidus. in which shading has been used 10 emphasise distinction between structures visible after Weigert and Nissl staining (modified from 279 and 280 Of METTLER. 1948). Key: 1. nucleus accumbens: 2. caudate nucleus; 3. internal capsule: 4. globus pallidus: 5. putamen.
None of the variables reported was correlated with the time lag between death and collection of the tissue. Although we have not made any detailed investigation of the post-mortem stability of each of the enzymes or substances measured, there exists a considerable volume of published information on stability which we have attempted to summarise in Table 2.
Age inhibitors' of DBH (for review. see LADURON. 1975) we The age of the patient was found to be negatively assayed samples of each region at five different homo- correlated with the DNA concentration in the amyggenate concentrations (1:25. 1:50. 1:75. l : l O and 1:150. dala (r = -0.90) and in the substantia nigra w/v). and further studied the eRects of 6 different concen- (r = -0.82) but there was otherwise no correlation trations of cupric ions (2pM. 1 0 ~ 4~0 1.1 ~ .6 0 ~ 8opM ~ . with a co-efficient greater than 0.8 between age and and 120pM) on each homogenate dilution. We found that maximal activities were obtained for all regions with a 1 :50 any of the variables reported. (wiv) homogenate and a copper concentration of 60 I'M. En:ynte acriciries The activities obtained under these conditions form the All results shown in Tables 3 1 0 are expressed as basis of the data in Table 9. Internal standards (phenylethanolamine; 182. 365 and 730 pmol/tube) and blanks percentages derived from the ratio of absolute values (boiled tissue homogenate) were included in each batch to an internal reference point. In an effort to compare of assays. our results with previously published reports we have. 5. MA0 activity was assayed using tyramine as substrate where possible. recalculated published data in order and the procedure of MCCAMAN er al. (1965). Tyramine to express values relative to one anatomical area. appears to be an appropriate substrate for measuring total Average values derived in this way are displayed in monoamine oxidase activity (WHITE& Wu, 1975).We have the appropriate tables. Expression of results in this not attempted to asscss the contributions to the total acway allows not only a simple appreciation of regional tivity made by the apparently distinct forms of this enzyme differences but also a comparison between labora(see YOUDIM, 1974). 6. ChATand AChE activities were assayed by the liquid tories whose methodology may differ. The values were (1969). The derived on a wet weight basis but the DNA and RNA ion exchange methods developed by FONNUM assay mixture contained (final concentration): 0.15 mM- values given in Table 3 provide an indication of the ['4C]acetyl-CoA. 8 mu-choline bromide. 250 mm-NaCI. cellularity of each of the twenty areas. The concen10 mwEDTA. 20 mM-sodium phosphate buffer (pH 7.4) tration of RNA provides an estimate of the density and 0.1 mM-physostigmine salicylate. of neuronal somata per volume of whole brain (HESS 7. D N A . R N A . Tissue was extracted in 0.5ml ( 5 - 2 0 , & THALHEIMER. 1965) and DNA, the most stable cell W / V ) b",, TCA and the DNA and R N A concentrations estiparameter. provides an estimate of total cell density mated as described by DEWAR & READING(1973).
(DAVIDSON, 1960).
Statistical analyses
Although an insufficient number of individual brains was available to test the assumption. it was assumed that all
ChA?; AChE acriiriries. These enzymes are among the most resistant to post-mortem deterioratiori (Table 2). ChAT activity appeared to be the highest
49
F
M
M
M
F
M
F
M
J.B.
R.R.
J.T.
F.S.
T.B.
W.D.
A.W.
A.G.
Myocardial infarction
Bronchopneumonia. oesophageal carcinoma Pancreatic carcinoma
Pulmonary embolism. cholecystit is Aspiration pneumonitis. colonic carcinoma
Pulmonary embolism Lymphoma Bronchopneumonia, bronchial carcinoma
Renal failure. chronic nephritis
Bronchopneumonia Aspiration pneumonitis, aortic valve replacement
Cause of death
Pentobarbitone (Digoxin) Morphine, Cyclizine, Nitrazepam. Dihydrocodeine. (Digoxin) Papaveretum. Diazepam. Nitrazepam, Dihydrocodeine Diazepam (Prednisolone)
Morphine, Cyclizine, Diazepam
(Digoxin) Chlorpromazine, Chlordiazepoxide, Nitrazepam, Dihydrocodeine Trimeprazine. Papaveretum, Morphine, Cyclizine Nil
Non-psychoactive drugs which might influence neurochemical variables are given in brackets
47
53
70
71
74
70
59
69 46
M M
E.W.
T.H.
Agdyrs)
Sex
Patient
Psychoactive medication. (within 1 week of death)
TABLE1. CLINICO-PATHOLOGICAL DATA
Normal
32
Tiny resolved infarct in outer aspect of putamen Normal
34
34
26
24
36
23
34
28 30
Time spent at 4 C ( h )
Normal
Several small old cystic infarcts in head and body of caudate and in depths of sulci on convexity of occipital lobe Normal
Normal
Normal
Normal Normal
Gross pathological assessment of hemi-brain
z
m
wm
5'
w
Y
A. V. P. MACKAY,P. DAVIES. A. 1. DEWAR and C. M. YATES
832
TABLE 2. SUMMARY OF PUBLISHED F I N D I N G S ON Stability Room temp.
Enzyme
GAD AChE MA0
ChAT DDC D/IH
-
4 c
Frozen
Stable Stable Stable
Stable Stable Stable
Stable Stable Stable
Moderately stable Unstable
Stable
-
2. 3. 7. 14. 16. 17, 22. 25. 7, 16, 17. 21 6. 7, 8. 9. 10, 18. 19, 20. 21, 23, 24. 1. 3, 5, 7. I I . 13. 16. 17.
Moderately stable Moderately unstable Unstable
-
4. 7. 12, 17, 24.
Stable
4. 10. 24. 26. 27.
Moderately unstable Very unstable
T-OH
POST-MORTEM STABILITY
References
-
4. 7. 10. 14. 15. 17, 24.
'
'
'
' ALQUILONIUSPI a/.. 1975; BIRD el ul.. 1973; BIRD & IVERSEN.1974; BLACK& GEEN,1975; BULL er al.. 1Y70: I, DOMINO er a/.. 1973: ' FAHN& C ~ T E ,1976; 'GANROTer 01.. 1962; 9 G er 01.. 1975; ~ "GROTE ~ er ~ ul.. 1974: I ' HEBB& SILVER. 1956; l 2 LLOYD& HORNYKIEWICZ. 1972; " MAHONEY er a/., 1971; l 4 MCGEERrr a/.. 1 9 7 1 ~ :I 5 MCGEER er ul.. 1971b: I b MCGEER er 01.. 1973; MCGEER& MCGEER.1976; I' ROBINSON er al.. 1971; '' ROBINSON cr ul.. 1972: SCHWARTZ el a/.. 19746; 2 1 SWANSON ef a/., 1973; 2 2 URQUARTel ul.. 1975: " UTENA Y I u/.. 1968: " VOGEL yr UI.. 1969: 2 5 VWEL cr a/.. 1975; z 6 WISE & STEIN, 1973: 2 7 WYAITer a/., 1975.
''
in the nigro-striatal and anterior limbic systems (Table 4) and was otherwise rather sparsely and evenly distributed. AChE activity was greatest in caudate nucleus. followed by the olfactory area and otherwise fairly evenly distributed throughout the 20 brain areas (Table 5). GAD ucririry. GAD appears to be stable after death (Table 2). Although the distribution is generally rather even throughout the brain (Table 6) certain areas stand out as having relatively high activity. These are nucleus accumbens. frontal cortex. temporal cortex. hypothalamus. substantia nigra and caudate nucleus. T-OH. DDC tirid D/IH acticiries. The enzymes in
the biosynthetic pathway for catecholamines appear to be the least stable of the enzymes studied, T-OH being particularly labile (Table 2). The activity of T-OH falls by approx 50% over 10 post-mortem hours in human brain (MCGEER& MCGEER.1976) and by a similar degree over 3 post-mortem hours at 20°C in rat brain (BLACK & GEEN, 1975). The greatest rate of decay seems to occur between the time of death and chilling to 4°C in the mortuary (MCGEER& MCGEER,1976). Instability of DDC and Dj3H is rather less than T-OH but still represents a problem to which no practical solution seems obvious. given the largely inevitable time delays
3. DISTRIBUTION OF DNA TABLE
AND
RNA
Concentration as percentage of caudate (mean f S.E.)* Area DNA Caudate Substantia nigra Pons Mid-brain Thalamus Hypothalamus Mamrnillary body Am ygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
100 (by definition) 160 f 24 (8)
137 & 9 ( I ) 121 f 6 (10) 127 f 8 (8) 155 f 28 (9) 93 f 12 (3) 147 f 14 (8) 108 f 8 (10) 94 f 7 (10) 129 f 6 (10) 119 f 5 (10) 114 f 8 (9) 120 f 5 (8) 117 f 13 (9) 122 f 10 (9) 114 f 7(10) 154 f 12 (9) 135 f 17 (4) 167 f I5 (6)
RNA
100 159 f 19 (7) 181 f 39 (9) 133 f 14 (9) 138 f 19 (7) 179 f 33 (8) 170 f 36 (3) 159 27 (8) 79 f 11 (9) 80 f 9 (9) 120 f lO(9) 97 f 14(9) 97 f 22 (9) 78 8 (8) 110 & 18 (9) 86 k 7 (7) 89 f 12 (8) 513 50 (8) 373 f 25 (2) 322 f 52 (6)
+
+
+
No. of samples is given in brackets. Absolute mean caudate concentrations were 0.03 (10) mg/g wet weight for R N A and 0.43 0.04 (9) mgjg wet weight for DNA.
0.96
~
Enzyme distribution in human brain TABLE4. DISTRIBUTION OF
833
CHOLINE ACETYLTRANSFERASE
Activity as percentage of caudate. Area
Mean of published results
Present study
Caudate (by definition) Substantia nigra Pons Mid-brain Thalamus Hypothalamus Mammillary body Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
+
(mean S.E. (n)) 100 61 f 28 (8) 36 14 (8) 28 f 6 (9) 23 f 7 (8) 53 f 37 (8) 14 f 2 (2) 48 f 9 (7) 18 f 7(9) 16 f 6 (9) 16 f 4 (9) 26 f 13 (9) 26 f 9 (9) 11 f 3 (9) 22 f 11 (9) 18 f 4 (9) 14 f 4 (9) 1 I f 3 (9) 110 f 24 (4) 92 f 18 (7)
(range) 100 I2 (2-36) I5 (10-21) I2 19 (3-81) 1 3 (2-42) LO (1-25) 18 (6-25) 4 13 (1-60) 1 1 (3-28) 16 (6-25) 21 ( 3 4 1 ) 3 (2-3) 3( 1 4 10 (8-11) 4 (1-6) I2 (1-4) 84 (78-90) 30 (7-44)
The mean absolute activity of the caudate in our study was 4.18 f 0.5 (9)pmol/h/g wet weight. Values from the cited papers have been recalculated as a percentage of the caudate values. Figures shown are mean percentages with the range of values shown in brackets. SOUrCeS: AQUlLONlUS ef a/., 1975: BIRD & IVERSEN. 1974: BULLef a/.. 1970: DOMINO er a[., 1973: HEBB& SILVER. 1956: MAHONEY ef a/., 1971: MCGEER& MCGEER.1971: MCGEERer a/.. 1973: MCGEER& MCGEER.1976: STAHL& SWANSON. 1974: ZETTLER & SCHLOSSER. 1955.
TABLE5. DISTRlBUTlON OF
ACETYLCHOLINESTERASE ~~
Activity as percentage of caudate
Area Caudate (by definition) Substantia nigra Pons Mid-brain Thalamus Hypothalamus Mammillary body Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
Present study (Mean f S.E. (n)) 100
43 f 2 (8) 34 f 3 (8) 33 f 2 (9) 37 f 4 (8) 41 f 4 (8) 44 f 2 (2) 38 f 3 (7) 29 f 3 (9) 25 f 4 (9) 26 f 2 (9) 25 f 3 (9) 26 f 3 (9) 30 f 2 (9) 27 f 2 (9) 24 i 2 (9) 31 f 2 (9) 29 f 2 (9) 26 i 2 (4) 52 f 5 (7)
Mean of published results (Range) 100 23 (20-25) 16 (10-22) 22 I I (10-12) 16 (10-23) 13 18 (10-25) 12 6 (1-10) 8 (4-12) 10 9 9 8 I 1 (9-14) 7 (2-11) 23 (20-25) 85 (80-89) 61
The mean absolute activity of the caudate in our study was 1.74 f 0.1 (9) mmol/h/g wet weight. Further details, see legend to Table 4. Sources: D O M I N O ef a/., 1973: MCGEER& MCGEER.1971: MCGEER& MCGEER.1976: STAHL& SWANSON, 1974.
A. V. P. MACKAY, P. D ~ V I EA. S . J. DEWAR and C. M. YATES
834
TABLE6. DISTRIBUTION OF GLUTAMIC
ACID DECARBOXYLASE
Activity as percentage of caudate Mean of published results (Range)
Present study (Mean S.E.(n))
Area Caudate (by definition) Substantia nigra Pons Mid brain Thalamus Hypothalamus Mammillary body Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
100
94 f 16 (8) 30 f 3 (8) 41 f 5 (9) 27 f 3 (8) 87 f 12 (7) 21 f 3 (2) 44 f 7 (7) 89 f 14 (10) 102 f 18(10) 63 f 8 (8) 79 f 12 (10) 71 f 9 (10) 63 f 12 (10) 52 f 6 (9) 50 f 6 (9) 105 f 18 (10) 48 f 11 (9) 183 f 32 (4) 70 f 16 (7)
100 150 (90-214) 80 (44100) 96 59 (31-100) 108 (51-141) 46 (30-67) 51 (43-59) 86 (50-1 20) 86 (43-122) 89 (8C98) 110 (90-136) 110 (100-136) 112 (92-131) 83 (5&147) 51 1-( 86 (60-115) 78 ( 6 1 0 0 ) 177 (15CL213) 96 (62-124)
The mean absolute activity of the caudate in our study was 0.71 f 0.2 (lO)pmol/h/g wet weight. Further details. see legend to Table 4. Sources: BIRDer of.. 1973: BIRD & IVERSEN. 1974: LLOYD& HORNYKIEWICZ, 1973: MCGEERer ul.. 1971a: MCGEERer af.. 1971b: MCGEERer of., 1973: MCGEER& MCGEER.1976: MULLER & LANCEMAN, 1962: STAHL& SWANSON. 1974: UTENAer (11.. 1968: VOGEL er a/.. 1969.
TABLE 7. DISTRIBUTION OF TYROSINE
HYDROXYLASE
Activity as percentage of caudate
Area Caudate (by definition) Substantia Nigra Pons Mid-brain Thalamus Hypothalamus Mammillary body Am ygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
Present study (Mean i S.E. (n)) 100 121 f 9 (8) 55 f 8 (8) 23 f 4 (9) 9 f 1 (8) 17 f 2 (8) 40 f 5 (2) 33 f 5 (7) 3 f 0.4 (9) 4 f 0.6 (9) 5 f 0.5 (9) 5 f 1.0 (91 5 f 0.8 (9) 7 f 1.0 (9) 6 f 0.6 (9) 6 1.0 (9) 7 f 0.9 (9) 5 f 1.0 (9) 42 f 6.0 (4) 5 f 0.5 (7)
+
Mean of published results (Range) 100 125 (40-190) 38 (1&90) 13 45 (1-90) 30 (1&59) 24 6 (1-11) -
30 10 20 40 -
12 (1-23) 19 (8-30) 96 (89-1031 85
The mean absolute activity of the caudate in our study was 90.7 & 3.9 (9) nmol/h/g wet weight. Further details. see legend to Table 4. Sources: GROTEer a/.. 1974: MCGEERel a/.. 1971b: MCGEERer ol.. 1973: MCGEER & MCGEER.1976: VOGEL er a/.. 1969.
835
Enzyme distribution in human brain between death of a patient and removal of the body to the mortuary, and the cooling time of the brain once the cadaver is placed in a chilled environment. The distribution of T-OH activity was uneven, the highest activity being observed in the brain stem and the lowest in cerebral cortex (Table 7). DDC activity was greatest in caudate nucleus but appreciable activity was found in the hypothalamus and substantia nigra (Table 8). The activity in the various cortical regions was uniformly low. Certain large discrepancies were observed between the distributions of DDC and T-OH activities. For example, DDC activity in substantia nigra is approx one third of that in caudate (Table 8) whereas T-OH activity in substantia nigra is 21% higher than caudate (Table 7). The absolute level of DDC activity in the caudate nucleus is 7-8 times higher than T-OH but in a number of'cortical areas the absolute activities appear to be similar. DFH activity was investigated in only 13 of the 20 areas of routine dissection (Table 9). The activity in the hypothalamus was considerably higher than any other area with the exception of the substantia nigra which had one quarter as much activity as hypothalamus. A pattern of fairly even distribution was found over the cerebral cortex. M A 0 acriuiry. The activity of this enzyme also appears to be remarkably stable after death (Table 2). We found high activity in every area investigated. the highest activity (olfactory area) differing from the lowest (calcarine cortex) by a factor of only 2 (Table 10).
DISCUSSION Since the original studies of human brain neurochemistry (ZETTLER& SCHLOSSER, 1955; EHRINGER & HORNYKIEWICZ, 1960; MULLER& LANGEMAN, 1962; GANROTer a/., 1962; VOGEL et a/., 1969) there has been a proliferation of reports in which reference is made to the normal brain. A problem facing the neuro: chemist is that of synthesising an overall pattern from the mass of individual reports which have often concentrated on a relatively limited number of variables in a few areas of normal brain relevant only to a comparison with a particular disease group. The past two decades have seen a steady modification and refinement of biochemical techniques which has meant an increasing ability to detect sparse or diffuse innervation by neurochemically discrete neuronal systems, but it has also meant that comparison of absolute values between reports is virtually impossible. We share with others (MCGEER& MCGEER,1976) the view that it may be more useful to consider relative contributions by different transmitter systems to particular brain areas than to consider absolute values. In our study we made no attempt to modify the usual procedure by which tissue is made available at necropsy following a death in a general hospital. This meant that although cadavers always reached the mortuary cold-room within 3 h, the time elapsed between death and dissection was never less than about 24 h. Post-mortem deterioration is a problem which is of varying importance according to the enzyme being studied: for example GAD. AChE and
8. DISTRIBUTION OF DOPA TABLE
DECARBOXYLASE
Activity as percentage of caudate
Area Caudate (by definition) Substantia nigra Pons Mid-brain Thalamus Hypothalamus Mammillary body Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area ~~~
~
Mean of published results (Range)
Present study (Mean f S.E. (n))
100
100
29 I 1 (8) 11 f 2 (8) 10 f 2 (9) 1 f 0.1 (8) 58 f 27 (8) 13 f 2(2) 6 f 2(7) 3 f 0.8 (9) 1 f 0.5 (9) 1 f 0.3 (9) 0.3 f 0.2 (9) 1 f 0.2 (9) 0.6 f 0.2 (9) 0.8 f 0.1 (9) 0.7 f 0.1 (9) 3 f 0.7 (9) 0.9 f 0.2 (9) 16 f 4.0 (4) 9 f 2 (7) ~~
6
10 40 6
44 (30-82) 24 (8-39) 8
6 6 6 (5-8) 6 (5-8) 91 40 (10-70) ~~~~
The mean absolute activity of the caudate in our study was 682 f 74 (9) nmol/h/g wet weight. Further details. see legend 10 Table 4. Sources: BOWENel at.. 1974: LLOYD& HORNYKIEWICZ, 197Oa: LLOYD& HORNYKIEWICZ, 1970h: LLOYD & HORNYKIEWICZ, 1972: MCGEER& MCGEER.1976: SHARPE ef al.. 1973.
836
A. V. P. MACKAY.P. DAVID. A. J. DEWAR and C. M. YATES TABLE 9. DISTRIBUTION
OF WPAMINE-8-HY OROXYLASE ~~~
~
Activity as percentage of substantia nigra Mean of published results (Range)
Present study (Mean S.E. (n))
+
Area
~-
~
Caudate Substantia nigra (by definition) Pons Mid-brain Thalamus Hypothalamus Mammillary body Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
ND 100
47 (27-66) 100
-
677 (64-1290)
-
-
-
468 & 62 (8) -
228 (73-383) 1039 (1 11-1967) 83
15 f 5 (9)
-
9 i 2 (9) 16 5 (9) 11 f 2 (9) 17 k 8 (9) 10 f 3 (9) 16 f 3 (9) IS f 6 (9) 18 & 8 (9) 12 f 5 (9)
I16 75 55 48 41 100
*
41
83 (24-141)
-.
-
DBH was not detectable (ND) in the caudate in our study. The mean absolute activity in substantia nigra was 56.7 f 8.9 (8) nmol/h/g wet weight. Further details. see legend to Table 4. Sources: GROTEuf a/.. 1974: VOGEL er a/.. 1969.
TABLE10. DISTRIBUTION
OF MONOAMINE OXIDASE
Activity as percentage of caudate
Area Caudate (by definition) Substantia nigra Pons Mid-brain Thalamus Hypothalamus Mammillary body Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Sensory cortex Motor cortex Parietal cortex Calcarine cortex Hippocampal cortex Temporal cortex Cerebellar cortex Nucleus accumbens Olfactory area
Present study Mean f S.E. - n ) 100
95 f 17 (8) 98 f 19 (8) 97 f 14 (9) 103 15(8) 88 f 7(8) 92 f 34(2) 113 2 21 (7) 92 f 16 (9) 95 f 14 (9) 81 f 12(9) 87 f 19 (9) 87 & 25 (9) 77 f 14 (9) 65 f 12 (9) 87 f 21 (9) 67 18 (9) 116 & 22 (9) 93 f 28 (4) 129 f 17 (7)
Mean of published results (Range) 100 1 I2 (90-130) 102 (50-190)
I07 115 (m-160) 45 (1W196) 14 (80-140) 82 (72-120) 88 (70-101) 83 ( S 1 0 0 ) 70 (5C80) -
86 (50-1 10) 99 (80- 150) 93 (80-1 10) 40 (30-60) -
120
The mean absolute activity of the caudate in our study was 1.40 f 0.19(9) pmol/ h/g wet weight. Further details. see legend to Table 4. 01.. 1974; GOTTFRIES SOUrCeS: DOMINO ef a/., 1973; GANROT Cf a/.. 1962; GOTTFRIES er 01.. 1975; GROTE ef a/.. 1974; ROBINSONef a/.. 1971 ; Robinson er al.. 1972: SCHWARTZ ef 01.. 1974a: SCHWARTZ el a/.. 19746: STAHL & SWANSON. 1974: UTENAef a/., 1968: VWEL er 01.. 1969.
Enzyme distribution in human brain
837
M A 0 appear to be remarkably stable even at room as that found in caudate nucleus. A more detailed temperature. This contrasts with T-OH which deter- grid or layered dissection of cerebral cortex might iorates rapidly, losing over half of its activity within reveal clear differences between areas but our results. 10 h of death in human brain (MCGEER& MCGEER, like those of others, suggest that the cortex is a site of fairly even representation by these neurotrans1976) and approximately half of its activity within 3 h of death in the rat brain kept at room temperature mitter systems. Clear differences do however exist between the con(BLACK& GEEN,1975). In general, it seems that the majority of losses in enzyme activity are incurred dur- tributions of transmitter systems to areas along the ing the immediate post-mortem period during which rostro-caudal axis of the brain. ChAT activity was time the core temperature will be high even if the highest in the caudate nucleus and nucleus accumbens intact cadaver is in a chilled environment. We can which agrees well with previous reports. Relatively see no way of avoiding this deterioration short of high activity was found also in limbic structures such excising the brain within minutes of death and clearly as the olfactory area amygdala and hypothalamus. such a proposal is fraught with problems. Accepting Otherwise the ChAT activity varied between approxithe routine procedure by which brains are usually mately one tenth and one half of that found in cauavailable between 24 h and 36 h after death means date. Our finding of relatively high ChAT activity in that tissue is obtained at a time when the rates of substantia nigra is at variance with previous reports deterioration of most enzymes are relatively low and we recognise that this may have arisen due to (MCGEER& MCGEER.1976; GROTEet a/.. 1974; FAHN contamination of our dissected samples with adjacent & C 6 r k 1976; BLACKGEEN,1975; BIRD& IVERSEN, ChAT-rich structures such as the interpeduncluar nu1974). The rapid deterioration of enzymes such as cleus and the roots of the occulomotor nerve. We T-OH and DPH probably explains the extremely low found a somewhat more even distribution of AChE levels of activity recorded in many of the areas inves- throughout the neuroaxis than previous workers. with tigated in the present study and although we have lowest activity in the hippocampal gyrus differing by expressed results as relative activities the reliability a factor of 4 from the highest activity in the caudate, of such data for these enzymes must be limited. whereas some published results show 10-fold differAnother problem is the possibility that certain ences between cortical areas and caudate nucleus. The interpretation of G A D activity based on enzymes such as DDC may deteriorate at different rates in different brain areas (BLACK& GEEN. 1975). I4CO2 evolution requires caution. It need not follow Although our method of expression of results that the quantity of I4CO2 evolved bears a strict stoireduced the inter-brain variation, the differences chiometric relationship to the amount of GABA probetween brains was considerable. with standard errors duced by decarboxylation of L-glutamic acid in the as high as 50% of the mean in some cases, and a 1-position. Exposure of tissue to glutamic acid in the number of factors can be suggested to account for presence of an inhibitor of mitochondria1 oxidative this. In the limited number of brains we have studied metabolism such as sodium arsenite (NEAL & 1969) and Triton X-100 has been shown to there was no apparent relationship between age, sex, IVERSEN, medication, cause of death or time lag between death abolish non-GAD-dependent decarboxylation of gluand dissection and any of the enzyme activities tamate in a range of mammalian tissues (MACDON1975) and thus we routinely inreported. The age range of the cases examined was NELL & GREENGARD. not large and this probably explains our failure to cluded both of these agents in our assay. It has been find age-related changes in enzyme activities as observed by HABERet a/. (1970) that apparent GAD reported by others (MCGEER& MCGEER,1976). Dis- activity in non-neuronal mammalian tissues is stimucrepancies in dissection and assay procedures must lated by AOAA. but this activity, as measured by be considered but they seem unlikely to have I4CO2 evolution, probably reflects non-GAD-depen& PHILLIPS, 1974; accounted for much of the variation since each brain dent decarboxylation (DRUMMOND was carefully handled in a similar way and assays WILSONet a/., 1973). Under our assay conditions we were usually performed in bulk on several brains at found that AOAA virtually abolished I4CO2 evoluthe one time. It seems plausible that there are indeed tion in three different regions of human brain. suglarge neurochemical differences between the brains of gesting that our estimates reflect GAD of neural orier a/. (1975) we observed a timenormal human beings which reflect genetic and en- gin. Like URQUHART vironmental heterogeneity and that we are using the dependent inactivation of GAD at 37 C and it thus yardstick of the pure-bred laboratory animal when seems worth emphasising that the incubation time we try to ascribe the bulk of such differences to arte- should be limited to around 20min when assaying GAD in human brain. fact. The greatest concentrations of GAD activity From the results we obtained it would appear that there is a roughly even distribution of ChAT, AChE. occurred in the extrapyramidal system, elements of T-OH, DDC and M A 0 activities between the nine cerebral cortex and one element of the limbic forecortical areas and that GAD activity is higher in con- brain. with the richest area being nucleus accumbens vexity frontal cortex and temporal cortex than else- and the poorest being amygdala mammillary body. where in cortex, with levels of activity at least as high hippocampal gyrus and calcarine gyrus. This picture
838
P. DAVIES. A. J. DEWAR and C. M. Y A T E ~ A. V. P. MACKAY,
of high GAD activity in the extrapyramidal system. nucleus accumbens and cerebral cortex is in agreement with previous reports (MCGEER& MCGEER, 1975). However. we detected lower relative GAD activity in substantia nigra than other workers and this may be partly explained by our sampling method which excluded pars reticulata and possibly the lateral edge of pars compacta which contain the highest GAD activity within the substantia nigra (FONN U M M er ol., 1974). T-OH activity was greatest in pons. substantia nigra. caudate nucleus. nucleus accumbens. amygdala and mammillary body. By far the highest DBH activity round in our series was in the hypothalamus but there was. rather surprisingly. one quarter as much activity in the dopamine-rich substantia nigra. This distribution agrees reasonably well with published reports. The presence of high DPH activity in samples of substantia nigra is puzzling; we are not satisfied that this merely reflects contamination by noradrenergic fibres 'en passant' from the locus coeruleus and investigation of amine and metabolite concentrations on aliquots of the same tissue failed to indicate the presence of significant quantities of norer a/., 1977; accompanying adrenaline (MACKAY paper). Like previous workers we found the highest DDC activity in caudate nucleus with intermediate activity in mid-brain and hypothalamus and generally low activity in cortical areas. According to MCGEER & MCGEER (1976) DDC activity in the olfactory area and nucleus accumbens was only slightly less than caudate but we found activity in these areas to be respectively one tenth and one sixth of the activity in caudate. M A 0 activity was found to be generally high throughout the brain and our findings agree with published data with the possible exception of the cerebellar cortex which has been reported to possess only one third of the M A 0 activity of the caudate nucleus (GROTE er a/., 1974; DOMINO er d.. 1973). A heuristic value of data from the normal human brain is the basis it provides for comparison with material obtained from clinically abnormal populations. We hope that the data we have reported here and our tabulated summary of previous reports will help in this respect, but the apparently general finding of large individual differences in biochemical values between individuals holds important implications for the neurochemical study of certain clinical disorders. For example. the functional psychoses may be associated with subtle changes in transmitter system interaction in the absence of detectable cellular loss and these changes could only be defined through the examination of a very large number of brains, no matter how reproducible the collection and processing methods might be. rlr~noH./cdgrmc,nr.~-We are very grateful to Professor
CURRYand his staff in the University Department of Pathology for the provision of necropsy material. to Dr. MALONEY and Dr. GORDON of the Department of Neuro-
pathology for their extensive assistance with dissection techniques and pathological assessment of brains. to Dr. DASof the Regional Blood Transfusion Centre and Dr. PEUTHERER of the University Department of Bacteriology for help and advice on safety procedures. to Professor ROMANS of the University Department of Anatomy for advice on anatomical matters, to KATHRYNGREENand GiLLiAN BARRONfor technical assistance, to Dr. G . HILL for computer analyses and to Dr. G. W. ASHCROFT for continual support and encouragement. We thank Mrs. GAILANDERSON for secretarial assistance. A. V. P. MACKAYwas a MRC Clinical Research Fellow.
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