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Regional distribution of muscarinic acetylcholine receptor in normal and Alzheimer's-type dementia brains
Brain Research, 138 (1978) 385-392 © Elsevier/North-HollandBiomedicalPress
385
Regional distribution of muscarinic acetylcholine receptor in normal and Alzheimer's-type dementia brains
PETER DAVIES and ANDREW H. VERTH MRC Brain Metabolism Unit, 1 George Square, Edinburgh EH8 9JZ (Great Britain)
(Accepted July 29th, 1977)
Neurochemical studies on human brains obtained at autopsy have over the past two decades contributed much to our understanding of the role of distinct neurotransmitter systems in human disease. Most notably, the studies of Hornykiewicz and coworkers 19 on Parkinson's disease have led to the concept that the clinical manifestations of this condition are the result of a degeneration of the nigrostriatal dopamine system, with a consequent deficiency of dopamine in the basal ganglia12. Two other conditions which have been studied neurochemically are Huntington's chorea, in which at least 3 putative neurotransmitter systems (7-aminobutyric acid1,2,25, acetycholine2,22 and substance p14) appear to show some degree of deficit, and Alzheimer'stype dementia, in which cortical cholinergic neurons appear to be very badly affected4, 6,24,26. The bulk of the neurochemical work on these disorders had concentrated on the measurement of neurotransmitters, their metabolites and/or the enzymes involved in their metabolism. More recently, techniques have been introduced which appear to allow quantitative measurement of the receptors for neurotransmitters in both animal and human braina,7,17,27,2~. These methods depend on measurement of that fraction of the binding of radioactively labelled transmitters, agonists or antagonists to particulate material in homogenates of brain tissue that is saturable and pharmacologically specific3. Such techniques have to date been applied only to Huntington's chorea, and in these studies some loss of the muscarinic acetylcholine receptor and of the 5hydroxytryptamine (5HT) receptor from the caudate nucleus/putamen has been observed s,l°,la. We have applied a technique for quantitation of the muscarinic receptor to the study of Alzheimer's-type dementia because of the evidence for extensive loss of enzymes of the cholinergic system, and report here that receptor concentrations in brains from 5 such cases do not appear to differ significantly in any area from those found in normal brains. A total of 18 brains were collected at routine autopsies performed between 4 and 36 h following death. All cadavers were placed at 4 °C within 4 h of death, and remained at this temperature until the post mortem examination was performed. The brains were sectioned down the midline immediately following removal, the right half being placed in formalin and dispatched for neuropathological examination, whilst
59 49 70 53 46 71 69 47 68 56 79
3 4 5 6 7 8 9 10 11 12 13
M F M F M F M M M F M
M M
Sex
Alzheimer's-type dementia 14 75 M 15 68 F 16 61 F 17 60 F 18 53 M
74 70
Age
Normal 1 2
Case no. and diagnosis
Bronchopneumonia Pulmonary embolism Bronchopneumonia Bronchopneumonia Bronchopneumonia
Pulmonary embolism Renal failure Bronchopneumonia Pancreatic carcinoma Aspiration pneumonitis Aspiration pneumonitis Bronchopneumonia Myocardial infarction Peritonitis Bronchopneumonia Myocardial infarction
Pulmonary embolism Bronchopneumonia
Cause O/death
21 23 33 12 30
23 34 32 34 30 26 28 34 4 24 21
24 36
nitrazepam, aminophylline dihydrocodeine, diazepam diazepam, nitrazepam nitrazepam, thioridizine nitrazepam, diazepam
nil morphine, trimeprazine diazepam, dihydrocodeine diazepam chlorpromazine, nitrazepam morphine, nitrazepam nil nil diacetylmorphine nil morphine
pentobarbitone morphine, diazepam
Delay between Medication death and (psycho-active drugs only) autopsy (h)
Numerous neurofibrillary tangles and senile plaques in sections of frontal, temporal, parietal and occipital cortex. No evidence for significant cerebrovascular disease
Normal Small old infarction in right caudate and occipital cortex Normal Normal Small old infarction in right putamen Normal Normal Normal Normal Normal Normal Normal Normal
Neuropathology
Listed are selected data from the clinical notes and pathology/neuropathology reports. The delay period between death and autopsy includes the delay before refrigeration of the cadaver (always less than 4 h) and the time taken for dissection (about 2 h).
Clinical and pathological data on cases from which brains were obtained
TABLE I
387 the left half was dissected for neurochemical studies. Dissected samples of tissue were chopped finely, mixed and stored in liquid nitrogen for up to one year prior to assay of the muscarinic receptor. Selected clinical and pathological data on the cases studied is presented in Table I. Briefly, 'normals' were defined as cases where there had been no evidence for neurological or psychiatric disorder prior to death, and no extensive pathological abnormality. Alzheimer's-type dementia describes cases with a progressive profound dementia with no focal neurological signs and pathological evidence of numerous senile plaques and neurofibrillary tangles in sections of the right frontal, temporal and parietal cortexS,11. Granulovacuolar degeneration was present in the right hippocampal formation in all 5 cases reported here. In accordance with recent developments, we do not draw any distinction between patients with dementia on the basis of agelL Samples of brain tissue (25-50 mg wet weight) were homogenised in KrebsHenseleit buffer, using about 1 ml buffer per 5 mg tissue. The protein concentration of the homogenates was determined by the method of Lowry et al. 2°. Triplicate 1 ml aliquots of the homogenates were incubated at 30 °C for 20 min in the presence of 10 nM [SH]quinucleodinyl benzilate ([aH]QNB) (13 Ci/mmole, batch 3 from the Radiochemical Centre, Amersham, U. K.) and a further 3 aliquots were incubated in the presence of 10 nM [aH]QNB and 10 #M atropine sulphate. Following incubation, the homogenates were centrifuged at 12,000 g for 2 rain, and the pellets were rapidly and superficially washed with Krebs-Henseleit buffer. The residues were dissolved in Protosol (New England Nuclear, Winchester, Hampshire, U. K.) and transferred to scintillation vials with 2 ml of ethanol. Toluene scintillant (10 ml, 0.425 ~ PPO and 0.0112 ~ POPOP (w/v) in toluene) was added, and the tritium content was assayed by liquid scintillation counting. The difference in [3H]QNB binding in the presence and absence of atropine was designated specific binding, and was used to calculate muscarinic receptor concentrations. This method is essentially that described in outline by Hulme et al. ~s. The same procedure was used for determination of affinity constants (see below), except that the [aH]QNB concentration was varied between 0.1 nM and 10 nM, and incubations were performed in duplicate rather than triplicate. Means, standard errors and the Student's t-test for the significance of differences were calculated as though the data was normally distributed, although this assumption was not tested. Brainsfrom normals. Significant levels of specific binding were detected in all the areas of brain examined (Table II). In agreement with published data, the highest muscarinic receptor concentration was found in the caudate nucleus7 although concentrations in the nucleus accumbens were almost as high. The number of samples of Accumbens assayed is rather small (3) as we have found this nucleus difficult to dissect from fresh brain. Receptor concentlations in the areas of cerebral cortex examined were fairly uniformly 60 ~ of the caudate levels. Midbrain, pons, substantia nigra, thalamus, hypothalamus and olfactory area all had receptor concentrations about 30 9/0 as high as in the caudate, whilst the cerebellar cortex had the lowest level encountered, about 15 ~ of caudate values. The pattern of distribution of the receptor in the normal brains agrees well with published data on human 7 and monkey27 brain.
388 TABLE II Distribution o f muscarinic receptor in normal and Alzheimer's-type dementia brains
Figures presented are mean receptor concentrations in picomoles per 100 mg protein, :k standard error, with the number of samples assayed in brackets. The limit of sensitivity of the method w~qs found to be 2-3 pmoles per 100 mg protein. Area
Normal
Alzheimer
Caudate Substantia nigra Pons Midbrain Thalamus Hypothalamus Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Precentral gyrus Postcentral gyrus Parietal cortex Calcarine cortex Hippocampal gyrus Midtemporal gyrus Cerebellar cortex Nucleus accumbens Olfactory area
74.0 -4- 3.7 (11) 76.2 -4- 6.5 (5) 20.6 -4- 5.1 (7) 13.9 -4- 1.7 (4) 20.0 ± 5.2 (2) 17.2 ± 2.1 (4) 16.0 -4- 3.7 (9) 19.1 -4- 3.6 (5) 21.8 -4- 2.4 (7) 27.0 ± 2.2 (5) 23.0 -4- 1.3 (2) 29.4 -4- 9.0 (3) 42.3 -4- 4.0 (5) 23.l ± 6.1 (3) 52.2 -4- 1.8 (11) 46.8 ± 5.4 (5) 51.7 -4- 5.2 (11) 52.4 -4- 7.5 (5) 42.7 -4- 1.5 (10) 44.4 -4- 5.0 (5) 43.3 -4- 5.1 (10) 48.0 4_ 8.1 (5) 36.8 -4- 2.7 (9) 48.3 -4- 7.9 (5) 44.8 -4- 3.1 (11) 54.4 -4- 2.9 (5) 46.3 -4- 2.7 (9) 44.0 -4- 5.7 (4) 45.1 -4- 2.6 (10) 40.2 ± 8.0 (4) 45.9 3z 3.3 (11) 51.1 :k 6.3 (5) 10.3 -4- 3.1 (7) 7.3 -4- 2.6 (4) 71.3 -4- 1.0 (3) 45.8 ± 3.7 (2) 27.7 ~ 3.1 (3) 44.5 -4- 10.9 (4)
R e c e p t o r c o n c e n t r a t i o n s did n o t seem to be affected by the m o d e o f death. F o r example, when the n o r m a l s were divided into two groups, those with a terminal lung c o n d i t i o n (n ~ 8) did n o t differ significantly in r e c e p t o r c o n c e n t r a t i o n in a n y area from the r e m a i n d e r (n -~ 5). In an a t t e m p t to evaluate the significance o f d r u g therapies given to n o r m a l individuals, we have divided up the cases into g r o u p s based on the use o f a p a r t i c u l a r d r u g type. M e a n r e c e p t o r c o n c e n t r a t i o n s in all areas were c o m p a r e d with c o n c e n t r a t i o n s in c o r r e s p o n d i n g areas from cases where the d r u g - t y p e in question h a d n o t been used. T h u s those given opiates (morphine, d i a c e t y l m o r p h i n e o r d i h y d r o c o d e i n e , n = 6) did n o t differ significantly f r o m those n o t so t r e a t e d (n = 7). Similarly, those treated with m i n o r tranquillizers (diazepam, nitrazepam, n - - 5) d i d n o t differ from the r e m a i n d e r (n ---- 8), n o r did the d r u g - t r e a t e d cases (n ~ 9) differ significantly from those w h o h a d died drug-free (n ~ 4). This r a t h e r crude analysis has been necessitated b y the considerable differences t h a t exist between i n d i v i d u a l cases in terms o f drugs used, dosage given a n d d u r a t i o n a n d timing o f a d m i n i s t r a t i o n in relation to time o f death. M o r e detailed a t t e m p t s to analyse the effects o f drugs could better be u n d e r t a k e n if brain d r u g concentrations were known. O u r crude analysis seems to indicate t h a t terminal therapeutic measures d o n o t greatly influence b r a i n muscarinic receptor c o n c e n t r a t i o n s in neurologically a n d psychiatrically n o r m a l individuals. E n n a et al. 7 have recently p o i n t e d o u t that studies to date on the muscarinic
389 0.6'SZPARIETAL CORTEX AFFINITY
CONSTANT
= 3 × i0 9M-1
~i 0'5a.
E
n,,
8
~
ffl w
•
NORMAL
"
[ POST-CENTRALOYRUS
A F F I N I T Y CONSTANT = 4 x 10 9M-1
_8 0"3-
O_ a Z
rn 0"2Z
?.-r0.1.(3
NORMAL ~SUBSTANTIA NIGRA A F F I N I T Y CONSTANT = 2 . 5 x 10 9M - 1 I
I
I
I
I
2
/~
6
8
10
3 FREE H-QNB CONCENTRATION (nM)
Fig. 1. The points plotted represent means of duplicate determinations of specific binding of pH]QNB at the indicated [aH]QNB concentrations. Values for free [3H]QNB were calculated by subtracting mean values for bound from the total added. receptor in human brains have not shown whether the observed difference in specific antagonist binding between two areas is due to the presence of different numbers of receptors, or to the presence of receptors which differ in affinity for the ligand used. We have examined this question by measuring the specific binding of [aH]QNB to particulate materials in a sample of normal brain postcentral gyrus and of substantia nigra, varying the [aH]QNB concentration from 0.1 to 10 n M (Fig. 1). We have used this data to estimate the affinity constant of [aH]QNB for the receptors in the two tissues (Fig. 1). Within the limits of accuracy of this methodology, the values for the affinity constants for the two areas of brain agree well enough to argue that the observed differences between the areas in specific [SH]QNB binding are due to the presence of different numbers of identical receptors. Brains from cases of Alzheimer's-type dementia. In all the areas of brain studied, the mean receptor concentrations appeared to be similar to those obtained for corresponding areas of normal brain (Table II). The receptor concentrations in the substantia nigra, amygdala and nucleus accumbens did appear to be lower than in corresponding areas of normal brain, but in no case did the difference achieve statistical significance. Similarly, the apparent elevation of receptor concentrations in the olfactory area was not significant. The affinity constant of [aH]QNB for the receptor in a
390 sample of parietal cortex from one of these cases was determined as described above (Fig. 1). The affinity constant obtained is almost identical to that obtained by studies on normal brain cortex. We were unable to determine whether the receptor concentrations in brains from these cases might be affected by drug treatments employed, because all 5 cases had received minor tranquiUisers for long periods (over 6 months) prior to death. There does not seem to be any compelling reason why techniques for the measurement of the concentration of neurotransmitter receptors should not be applied to human brains obtained at routine autopsy. Data from animal experiments indicates that the receptors remain intact for several hours after death 7,s and we have been unable to find any correlation between the concentration of the muscarinic receptor in normal brain and the time elapsing between death and autopsy. Further, there is no evidence for age-related changes in receptor concentration amongst our normal cases (between the age of 46 and 79), neither were any sex-related differences obvious. The principal prolbem with studies such as this one lies in the interpretation of the results obtained. While it does seem reasonable to assume that we are measuring the concentrations of the physiologically relevant muscarinic acetylcholine receptor (see above), we do not yet have adequante information about the precise cellular location of these receptors to make clear statements concerning our results. Muscarinic receptors seem to be found in greatest numbers where cholinergic terminals are most dense1% but we know veIy little about the nature of the cells that carry these receptors. The use of ligands labelled to high specific radioactivity in in vivo experiments, and of autoradiography to localise the bound ligand in brain slices may be very useful in this respect Is. There is an additional complication which must be considered in attempts to interpret our results on Alzheimer's-type dementia brains. In this condition, there is gross neuronal degeneration, especially in cerebral and limbic cortex a,ll and an apparently specific loss of enzymes of the cholinergic system 4,6,24,26. The lack of any apparent loss of muscarinic receptors can be interpreted in basically two ways. If the assumption is made that cholinergic neurons are selectively lost in this condition, the lack of apparent changes in the number of muscarinic receptors could be seen as indicating that cholinergic cells do not carry a significant proportion of these receptors. Thus it could be argued that the normal levels of these receptors is a further indication of a selective degeneration of one neuronal subpopulation, that is, a degeneration of cholinergic, but not cholinoceptive neurons. However, it is well known that degeneration of peripheral cholinergic nerves is followed by increases in the number of actylcholine receptors in the cholinoceptive tissue (although the majority of the well-studied systems of this type have nicotinic acetylcholine receptorsg,2t,~3). It may well be that in Atzheimer's-type dementia some loss of muscarinic receptors occurs because of loss of cholinergic (and perhaps other) neurons, but that surviving neurons attempt to compensate for the deficiency of acetylcholine by producing increased numbers of receptors. This suggestion has been put forward in one of two reports in which muscarinic receptor concentrations in parietal cortex 24 and
391 frontal cortex 26 from Alzheimer's-type dementia cases were reported to be normal. W h a t e v e r the reason for the apparently n o r m a l levels of muscarinic receptors in this condition, perhaps the most significant p o i n t which can be made is that this c o n d i t i o n m a y be a m e n a b l e to t r e a t m e n t with centrally active muscarinic agonists. O u r results together with those showing an apparently specific loss of enzymes of the cholinergic n e u r o n should encourage efforts in this direction.
This work would not have been possible but for the most generous co-operation of Dr. A. J. F. Maloney and Dr. A. Gordon, who performed the post mortem examinations and neuropathology. Drs. Boyd, Gray, Banyard and Gallimore provided access to clinical information concerning the cases of dementia, and Dr. G. W. Ashcroft and Dr. J. McQueen gave continual support and encouragement. Dr. E. C. Hulme and Dr. N. J. Birdsall provided practical advice regarding the receptor assay. To these colleagues and friends, we offer our most sincere thanks.
1 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, 1 (1973) 1090-1092. 2 Bird, E. D. and Iversen, L. L., Huntington's Chorea: post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia, Brain, 97 (1974) 457-472. 3 Birdsall, N. J. M. and Hulme, E. C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 4 Bowen, D. M., Smith, C. B., White, P. and Davison, A. N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-496. 5 CorseUis, J. A. N., Ageing and the dementias. In W. Blackwood and J. A. N. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1976, pp. 796-848. 6 Davies, P. and Maloney, A. J. F., Selective loss of central cholinergic neurons in Alzheimer's Disease, Lancet, II (1976) 1403. 7 Enna, S. J., Bennett, J. P., Bylund, D. B., Crease, I., Burt, D. R., Charnes, M. E., Yamamura, H. I., Simantov, R. and Snyder, S. H., Neurotransmitter receptor binding: regional distribution in human brain, J. Neurochem., 28 (1977) 233-236. 8 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 in neurotransmitter receptors in the brain, New Eng. J. Med., 294 (1976) 1305-1309. 9 Guth, L., 'Trophic' influences of nerve on muscle, Physiol. Rev., 48 (1968) 645-687. 10 Hiley, C. R. and Bird, E. D., Decreased muscarinic receptor concentration in post-mortem brain in Huntington's Chorea, Brain Research, 80 (1974) 355-358. 11 Hooper, M. W. and Vogel, F. S., The limbic system in Alzheimer's Disease: a neuropathological investigation, Amer. J. Pathol., 85 (1976) 1-20. 12 Hornykiewicz, O., Dopamine in basal ganglia, Brit. reed. Bull., 29 (1973) 172-178. 13 Hulme, E. C., Burgen, A. S. V. and Birdsall, N. J. M., Interactions of agonists and antagonists with the muscarinic receptor. In Proc. 1NSERM Colloquium on the Physiology and Pharmacology oJSmooth Muscle, INSERM, Vol. 50, 1976, pp. 49-69. 14 Kanzawa, I., Bird, E. D., O'Connell, R. and Powell, D., Evidence for a decrease in substance P content of substantia nigra in Huntington's Chorea, Brain Research, 120 (1977) 387-391. 15 Katzman, R., The prevalence and malignancy of Alzhei mer Disease: a major killer, Arch. Neurol., 33 (1976) 217-218. 16 Kuhar, M. J., The anatomy of cholinergic neurons. In A. M. Goldberg and I. Hanin (Eds.), Biology of Cholinergic Function, Raven Press, New York, 1976, pp. 3-28. 17 Kuhar, M. J., Pert, C. B. and Snyder, S. H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 447-450. 18 Kuhar, M. J. and Yamamura, H. I., Localization of cholinergic muscarinic receptors in rat brain by light microscopic autoradiography, Brain Research, 110 (1976) 229-243.
392 19 Lloyd, K., Davidson, L. and Hornykiewicz, O., The neurochemistry of Parkinson's Disease, J. PharmacoL exp. Ther., 195 (1975)453--464. 20 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with IFolin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 21 McConneU, M. G. and Simpson, L. L., The role of acetylcholine receptors and acetylcholincsterase activity in the development of denervation supersensitivity, J. Pharmacol. exp. Ther., 198 (1976) 507-517. 22 McGeer, P. L. and McGeer, E. G., Epzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's Disease and Huntington's Chorea, J. Neurochem., 26 (1976) 65-76. 23 Miledi, R. and Potter, L. T., Acetylcholine receptors in muscle fibres, Nature (Lond.), 233 (1971) 599-603. 24 Perry, E. K., Perry, R. H., Blessed, G. and Tomlinson, B. E., Necropsy evidence of central cholinergic deficits in senile demantia, Lancet, 1 (1977) 189. 25 Perry, T. L., Hansen, S., Lesk, D. and Klaster, M., Amino acids in plasma, cerebrospinal fluid and brain of patients with Huntington's Chorea. In Huntington's Chorea 1872-1972, A. Barbeau, T. N. Chase and G. W. Paulson (Eds.), Raven Press, New York, 1973, pp. 609-618. 26 White, P., Goodhardt, M. J., Keet, J. P., Hiley, C. R., Carasco, L. H., Williams, I. E. I. and Bowen, D. M., Neocortical cholinergic neurons in elderly people, Lancet, 1 (1977) 668-671. 27 Yamamura, H. I., Kuhar, M. J., Greenberg, D. and Snyder, S. H., Muscarinic cholinergic receptor binding: regional distribution in monkey brain, Brain Research, 66 (1974) 541-546. 28 Yamamura, H. I. and Snyder,. Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729.
385
Regional distribution of muscarinic acetylcholine receptor in normal and Alzheimer's-type dementia brains
PETER DAVIES and ANDREW H. VERTH MRC Brain Metabolism Unit, 1 George Square, Edinburgh EH8 9JZ (Great Britain)
(Accepted July 29th, 1977)
Neurochemical studies on human brains obtained at autopsy have over the past two decades contributed much to our understanding of the role of distinct neurotransmitter systems in human disease. Most notably, the studies of Hornykiewicz and coworkers 19 on Parkinson's disease have led to the concept that the clinical manifestations of this condition are the result of a degeneration of the nigrostriatal dopamine system, with a consequent deficiency of dopamine in the basal ganglia12. Two other conditions which have been studied neurochemically are Huntington's chorea, in which at least 3 putative neurotransmitter systems (7-aminobutyric acid1,2,25, acetycholine2,22 and substance p14) appear to show some degree of deficit, and Alzheimer'stype dementia, in which cortical cholinergic neurons appear to be very badly affected4, 6,24,26. The bulk of the neurochemical work on these disorders had concentrated on the measurement of neurotransmitters, their metabolites and/or the enzymes involved in their metabolism. More recently, techniques have been introduced which appear to allow quantitative measurement of the receptors for neurotransmitters in both animal and human braina,7,17,27,2~. These methods depend on measurement of that fraction of the binding of radioactively labelled transmitters, agonists or antagonists to particulate material in homogenates of brain tissue that is saturable and pharmacologically specific3. Such techniques have to date been applied only to Huntington's chorea, and in these studies some loss of the muscarinic acetylcholine receptor and of the 5hydroxytryptamine (5HT) receptor from the caudate nucleus/putamen has been observed s,l°,la. We have applied a technique for quantitation of the muscarinic receptor to the study of Alzheimer's-type dementia because of the evidence for extensive loss of enzymes of the cholinergic system, and report here that receptor concentrations in brains from 5 such cases do not appear to differ significantly in any area from those found in normal brains. A total of 18 brains were collected at routine autopsies performed between 4 and 36 h following death. All cadavers were placed at 4 °C within 4 h of death, and remained at this temperature until the post mortem examination was performed. The brains were sectioned down the midline immediately following removal, the right half being placed in formalin and dispatched for neuropathological examination, whilst
59 49 70 53 46 71 69 47 68 56 79
3 4 5 6 7 8 9 10 11 12 13
M F M F M F M M M F M
M M
Sex
Alzheimer's-type dementia 14 75 M 15 68 F 16 61 F 17 60 F 18 53 M
74 70
Age
Normal 1 2
Case no. and diagnosis
Bronchopneumonia Pulmonary embolism Bronchopneumonia Bronchopneumonia Bronchopneumonia
Pulmonary embolism Renal failure Bronchopneumonia Pancreatic carcinoma Aspiration pneumonitis Aspiration pneumonitis Bronchopneumonia Myocardial infarction Peritonitis Bronchopneumonia Myocardial infarction
Pulmonary embolism Bronchopneumonia
Cause O/death
21 23 33 12 30
23 34 32 34 30 26 28 34 4 24 21
24 36
nitrazepam, aminophylline dihydrocodeine, diazepam diazepam, nitrazepam nitrazepam, thioridizine nitrazepam, diazepam
nil morphine, trimeprazine diazepam, dihydrocodeine diazepam chlorpromazine, nitrazepam morphine, nitrazepam nil nil diacetylmorphine nil morphine
pentobarbitone morphine, diazepam
Delay between Medication death and (psycho-active drugs only) autopsy (h)
Numerous neurofibrillary tangles and senile plaques in sections of frontal, temporal, parietal and occipital cortex. No evidence for significant cerebrovascular disease
Normal Small old infarction in right caudate and occipital cortex Normal Normal Small old infarction in right putamen Normal Normal Normal Normal Normal Normal Normal Normal
Neuropathology
Listed are selected data from the clinical notes and pathology/neuropathology reports. The delay period between death and autopsy includes the delay before refrigeration of the cadaver (always less than 4 h) and the time taken for dissection (about 2 h).
Clinical and pathological data on cases from which brains were obtained
TABLE I
387 the left half was dissected for neurochemical studies. Dissected samples of tissue were chopped finely, mixed and stored in liquid nitrogen for up to one year prior to assay of the muscarinic receptor. Selected clinical and pathological data on the cases studied is presented in Table I. Briefly, 'normals' were defined as cases where there had been no evidence for neurological or psychiatric disorder prior to death, and no extensive pathological abnormality. Alzheimer's-type dementia describes cases with a progressive profound dementia with no focal neurological signs and pathological evidence of numerous senile plaques and neurofibrillary tangles in sections of the right frontal, temporal and parietal cortexS,11. Granulovacuolar degeneration was present in the right hippocampal formation in all 5 cases reported here. In accordance with recent developments, we do not draw any distinction between patients with dementia on the basis of agelL Samples of brain tissue (25-50 mg wet weight) were homogenised in KrebsHenseleit buffer, using about 1 ml buffer per 5 mg tissue. The protein concentration of the homogenates was determined by the method of Lowry et al. 2°. Triplicate 1 ml aliquots of the homogenates were incubated at 30 °C for 20 min in the presence of 10 nM [SH]quinucleodinyl benzilate ([aH]QNB) (13 Ci/mmole, batch 3 from the Radiochemical Centre, Amersham, U. K.) and a further 3 aliquots were incubated in the presence of 10 nM [aH]QNB and 10 #M atropine sulphate. Following incubation, the homogenates were centrifuged at 12,000 g for 2 rain, and the pellets were rapidly and superficially washed with Krebs-Henseleit buffer. The residues were dissolved in Protosol (New England Nuclear, Winchester, Hampshire, U. K.) and transferred to scintillation vials with 2 ml of ethanol. Toluene scintillant (10 ml, 0.425 ~ PPO and 0.0112 ~ POPOP (w/v) in toluene) was added, and the tritium content was assayed by liquid scintillation counting. The difference in [3H]QNB binding in the presence and absence of atropine was designated specific binding, and was used to calculate muscarinic receptor concentrations. This method is essentially that described in outline by Hulme et al. ~s. The same procedure was used for determination of affinity constants (see below), except that the [aH]QNB concentration was varied between 0.1 nM and 10 nM, and incubations were performed in duplicate rather than triplicate. Means, standard errors and the Student's t-test for the significance of differences were calculated as though the data was normally distributed, although this assumption was not tested. Brainsfrom normals. Significant levels of specific binding were detected in all the areas of brain examined (Table II). In agreement with published data, the highest muscarinic receptor concentration was found in the caudate nucleus7 although concentrations in the nucleus accumbens were almost as high. The number of samples of Accumbens assayed is rather small (3) as we have found this nucleus difficult to dissect from fresh brain. Receptor concentlations in the areas of cerebral cortex examined were fairly uniformly 60 ~ of the caudate levels. Midbrain, pons, substantia nigra, thalamus, hypothalamus and olfactory area all had receptor concentrations about 30 9/0 as high as in the caudate, whilst the cerebellar cortex had the lowest level encountered, about 15 ~ of caudate values. The pattern of distribution of the receptor in the normal brains agrees well with published data on human 7 and monkey27 brain.
388 TABLE II Distribution o f muscarinic receptor in normal and Alzheimer's-type dementia brains
Figures presented are mean receptor concentrations in picomoles per 100 mg protein, :k standard error, with the number of samples assayed in brackets. The limit of sensitivity of the method w~qs found to be 2-3 pmoles per 100 mg protein. Area
Normal
Alzheimer
Caudate Substantia nigra Pons Midbrain Thalamus Hypothalamus Amygdala Orbital frontal cortex Convexity frontal cortex Cingulate gyrus Precentral gyrus Postcentral gyrus Parietal cortex Calcarine cortex Hippocampal gyrus Midtemporal gyrus Cerebellar cortex Nucleus accumbens Olfactory area
74.0 -4- 3.7 (11) 76.2 -4- 6.5 (5) 20.6 -4- 5.1 (7) 13.9 -4- 1.7 (4) 20.0 ± 5.2 (2) 17.2 ± 2.1 (4) 16.0 -4- 3.7 (9) 19.1 -4- 3.6 (5) 21.8 -4- 2.4 (7) 27.0 ± 2.2 (5) 23.0 -4- 1.3 (2) 29.4 -4- 9.0 (3) 42.3 -4- 4.0 (5) 23.l ± 6.1 (3) 52.2 -4- 1.8 (11) 46.8 ± 5.4 (5) 51.7 -4- 5.2 (11) 52.4 -4- 7.5 (5) 42.7 -4- 1.5 (10) 44.4 -4- 5.0 (5) 43.3 -4- 5.1 (10) 48.0 4_ 8.1 (5) 36.8 -4- 2.7 (9) 48.3 -4- 7.9 (5) 44.8 -4- 3.1 (11) 54.4 -4- 2.9 (5) 46.3 -4- 2.7 (9) 44.0 -4- 5.7 (4) 45.1 -4- 2.6 (10) 40.2 ± 8.0 (4) 45.9 3z 3.3 (11) 51.1 :k 6.3 (5) 10.3 -4- 3.1 (7) 7.3 -4- 2.6 (4) 71.3 -4- 1.0 (3) 45.8 ± 3.7 (2) 27.7 ~ 3.1 (3) 44.5 -4- 10.9 (4)
R e c e p t o r c o n c e n t r a t i o n s did n o t seem to be affected by the m o d e o f death. F o r example, when the n o r m a l s were divided into two groups, those with a terminal lung c o n d i t i o n (n ~ 8) did n o t differ significantly in r e c e p t o r c o n c e n t r a t i o n in a n y area from the r e m a i n d e r (n -~ 5). In an a t t e m p t to evaluate the significance o f d r u g therapies given to n o r m a l individuals, we have divided up the cases into g r o u p s based on the use o f a p a r t i c u l a r d r u g type. M e a n r e c e p t o r c o n c e n t r a t i o n s in all areas were c o m p a r e d with c o n c e n t r a t i o n s in c o r r e s p o n d i n g areas from cases where the d r u g - t y p e in question h a d n o t been used. T h u s those given opiates (morphine, d i a c e t y l m o r p h i n e o r d i h y d r o c o d e i n e , n = 6) did n o t differ significantly f r o m those n o t so t r e a t e d (n = 7). Similarly, those treated with m i n o r tranquillizers (diazepam, nitrazepam, n - - 5) d i d n o t differ from the r e m a i n d e r (n ---- 8), n o r did the d r u g - t r e a t e d cases (n ~ 9) differ significantly from those w h o h a d died drug-free (n ~ 4). This r a t h e r crude analysis has been necessitated b y the considerable differences t h a t exist between i n d i v i d u a l cases in terms o f drugs used, dosage given a n d d u r a t i o n a n d timing o f a d m i n i s t r a t i o n in relation to time o f death. M o r e detailed a t t e m p t s to analyse the effects o f drugs could better be u n d e r t a k e n if brain d r u g concentrations were known. O u r crude analysis seems to indicate t h a t terminal therapeutic measures d o n o t greatly influence b r a i n muscarinic receptor c o n c e n t r a t i o n s in neurologically a n d psychiatrically n o r m a l individuals. E n n a et al. 7 have recently p o i n t e d o u t that studies to date on the muscarinic
389 0.6'SZPARIETAL CORTEX AFFINITY
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3 FREE H-QNB CONCENTRATION (nM)
Fig. 1. The points plotted represent means of duplicate determinations of specific binding of pH]QNB at the indicated [aH]QNB concentrations. Values for free [3H]QNB were calculated by subtracting mean values for bound from the total added. receptor in human brains have not shown whether the observed difference in specific antagonist binding between two areas is due to the presence of different numbers of receptors, or to the presence of receptors which differ in affinity for the ligand used. We have examined this question by measuring the specific binding of [aH]QNB to particulate materials in a sample of normal brain postcentral gyrus and of substantia nigra, varying the [aH]QNB concentration from 0.1 to 10 n M (Fig. 1). We have used this data to estimate the affinity constant of [aH]QNB for the receptors in the two tissues (Fig. 1). Within the limits of accuracy of this methodology, the values for the affinity constants for the two areas of brain agree well enough to argue that the observed differences between the areas in specific [SH]QNB binding are due to the presence of different numbers of identical receptors. Brains from cases of Alzheimer's-type dementia. In all the areas of brain studied, the mean receptor concentrations appeared to be similar to those obtained for corresponding areas of normal brain (Table II). The receptor concentrations in the substantia nigra, amygdala and nucleus accumbens did appear to be lower than in corresponding areas of normal brain, but in no case did the difference achieve statistical significance. Similarly, the apparent elevation of receptor concentrations in the olfactory area was not significant. The affinity constant of [aH]QNB for the receptor in a
390 sample of parietal cortex from one of these cases was determined as described above (Fig. 1). The affinity constant obtained is almost identical to that obtained by studies on normal brain cortex. We were unable to determine whether the receptor concentrations in brains from these cases might be affected by drug treatments employed, because all 5 cases had received minor tranquiUisers for long periods (over 6 months) prior to death. There does not seem to be any compelling reason why techniques for the measurement of the concentration of neurotransmitter receptors should not be applied to human brains obtained at routine autopsy. Data from animal experiments indicates that the receptors remain intact for several hours after death 7,s and we have been unable to find any correlation between the concentration of the muscarinic receptor in normal brain and the time elapsing between death and autopsy. Further, there is no evidence for age-related changes in receptor concentration amongst our normal cases (between the age of 46 and 79), neither were any sex-related differences obvious. The principal prolbem with studies such as this one lies in the interpretation of the results obtained. While it does seem reasonable to assume that we are measuring the concentrations of the physiologically relevant muscarinic acetylcholine receptor (see above), we do not yet have adequante information about the precise cellular location of these receptors to make clear statements concerning our results. Muscarinic receptors seem to be found in greatest numbers where cholinergic terminals are most dense1% but we know veIy little about the nature of the cells that carry these receptors. The use of ligands labelled to high specific radioactivity in in vivo experiments, and of autoradiography to localise the bound ligand in brain slices may be very useful in this respect Is. There is an additional complication which must be considered in attempts to interpret our results on Alzheimer's-type dementia brains. In this condition, there is gross neuronal degeneration, especially in cerebral and limbic cortex a,ll and an apparently specific loss of enzymes of the cholinergic system 4,6,24,26. The lack of any apparent loss of muscarinic receptors can be interpreted in basically two ways. If the assumption is made that cholinergic neurons are selectively lost in this condition, the lack of apparent changes in the number of muscarinic receptors could be seen as indicating that cholinergic cells do not carry a significant proportion of these receptors. Thus it could be argued that the normal levels of these receptors is a further indication of a selective degeneration of one neuronal subpopulation, that is, a degeneration of cholinergic, but not cholinoceptive neurons. However, it is well known that degeneration of peripheral cholinergic nerves is followed by increases in the number of actylcholine receptors in the cholinoceptive tissue (although the majority of the well-studied systems of this type have nicotinic acetylcholine receptorsg,2t,~3). It may well be that in Atzheimer's-type dementia some loss of muscarinic receptors occurs because of loss of cholinergic (and perhaps other) neurons, but that surviving neurons attempt to compensate for the deficiency of acetylcholine by producing increased numbers of receptors. This suggestion has been put forward in one of two reports in which muscarinic receptor concentrations in parietal cortex 24 and
391 frontal cortex 26 from Alzheimer's-type dementia cases were reported to be normal. W h a t e v e r the reason for the apparently n o r m a l levels of muscarinic receptors in this condition, perhaps the most significant p o i n t which can be made is that this c o n d i t i o n m a y be a m e n a b l e to t r e a t m e n t with centrally active muscarinic agonists. O u r results together with those showing an apparently specific loss of enzymes of the cholinergic n e u r o n should encourage efforts in this direction.
This work would not have been possible but for the most generous co-operation of Dr. A. J. F. Maloney and Dr. A. Gordon, who performed the post mortem examinations and neuropathology. Drs. Boyd, Gray, Banyard and Gallimore provided access to clinical information concerning the cases of dementia, and Dr. G. W. Ashcroft and Dr. J. McQueen gave continual support and encouragement. Dr. E. C. Hulme and Dr. N. J. Birdsall provided practical advice regarding the receptor assay. To these colleagues and friends, we offer our most sincere thanks.
1 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, 1 (1973) 1090-1092. 2 Bird, E. D. and Iversen, L. L., Huntington's Chorea: post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia, Brain, 97 (1974) 457-472. 3 Birdsall, N. J. M. and Hulme, E. C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 4 Bowen, D. M., Smith, C. B., White, P. and Davison, A. N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-496. 5 CorseUis, J. A. N., Ageing and the dementias. In W. Blackwood and J. A. N. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1976, pp. 796-848. 6 Davies, P. and Maloney, A. J. F., Selective loss of central cholinergic neurons in Alzheimer's Disease, Lancet, II (1976) 1403. 7 Enna, S. J., Bennett, J. P., Bylund, D. B., Crease, I., Burt, D. R., Charnes, M. E., Yamamura, H. I., Simantov, R. and Snyder, S. H., Neurotransmitter receptor binding: regional distribution in human brain, J. Neurochem., 28 (1977) 233-236. 8 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 in neurotransmitter receptors in the brain, New Eng. J. Med., 294 (1976) 1305-1309. 9 Guth, L., 'Trophic' influences of nerve on muscle, Physiol. Rev., 48 (1968) 645-687. 10 Hiley, C. R. and Bird, E. D., Decreased muscarinic receptor concentration in post-mortem brain in Huntington's Chorea, Brain Research, 80 (1974) 355-358. 11 Hooper, M. W. and Vogel, F. S., The limbic system in Alzheimer's Disease: a neuropathological investigation, Amer. J. Pathol., 85 (1976) 1-20. 12 Hornykiewicz, O., Dopamine in basal ganglia, Brit. reed. Bull., 29 (1973) 172-178. 13 Hulme, E. C., Burgen, A. S. V. and Birdsall, N. J. M., Interactions of agonists and antagonists with the muscarinic receptor. In Proc. 1NSERM Colloquium on the Physiology and Pharmacology oJSmooth Muscle, INSERM, Vol. 50, 1976, pp. 49-69. 14 Kanzawa, I., Bird, E. D., O'Connell, R. and Powell, D., Evidence for a decrease in substance P content of substantia nigra in Huntington's Chorea, Brain Research, 120 (1977) 387-391. 15 Katzman, R., The prevalence and malignancy of Alzhei mer Disease: a major killer, Arch. Neurol., 33 (1976) 217-218. 16 Kuhar, M. J., The anatomy of cholinergic neurons. In A. M. Goldberg and I. Hanin (Eds.), Biology of Cholinergic Function, Raven Press, New York, 1976, pp. 3-28. 17 Kuhar, M. J., Pert, C. B. and Snyder, S. H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 447-450. 18 Kuhar, M. J. and Yamamura, H. I., Localization of cholinergic muscarinic receptors in rat brain by light microscopic autoradiography, Brain Research, 110 (1976) 229-243.
392 19 Lloyd, K., Davidson, L. and Hornykiewicz, O., The neurochemistry of Parkinson's Disease, J. PharmacoL exp. Ther., 195 (1975)453--464. 20 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with IFolin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 21 McConneU, M. G. and Simpson, L. L., The role of acetylcholine receptors and acetylcholincsterase activity in the development of denervation supersensitivity, J. Pharmacol. exp. Ther., 198 (1976) 507-517. 22 McGeer, P. L. and McGeer, E. G., Epzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's Disease and Huntington's Chorea, J. Neurochem., 26 (1976) 65-76. 23 Miledi, R. and Potter, L. T., Acetylcholine receptors in muscle fibres, Nature (Lond.), 233 (1971) 599-603. 24 Perry, E. K., Perry, R. H., Blessed, G. and Tomlinson, B. E., Necropsy evidence of central cholinergic deficits in senile demantia, Lancet, 1 (1977) 189. 25 Perry, T. L., Hansen, S., Lesk, D. and Klaster, M., Amino acids in plasma, cerebrospinal fluid and brain of patients with Huntington's Chorea. In Huntington's Chorea 1872-1972, A. Barbeau, T. N. Chase and G. W. Paulson (Eds.), Raven Press, New York, 1973, pp. 609-618. 26 White, P., Goodhardt, M. J., Keet, J. P., Hiley, C. R., Carasco, L. H., Williams, I. E. I. and Bowen, D. M., Neocortical cholinergic neurons in elderly people, Lancet, 1 (1977) 668-671. 27 Yamamura, H. I., Kuhar, M. J., Greenberg, D. and Snyder, S. H., Muscarinic cholinergic receptor binding: regional distribution in monkey brain, Brain Research, 66 (1974) 541-546. 28 Yamamura, H. I. and Snyder,. Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729.