Metabolic and functional studies on post-mortem human brain

Neurochemistry International, Vol. 5, No. 3, pp. 253-266, 1983 Printed in Great Britain. All rights reserved

0197-0186/83/030253-14503.00/0 © 1983 Pergamon Press Ltd

COMMENTARY METABOLIC A N D F U N C T I O N A L STUDIES ON P O S T - M O R T E M H U M A N BRAIN J. A. HARDY and P. R. DODD* MRC Neuroendocrinology Unit, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, U.K. (Received 11 November 1982; accepted 18 November 1982)

Abstract--The evidence that samples of human brain tissue obtained at autopsy may be used as starting material for the isolation of cellular and subcellular preparations which exhibit metabolic and functional activity when incubated in vitro has been reviewed. Supporting evidence has been found in data from model experiments which used animal brain as the source material. Active preparations have been obtained after considerable (up to 24 h) post mortem delays. Such findings are less surprising when the post mortem stability of key tissue components (enzymes, receptors, nucleic acids) and the retention of cellular integrity are examined. The data from these fields have been reviewed and their relevance to functional studies assessed. Studies which use human autopsy material must consider many additional sources of variation not found in experiments with animal brain and the major problems are briefly discussed. It is argued that functional experiments present few, if any, difficulties not already inherent in static analyses of autopsy material and some procedures which help to minimise these difficulties are outlined. Experimentation in this area is greatly aided by the finding that metabolically and functionally active preparations may be obtained from frozen tissue pieces. Dynamic studies provide a new approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of neuroactive drugs in man.

The ultimate goals of biochemical studies on postm o r t e m (p.m.) h u m a n brain are to explain the processes which underly integrated cellular activity, to delineate the modes of action of neuroactive drugs, and to determine the chemical pathology of neurological and psychiatric disorders. Investigations in this field have generally utilised measurements of the concentrations or specific activities of particular constituents such as enzymes, intermediary metabolites, cofactors and neurotransmitters. This simple a p p r o a c h reflects the widespread misconception that p.m. brain c a n n o t be used as source material for preparations such as tissue slices, m i t o c h o n d r i a or synaptosomes which will exhibit integrated dynamic activity when incubated in vitro. The purpose of this review is to show that contrary to popular belief. preparations isolated m a n y h o u r s p.m. are indeed capable of such activity and that metabolic a n d functional studies afford a new a p p r o a c h to h u m a n neurochemical pharmacology and pathology.

* To whom correspondence should be addressed.

STUDIES ON METABOLISM IN POST-MORTEM TISSUE Transmitter uptake and release require the maintenance of m e m b r a n e potentials a n d therefore the presence of the A T P formed during glycolysis and oxidative phosphorylation. These processes, in turn, are supported by oxygen uptake (respiration). Respiration has been demonstrated in p.m. preparations of mitochondria and tissue slices ( M a n n et al., 1978; Tower and Young, 1973). In addition, synaptosomal oxygen uptake has been demonstrated in preparations from guinea-pig brain stored for 3 h at 20°C p.m. (Stahl a n d Swanson, 1976), and in preparations from rat brain after overnight storage of carcasses at 4°C ( D o d d et al., 1979), using glucose as substrate. In the latter two cases, respiratory rates were a b o u t 80% of those of the appropriate fresh tissue-derived preparation and, as in the fresh preparation, were increased by depolarising treatments. Moreover, preparations from rat tissue stored for 1 h at 20°C then overnight at 4°C had tissue potassium concentrations, after incubation with glucose, which were similar to those in preparations from fresh tissue, indicating the 253

254

J.A. HARDYand P. R. DDDD

•~ 1.

25

•

_

°~ O

il

I

1

I

1

IO

15

20

25

p.m. ~ t o y ,

h

Fig. 1. Respiration rates of human synaptosomes against p.m. delay. Squares, rate in control medium: triangles, corresponding rate in the presence of 75 ~M veratrine. The solid circle encloses values from patients dying with brain trauma, while the dashed circle encloses values from patients dying after diamorphine treatment; these were excluded from subsequent calculations. *, values from surgical biopsy material. The solid line shows the regression of control (including fresht values on p.m. delay (r = 0.78 [df 12], P < 0.01; intercept = 58 #mol/h per 100 mg protein, slope = -2.0/~mol/h per 100 mg protein/h); the dashed line shows the corresponding regression of veratine-stimulated values on p.m. delay (r = 0.77 [df 12], P < 0,01; intercept = 82 #mol/h per 100 mg protein, slope = -2.9 Hmol/h per 100 mg protein,/h). Adapted from Hardy et al. (1982ai.

maintenance of a membrane potential (Dodd et al., 1979, 1981b). Synaptosomes isolated from human brain up to 24 h p.m. also showed a linear oxygen uptake rate with glucose as substrate which was stimulated by depolarisation (Fig. 1). The rates of respiration (control and stimulated) of these preparations fell steadily with increasing p.m. delay, from levels initially similar to the corresponding values obtained with biopsy material ( D o d d e t al., 1981a). The preparations also showed high tissue potassium concentrations and thus maintained a membrane potential (Hardy et al., 1982). (Preparations derived from tissue from patients dying after brain injury were inactive (Fig. 1): this will be discussed below.) In agreement with these findings, Bowen et al. {1982) have shown that in tissue slices prepared from human brain excised within 1 h of death, the glucose oxidation rates were within the range of values shown by similar preparations from surgical biopsy material {Sims et al., 1981). Both fresh and p.m. preparations responsed to depolarisation by exhibiting increased rates of substrate oxidation (Bowen et al., 1982; Sims et al., 1981). Hence, preparations from p.m. brain are clearly able to metabolise glucose, take up oxygen and accumulate potassium against a concentration gradient, thereby maintaining a membrane potential. Taken with reports of the successful culturing of neurons from human trigeminal and superior cervical nuclei

4 - 6 h p.m., from which action potentials were recorded (Kim et al., 1979), this evidence would indicate that such preparations are likely to be capable of functional responses. TRANSMITTER UPTAKE, SYNTHESIS AND RELEASE BY POST-MORTEM TISSUE PREPARATIONS Uptake of transmitters and their precursors by p.m. preparations has been demonstrated by several groups, using both human and animal tissue. Thus, human synaptosomes isolated 6 h p.m. took up dopamine and noradrenaline by high affinity mechanisms, as did rat synaptosomes prepared after 12h tissue storage at room temperature (Garey and Heath, 19741. Comparison of the kinetic parameters of the uptake processes in fresh tissue with those of the p.m.stored preparations revealed that the affinity of the uptake site (Kin) was unchanged by storage, whereas the maximum velocity (Vmax) was reduced. This probably indicates that the number of uptake sites is reduced during p.m. storage, but that the characteristics of the remaining sites are unchanged (Garey and Heath, 1974). Uptake of catecholamines and serotonin has also been demonstrated in slices prepared from autopsy material up to 7 h p.m. (Olsen et al.. 19731. In concordance with this, synaptosomal serotonin uptake has been shown to be stable in rat tissue

Metabolic and functional studies on post-mortem human brain

255

40F E

_8 E

o

=E o

0

I

3

2

UptoRe/[GABA]

x IO-3

4C

.=_

~]

Krebs-phosptmte

]]]] + nil~cotic acid

T

r _8 == 2o

Tris- pho~ohote

[~

+ nipecotic acid

E

Fresh

P.M.

I~M.

Frozen

m

0

Fig. 2. GABA uptake in human brain synaptosomes. (A) Eadie Hofstee plot of [3H]GABA uptake by synaptosomes prepared from human brain obtained 5 h p.m. and stored frozen (see text and Hardy et al., 1983). Results are expressed as means_+S.E.M, of three experiments each. K m = 11.64/*M. V,,~ = 32.4 nmol/min per 100 mg protein. Incubations were carried out in Krebs phosphate medium containing glucose, for 30 s at 37C. (B) Sodium dependance of [-3H]GABA uptake and effect of the inhibitor nipecotic acid, in synaptosomes prepared from human brain obtained at surgery, at autopsy and used immediately (5 h p.m. delay), and from the same autopsy brain after freezing and rethawing. The GABA concentration was 20 #m, that of nipecotic acid was 100/~M. Incubation conditions as in (A), except that equimolar Tris HCI replaced NaCI for the sodium-free case. Results are means _+ S.E.M. of three experiments each. for up to 7 h p.m. at 22cC (Weiner et al., 1979). The high affinity uptake system for glutamate was also found to be fairly stable in rat tissue p.m. (Schwarcz, 1981) and its presence in h u m a n autopsy-derived synaptosomes d e m o n s t r a t e d (Schwarcz and Whetsell, 19821. We have found that synaptosomes from autopsy material posses a high affinity uptake system for GABA (Fig. 2). The glutamate and G A B A uptake systems in p.m. preparations both exhibited similar inhibitor characteristics and sodium dependence to those in fresh preparations (Schwarcz, 1981; Schwarcz and Whetsell, 1982; cf. Fig. 2). Choline uptake in rat synaptosomes after storage of the tissue for 24 h at 4~'C has been demonstrated to be similar to that in the fresh preparation (Klemm and Kuhar, 1979).

Synaptosomes isolated from rat brain after storing carcasses for 1 h at 4 C plus 16h at 20:'C, and from p.m. (up to 20 h) h u m a n brain have been shown to release the transmitter amino acids, as well as the n e u r o m o d u l a t o r somatostatin, in response to depolarising stimuli (Dodd et al., 1979; Hardy et al., 1982). The responses in the p.m. preparations were similar to, although generally reduced from, values found using fresh tissue material (Bennett et al.. 1979; Dodd et al., 1981a,b). In agreement with these findings, Bowen et al. (1982) have shown that tissue prisms from rapid autopsies (ca. I h p.m. delay) synthesised acetylcholine from glucose and released it in response to depolarisation at rates within the range of values obtained with the equivalent flesh tissue preparation.

256

J.A. HARDY and P. R. DODD THE UNDERLYING ENZYMIC AND STRUCTURAL INTEGRITY OF POST-MORTEM BRAIN

The isolation and incubation of metabolically and functionally active preparations (e.g. synaptosomes, mitochondria, isolated glia and neurons, tissue slices, or tissue culture preparations) from p.m. brain requires, in addition to the usual provisos associated with the use of in vitro preparations, two further conditions: (I) that the enzymes, receptors and nucleic acids involved in the processes to be studied are sufficiently stable p.m. ; (2) that the structural integrity of the tissue is maintained to such an extent that any damage occurring p.m. does not significantly affect the process under study or is sufficiently repaired during the course of the preparation or incubation procedures. Losses of transmitters or intermediary metabolites p.m. need not prevent the isolation of active preparations, so long as they can be resynthesised in vitro from added substrate: as examples, dopamine and many amino acids are synthesised in fresh brain synaptosomes from tyrosine and glucose respectively (De Belleroche et al., 1976; Bradford and Thomas, 1969). Note that there is no evidence for synaptosomal biosynthesis of neuroactive peptides #1 vitro; however, most peptides are thought to be stable p.m. (for review see Edwardson and McDermott, 1982). Eltzymes

A large number of studies have been carried out using p.m. brain from patients dying with various neurological and psychiatric disorders, as well as from normal patients. Frequently in these studies, comparisons have been made with 'model' animal experiments to assess the p.m. stability of brain enzymes. The latter experiments have involved either leaving the animal brain at 4'~C, ca. 20°C, or 37°C, for varying time periods; or by taking such tissue down the human brain cooling curve (Perry et al., 1977; Spokes and Koch, 1978). In addition, the stability of enzymes has been assessed in both human biopsy and autopsy samples by incubating such tissue at different temperatures and sampling over time, or by comparing enzyme activities in brains obtained after differing p.m. delay periods. Results from studies on general metabolic enzymes are summarised in Table 1, while the corresponding data for transmitter-related enzymes are given in Table 2. For most practical purposes, an enzyme may be considered to be stable if less than 3°~ of its activity is

lost per hour of p.m. storage, but unstable if more than 5°~ of its activity is lost per hour. By these definitions, more than 70°,4 of the original activity ot" a stable enzyme would be present in brain obtained at 10 h p.m. Using these criteria, all enzymes involved in central metabolism which have been studied so far are stable p.m. (GABA transaminase is discussed below), although hexokinase and glucose 6-phosphate dehydrogenase seem to show artifactually high concentrations under some storage conditions (Bird et ul.. 1977). Moreover, most enzymes which are localised predominantly in the brain and specifically related to neurotransmission are also stable p.m. However, there are serious discrepancies in the reported stabilities of tyrosine hydroxylase, monoamine oxidase, aromatic amino acid decarboxylase and glutamate decarboxylase. Adenylate cyclase is unstable p.m. (Witte and Matthaei, 1979), and GABA transaminase seems to be almost completely absent from rat brain after p.m. storage (Vogel et al., 1975) although its presence in p.m. human brain has been reported (Urquhart et al., 1975}. Receptors

Few studies of the p.m. stability of receptors have been undertaken. The majority of the receptors investigated appear to be remarkably stable p.m. (Table 3). Nucleic acids

The total content of nucleic acids of brain has been found to be constant in incubated (4 or 16'C) autopsy material (Naber and Dahnke, 1979; Mann et al., 1978), although a decrease in cytoplasmic RNA was reported (15°~ loss over 10h at 4c'C; Mann et al., 1978). In addition, biologically active mRNA and polysomes have been isolated from autopsy brain and used to transcribe human brain proteins (Gilbert et al., 1981 : Marrota et al., 1981). The mRNA and polysomes were obtained in reduced yield cf. rat brain, in agreement with the reduction in cytoplasmic RNA mentioned above. However, there was no evidence of a selective loss of higher molecular weight mRNA. Structure

Electron microscopy of p.m. monkey and human brain (Rees, 1976, 1977) has shown that several structural changes occur after p.m. storage. These include swelling of endoplasmic reticulum and dendritic and axonal processes, some damage to internal mitochondrial membranes and some loss and clumping of synaptic vesicles; synaptic structure is, on the other hand, generally well maintained (Rees, 1977). Provided care is taken when handling the p.m. material,

Metabolic and functional studies on post-mortem human brain remarkably good preservation of structure may be obtained up to at least 5h p.m. in human brain (Fig. 3). At the light microscope level, fluorescence histochemistry revealed that structure is well preserved for many hours p.m. in the rat (Oehmichen and Gencic, 1980). Morphological examination of mitochondrial and synaptosomal fractions from p.m. guinea-pig and human brain has shown that these components can be isolated in a similar manner to preparations derived from fresh brain (Swanson et al., 1973; Garey et al., 1974; Van Kempen and Vrensen, 1974; Hickey et al., 1976). Such fractions were of reduced purity cf. fresh-derived preparations~ contained some artifacts due to p.m. storage and showed p.m. degeneration similar to that seen in intact tissue (ibid.; cf. Rees, 19761. Thus, there was some disorganisation of the internal mitochondrial membrane, as well as clumping of synaptic vesicles (Hickey et al., 1976). Subcellular enzyme distribution studies on these fractions indicated that, in addition to relatively good preservation of gross morphology, the enzymes associated with particular fractions in fresh brain remained localised during p.m. storage. Thus, succinate dehydrogenase and cytochrome c oxidase were enriched in the mitochondrial fraction (Swanson et al., 1973; Garey et al.. 1974: Van Kempen and Vrensen, 1974; Hickey et al., 1976), whereas acetylcholinesterase, choline acetyl transferase and Na~/K + ATPase were associated with synaptosome profiles (ibid.). In addition, fl-glucosaminidase and fl-galactosidase remained associated with the lysosomal fraction in rat brain stored for 6h at 21~C p.m. (McKeown, 1979). There is thus considerable evidence that the majority of brain enzymes are stable p.m., and that these enzymes remain associated with particular structures whose integrity is well maintained during p.m. storage. This evidence, which indicates the retention of cellular and subcellular organisation during p.m. storage, supports the data on the isolation of preparations capable of integrated metabolic and functional performance which has been presented above. STORAGE OF TISSUE WITH RETENTION OF ACTIVITY As outlined above, preparations from human autopsy material have been demonstrated to take up and release transmitters in the same way as fresh preparations when incubated in vitro. However, since the average human brain weighs more than I kg whereas most experiements require less than 5 g of starting material, it would clearly be desirable if

257

methods for storing this material without drastic loss of activity could be found. The development of a method for freezing and storing brain tissue with retention of metabolic activity would aid in the use of human brain material for medical research in four main ways: (1) by allowing the more efficient use of brain material in such experiments; (2) by allowing transfer of specimens between different laboratories; (3! by making it possible to compare abnormal to matched control tissue from the same brain in experiments carried out on different days, thereby reducing experimental variation; (4) by allowing multiple studies on the same brain, for example when comparing the effects of different drugs. Frozen brain has been routinely used in the study of enzyme and receptor levels p.m. Most brain enzymes are thought to be stable at - 7 0 ' C , although there have been few studies on this point (see Puymirat et al., 1979). Early work indicated that it might be possible to isolate preparations which retained integrated activity after freezing. Thus, electrophysiological activity has been reported in cat brain after a complicated freezing and thawing procedure (Suda et al., 1966!, and metabolic activity has been observed in incubated tissue slices prepared from frozen brain (Tower and Young, 1973; Tower et al., 1976). In addition, studies on the subcellular distribution of a variety' of marker enzymes in frozen guinea-pig and hum;,,n brain revealed that neither the distribution of tota: protein, nor the localisation of any of the enzymes, was significantly altered cf. fresh-derived material (Stahl and Swanson, 1975). It thus seemed possible that active preparations might be isolable from frozen brain. We compared a variety of methods of freezing and thawing rat and human brain with the aim of producing viable synaptosomes (Hardy et al., 19831. The most effective method was one of slow freezing and fast thawing of the tissue (Hardy et al., 1983), similar to that routinely used for the preservation of tissue culture stocks (Coriell, 1979). This procedure yielded preparations with about 70°;i of the activity and purity of fresh preparations, as judged by electron microscopy, oxygen uptake, potassium content and the ability to release transmitter amino acids. A similar, though not identical, slow freezing/fast thawing procedure (Schwarcz, 19811 has been used in the study of glutamate uptake by p.m.-prepared human synaptosomes (Schwarcz and Whetsell, 1982). Similarly, meta-

258

J.A. HARDY and P. R. DODD

bolically and functionally active tissue prisms have been used after slow (stepwise) freezing in the presence of a c r y o p r o t e c t a n t followed by fast t h a w i n g ( H a a n a n d Bowen, 1981). Thus, consistent and g o o d retention of m e t a b o l i c and functional activity can be achieved by freezing a n d t h a w i n g brain tissue under c o n t r o l l e d conditions. PROBLEMS E N C O U N T E R E D WITH THE t:SE O F H U M A N BRAIN TISSUE FOR BIOCHEMICAL RESEARCH

p r e p a r a t i o n s from h u m a n brain. Moreover, there are no p r o b l e m s unique to flmctional experiments, i.e. which need not also be c o n s i d e r e d when any work with p.m. brain is to be u n d e r t a k e n . T h e following section provides a brief resume o f the factors which need to be taken into a c c o u n t in e x p e r i m e n t s using such tissue. A.qonal state of the patient

P.m. delay and tissue storage are not i n s u r m o u n t able p r o b l e m s for the isolation of metabolically active

S y n a p t o s o m e s isolated from patients dying of brain t r a u m a had no metabolic activity' despite short p.m. delays (Fig. 1) and therefore did not release transmitters in response to depolarising stimuli {unpub-

Table 1. P.m. stabilities of general metabolic enzymes Enzyme Hexokinase f!C 2.7.1.1

l-'iss ue Mouse Human biopsy Human autopsy Human autopsy

Glucose 6phosphate dehydrogenase EC 1.1.1.49

Phosphofructokinase EC2.7.1.11

Storage conditions 4 I1 at 4 h tit 4 h at 4 h at 10h at

26C +

% Stability

Reference

210

Bird et al., 1977

85

Bird el al., I977

7{'1

Bird et al., 1977

4C

26C + 4 C 4 C

10 h at 4 C

I00

Mann et al., 1978

Mouse

4 h at 2 6 C + 4 h at 4 C

161'1

Bird et al., 1977

Human biopsy Human autopsy Human autopsy

4 h at 2 6 C + 4 h at 4 C 10h at 4 C

80

Bird et al., 1977

9(I

Bird el al.. 1977

Mouse Cat Human biopsy Human autopsy Human antopsy Human autopsy

11) h at 4 C

111)

Mann et al.. 1978

4 11 at 4hat I1"1h at 4 h at 4 h at 10 h at

11)5

Bird et al., 1977

103 75

lwangoff et al.. 1980 Bird et al., 1977

26:'C +

4C 2@C 26 C + 4C 4C

90

Bird et al., 1977

10 h at 4 C

74

Mann.et al., 1978

Calculated activity at 10 h from different specimens

46

lwangoff et al,, 1981)

Pyruvatc kmase EC 2.7.1.40

Human autopsy

10 h at 4 C

118

Mann et al.. 1978

Lactate dehydrogenase EC 1.1.1.27

Rat

10h at 21 C

101

Fahn and Cote, 1976

Human autopsy

10 h at 4 C

150

Mann el al., 1978

Succmate dehydrogenase EC 1.3.99.1

Guinea-pig

3 h at 20'C +

76

Swanson et al., 1973

65

Swanson et a l . 1973

16 h at 4"C

Guinea-pig

19 h at 2OC

Metabolic and functional studies on post-mortem human brain

259

Table 1 c o n t i n u e d

Enzyme

Tissue

Storage conditions

% Stability

Re~rence

Glutamatc dehydrogenase EC 1.4.1.2

Rat

3 h at 2 0 C + 12 h at 4 C

8(I

Vogel e t a l . , 1969

Na+/K~

Rat

10 h at 21 C

95

Fahn and Cote, 1976

Mg e ~ ATPase EC 3.6.1.4

Rat

10h at 21'C

82

Fahn and Cote, 1976

2.3-Cyclic n ucleotide 3-phosphodiesterase EC 3.1,4.37

Human atttopsy

Calculated activity at 10 h from different specimens

105

Rand et al.. 1979

Protein kinase EC 2.7.1.37

Rat

1 h at 20"C + 15 h at 10'C

118

Schmidt et al., 1978

Acid proteinase{sl EC 3.4.2Yx

Ox

4h 16h 4h 16h

at at at at

20C + 4C 20'C + 4C

100

Roytta et al., 1980

120

Roytta et al., 1980

4h 16 h 4h 16 h

at at at at

2OC + 4C 20C + 4 C

87

Roytta et al., 1980

100

Roytta et al., 1980

100

Roytta et al., 1980

106

Annunziata and Federico. 1979

100

Roytta et al., 1980

88

Roytta et al., 1980

116

Roytta et al., 1980

Mg 2+

ATPase EC 3.6.1.3

Acid phosphatase EC 3, 1.3,3 /~-Glucuronidase EC 3.2.1.31

Human autopsy Ox Human autopsy Ox Rat

4 h at 20'C + 16 h at 4'C 12 h at 2 0 C

Human autopsy

4 h at 2 0 C + 16h at 4 C

Leucine aminopeptidase EC 3.4.1.1

Ox Human autopsy

4h 16h 4h 16h

fl-glucosaminidase EC 3.2.1.30

Rat Rat

6 h at 3 7 C 12 h at 2 0 C

98 83

McKeown, 1979 Annunziata and Federico, 1979

/:t-galactosidase EC 3.2.1.23

Rat Rat

6 h at 37'C 12 h at 20°C

107 73

McKeown, 1979 Annunziata and Federico, 1979

N-acetylglucosaminidase EC 3.2.1.51)

Rat

12 h at 20:C

94

Annunziata and Federico, 1979

~-Glucosidase EC 3.2,1.20

Rat

12hat20C

129

Annunziata and Federico, 1979

/#Glucosidase EC 3.2.1.21

Rat

12 h at 20°C

60

Annunziata and Federico, 1979

:~-Fucosidase EC 3.2.1.51

Rat

12 h at 2 0 C

88

Annunziata and Federico, 1979

~-Mannosidase EC 3.2.1.24

Rat

12h at 20'-'C

77

Annunziata and Federico, 1979

Arylsulphatase EC 3.1.6.1

Rat

12h at 20"C

Ill

Annunziata and Federico, 1979

at 2 0 C + at 4 C at 2 0 C + at4C

Thc values quoted are percentages of the activity in fresh tissue after storage of tissue under the stated conditions. They were given in, or calculated from, data found in the quoted references: see atso Oehmichen (1980).

260

J.A. HARDYand P. R. DODD

lished data). This illustrates that the agonal state of the patient influences the activity of in vitro preparations. The effect of agonal state on enzyme concentrations is well established: the enzymes glutamate decarboxylase, phosphofructokinase and aromatic amino acid decarboxylase are lower in patients dying after prolonged illness than in patients dying suddenly after good health (Bowen et al., 1976; Perry et al., 1977a, 1982). Although these effects of agonal state are a problem when comparing normal and diseased groups of patients, they can be overcome by careful matching. In experiments to determine the effects of neuroactive drugs on human preparations, tissue from patients dying after coma or long illness should probably not be used. Moreover, the fact that agonal state affected some, but not all, biochemical parameters measured p.m. (Perry et al., 1977a), indicates that results from in vitro experiments may sometimes reflect differences due to the state of the patient ante-mortem. Medication

Respiratory rates of synaptosomes prepared from patients dying after prolonged treatments with opiate analgesics were slightly lower than expected in comparison with 'no drug' patients (Fig. 1). This may be an example of an effect of preterminal medication, although many more experiments are needed to confirm this. Medication with opiates has been reported to decrease the serotonin concentration in autopsy brain (Bucht et al., 1981), while neuroleptics increased choline acetytransferase and dopamine receptor con-

centrations and decreased the dopamine fl-hydroxylase concentration (see Crow et al., 1980). For experiments studying the effects of neuroactive drugs on human brain, tissue from patients who have recently received neuroactive drugs should obviously be used with caution. However, in experimental series comparing normal with diseased brain, the use of such material is frequently unavoidable since many psychiatric disorders are treated with specific medication as a routine. Three approaches may be used to circumvent this problem: (i) separating any 'no drug' diseased patients when analysing results; (ii) using patients without the disease who have received similar medication as an extra control group (some patients with Huntingdon's chorea, for example, receive neuroleptics, as do most schizophrenics; Lloyd et al., 1980); (iii) using animal experiments to determine the effects of the drug regime on the parameter/sl of interest.

A g e o f patient at death

Choline acetyltransferase and tyrosine hydroxylase have been reported to decrease with age (McGeer and McGeer, 1976: Perry et al., 1977a). In experiments involving 'between group' comparisons, groups should be age- (and also sex-) matched as far as possible.

Table 2. P.m. stabilities of transmitter-related enzymes Enzyme Choline acetyltransferase EC 2.3.1.6

Tissue Mouse Rat Rat Rat Rat Human autopsy Human autopsy

Acetylcholine esterase EC 3.1.1.7

Rat Rat Guinea-pig

Storage conditions l(I h on human cooling curve 8 h at 20°C 10h at 21"C 10h at 20"C 10h at 37°C Calculated activity at 10 h from different specimens Calculated activity at 10 h from different species

o/ Stability 100

Reference Spokes and Koch, 1978

93 73 95 75 99

McGeer and McGeer, 1976 Fahn and Cote, 1976 Puymirat et al., 1979 Bowen et al., 1976 Aquilonius et al., 1975

88

Perry et al., 1977

8 h at 20°C

87

McGeer and McGeer, 1976

lOh at 210C 3 h at 20°C + 16 h at 4°C

84 80

Fahn and Cote, 1976 Swanson et al., 1973

Metabolic and functional studies on post-mortem h u m a n brain

261

Table 2 - - c o n t i n u e d

Enzyme Tyrosine hydroxylase EC 1.14.16.2

Tissue Rat Rat Rat Rat Rat Rabbit Rat

Storage conditions 8 h at 20°C

~o Stability 38

t0h 10 h 10 h 16h 10 h 2h 12 h 8h 10 h 10 h 16 h 10b 2h 12 h

at at at at at at at at at at at at at at

21°C 20°C 20C 20°C 20°C 20°C + 4°C 2ff'C 21°C 20°C 20'C 37°C 20"C + 4~C

64 78 25 133 78 85

at at at at

McGeer and McGeer, 1976 Fahn and Cote, 1976 Puymirat et al.. 1979 Black and Geen, 1975 Vogel et al., 1969 Grote et al., 1974 Roytta et al., 1980

DOPA decarboxylase (Aromatic amino acid carboxylase) EC4.1.1.28

Rat Rat Rat Rat Rat Rat

Dopamine /~-hydroxytase EC 1.14.2.1

Rat Rat Rat Rat

10 h 10h 10 h 16h

20:C 20°C 20~C 20C

100 85 85 125

Grote Black Black Vogel

Catechol-omethyltransferase EC2.1.1.6

Rabbit Rat

10 h at 20°C 16 h at 20°C

93 115

Grote et al., 1974 Vogel et al., 1969

Monoamine oxidase EC 1.4.3.4.

Rat Rabbit Guinea-pig

10h 10 h 3h 8h

87 100 150 67

Fahn and Cote, 1976 Grote et al., 1974 Swanson et al., 1973 Parkinson and Callaghan, 1978

Adenylate cyclase (DA & NA Stim.) EC 4.6.1.1

Rat

9 h at 22°C

30

Witte and Matthaei, 1979

Glutamine synthetase EC6.3.1.2

Rat

3 h at 20°C 12 h at 4°C

97

Witte and Matthaei, 1979

7-Glutamyl transpeptidase EC 2.3.2.2

Rat

10h at 37°C

100

Glutamic acid decarboxylase EC 4.1.1.5

Mouse

10h on h u m a n cooling curve 2 h at 20°C + 12 h at 4°C 3 h at 20~C + 12 h at 4°C 8 h at 20°C 1011 at 4°C 10 h at 20°C 10h at 21°C 10h at 37°C Calculated activity at 10 h from different specimens

Rat Rat Rat Rat Rat Rat Rat Human autopsy

GABA transaminase EC 2.6.1.19

Rat

at at at at

21cC 20"C 20°C + 4°C

3 h at 20'JC + 12 h at 4°C

Legend as for Table 1 ; see also Oehmichen (1980).

58 79 25 86 65 96

Reference

87

McGeer and McGeer, 1976 Fahn and Cote, 1976 Black and Geen, 1975 Vogel et al., 1969 Bowen et al., 1976 Roytta et al., 1980 et al., 1974 and Geen, 1975 and Geen, 1975 et aL, 1969

Bowen et al., 1976

Spokes and Koch, 1978

100

Roytta et al., 1980

92

Vogel et al., 1975

36 90 80 91 75 95

McGeer and McGeer, 1976 Crow et al., 1978 Puymirat et al., 1979 Fahn and Cote, 1976 Bowen et al., 1976 Perry et al., 1977

3 (but see text)

Vogel et al., 1975

262

J.A. HARDY and P. R. DODD Table 3. P.m. stabilities of transmitter receptors Receptor (ligand) M uscarinic (QNB)

GABA (GABA)

Serotonin {LSD)

Tissue

% Stability

Reference

Rat Rat

6 h at 27 C 3 h at 21 C + 30 h at 4 C

80+ I00

Enna et al., 1976a,b Owen et al., 1983

Rat Rat

6h at 2TC 3 h at 21 C + 30 h at 4 U

80+ 100

Enna et al., 1976a.b Owen et al., 1983

Rat

(Serotonin}

Storage conditions

6h at 2 7 C

80+

Rat Rat

6 h at 22 C 3 h at 21 C + 30 h at 4 C

Opiate (Dihydromorphine)

Rat

O h at 22 C

Dopamine (Spiperone)

Rat

3 h at 21 C + 30 h at 4 C

60 100

65 + 100

Enna et al.. 1976a,b Weiner et al., 1979 Owen et al., 1983

Kuhar et al.. 1973 Owen et al., 1983

Legend as Table 1 : receptor binding was assayed using the ligand indicated. QNB, quinuclidinyl benzilate: LSD. lysergic acid diethylamide.

Circadian and circammal

rhythms

Circadian fluctuations in the concentrations of choline acetyltransferase and glutamate decarboxylase in autopsy brain (Perry et al., 1977c) and circannual changes in concentrations of serotonin and noradrenaline (Carlsson et al., 1980) have been reported. A record of the date and time of death should therefore be kept and the data checked against such possibilities. Dissection procedures

Differences in dissection protocols are thought to have been responsible for inconsistences in reports concerning the role of the nucleus accumbens in schizophrenia (see Matthysse, 1980). Careful and reproducible dissection procedures are therefore important (see Niewenhuys et al., 1979). The above problems relate to all work on p.m. h u m a n brain and are not specific to the isolation of metabolically active preparations. All of them can be largely overcome by careful experimentation and good documentation of specimens. CONCLUSIONS AND FUTURE PROSPECTS This review has brought together the evidence pertaining to the use of p.m. h u m a n brain tissue for rune-

tional studies. Together with brain scanning techniques, this approach provides a major new direct method for studying h u m a n brain dynamics in health and disease. It is pertinent in this context to remember that many key enzymes are present to considerable excess in brain. For instance, the extractable activity of phosphofructokinase, the main rate-limiting enzyme of the glycolytic chain, is some 20-fold greater than the resting glucose flux in vivo (Scrutton and Utter, 1968: glucose flux may increase 2 3 fold during tetanic stimulation): while pyruvate kinase, which is also considered to be a control-point enzyme in glycolysis, is present at 300-fold more than the glucose flux (Scrutton and Utter, 1968). Similarly, choline acetyltransferase activity widely used as a marker for cholinergic pathways is some 20-fold greater than the measured rate of acetylcholine synthesis [Racagni et al., 1976: Cooper et al., 19781. Two comments may be made about these observations: firstly, if such enzyme activities are accessible to their appropriate substrate within the cell, there are more than adequate levels of the "stable' enzymes (as defined above) to support the metabolic rates observed in p.m. preparations: secondly, even if quite a large difference is found in the extractable activity of a given enzyme when two groups of patients are compared, any inference that this would cause a large difference in corn-

Metabolic and functional studies on post-mortem human brain

263

Fig. 3. Electron micrograph of p.m. human temporal cortex. P.m. delay 5 h" cause of death: ischaemic heart disease; × 11.500. a, axon: d, dendrite: mr, mitochondrion: my, myelin: s, synaptic contact. Synaptosomes prepared from this brain respired, showed depolarisation-induced release of transmitter amino acids, accumulated potassium against a concentration gradient and possessed a sodium-dependent high-affinity GABA uptake mechanism.

parative metabolic flux should be treated with caution. While it c a n n o t be denied that there are drawbacks inherent in all i1~ vitro experiments, measuring metabolic flux is usually straightforward in incubated preparations, whereas it is often difficult or impossible il~ vivo, especially in man. A n u m b e r of metabolic and functional parameters {including transmitter uptake, metabolism and release) have already been shown to be well retained in p.m. h u m a n preparations and these compare quite favourably with the corresponding data from laboratory animals. Of course, the use of p.m. material presents many difficulties, but none which is not already present when static studies (such as measurement of an enzyme concentration) are carried out. The fact that h u m a n material is not obtained from heavily inbred, genetically defined individuals adds further complications, but these may be offset by the opportunity for studying disease states for which ant-

mal models are either unavailable or inadequate. For example, an hypothesis of the aetiology of a neurological or psychiatric disorder which involed a specific derangement of neurotransmission, perhaps in a discrete brain area, might now be subjected to direct tests. Finally, it can be pointed out that there are often quite large inter-species differences in the potencies of drugs. The ability to study the actions of neuroactive drugs directly on h u m a n tissue may prove to be an i m p o r t a n t aspect of dynamic experiments with p.m. brain preparations. Ackllowledgements The authors would like to thank Mr A. E. Oakley of the MRC Neuroendocrinology Unit for permission to reproduce Figure 2. Mss A. M. Kidd and S. C. Cheetham were involved with several of the experiments described herein. Drs J. A. Edwardson, C. R. Snell, E. K Perry and J. M. Candy contributed critical discussion of the manuscript. We are grateful to Drs D. M. Bowen, C A. Marotta, F. Owen and P. Riederer for providing us with

264

J.A. HARDY and P. R. DODD

preprints and reprints of their work. The Medical Research Council provided financial support.

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