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Pre- and postsynaptic neurochemical alterations in Alzheimer's disease
Brain Research, 159 (1978) 477-481 © Elsevier/North-Holland Biomedical Press
477
Pre- and postsynaptic neurochemical alterations in Alzheimer's disease
TERRY D. REISINE, HENRY |. YAMAMURA, EDWARD D. BIRD, ERNEST SPOKES and S. J. ENNA
Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Ariz. 85724; ( E.D.B. and E.S.) Department of Neurological Surgery and Neurology, Addenbrookes Hospital, Cambridge (Great Britain) and (S.J,E.) Departments of Pharmacology, Neurobiology and Anatomy, University of Texas Medical School at Houston, P.O. Box 20708, Houston Texas 77025 (U.S.A.) (Accepted September 7th, 1978)
Although Alzheimer's disease (AD) is the most common form of senile dementia there is as yet no effective therapy for this disorder. The most profound neuropathological changes observed in this disease are gross atrophy of the cerebral cortical sulci, degeneration of neurons in the hippocampus and the presence of senile plaques, neurofibrillary tangles and argyrophilic inclusion bodies 3. Neurochemically the most significant alteration observed is a marked decrease in choline acetyltransferase (ChAc) activity within the hippocampus, caudate nucleus, amygdala, and cerebral cortex suggesting a selective loss of cholinergic cells in these brain areas 1,1°,14. Interestingly however, as opposed to some other neurological disorders, many of the neuropathological alterations observed in AD are seen, though to a much lesser extent, in a significant number of non-demented elderly individuals, suggesting that these changes are part of the normal aging process. Thus, studies on AD, in addition to better defining this disorder, may yield more general insights into the neurobiology of aging. With the advent of biochemical techniques to measure the presence of synaptic receptor sites for neurotransmitters in brain la it has been possible to obtain new information about the neuropathology of disorders such as Huntington's disease 4,5, 12,12 and Parkinson's diseaseS, 11. This information may be useful in designing rational therapeutic treatments for these disorders and, coupled with other neurochemical studies, has contributed significantly to the understanding of the interneuronal relationships which exist in the normal human brain. In the present investigation, these neurotransmitter binding techniques are used to study possible receptor alterations in selected brain areas obtained at autospy from individuals diagnosed as having had AD. In addition, the activity of ChAc was determined in an attempt to correlate receptor changes with neuronal cell type. Postmortem brain tissue was obtained from 6 individuals with AD having a mean age of 68 years (59-79) and 5 control individuals with a mean age of 69 years (52-82). No control tissue was used from individuals who suffered from an infection or malignancy of the central nervous system. All AD subjects were receiving either a
1454 (6) 267 (4) 24 (6) 67 (6) 2_ 12"
± 12"
± 37*
± 340
1901 (3) 463 (3) 82 (3) 148 (3) ± 10
:i: 12
~ 66
4- 368
1428 (5) 357 (4) 30 (5) 150 (5)
A
~: 23
2_ 8*
± 50
± 214
832 (3) 1800 (3) 5 f3) 24 (3)
C
± 5
± I
:[: 79
-4- 33
Frontal cortex
789 (4) 913 (4) 2 (4) 17 (4)
A
± 4
± 0.3*
2_ 86*
± 34
353 (5) 765 (5) 5 (5) 7 (5)
C
129
± 2
± 1
i
2_ 61
Hippocampus
153 (4) 453 (5) 1 (4) 7 (4)
A
~ 2
:i: 0.1"
± 77
± 41"
* For the neurotransmitter receptor binding assays, the final concentrations of [aH]ligand was 140 p M for [3H]QNB, 25 n M for [3H]GABA and 80 p M for [3H]spiroperidol. ** P < 0.05.
± 12
± 5
± 11
± 112
C
C
A
Putamen
Caudate nucleus
1631 (4) [ZH]GABA 468 (4) Choline acetyltransferase 68 (4) [ZH]spiroperidol 128 (4)
[3H]QNB
Receptor or enzyme*
The receptor binding data are expressed as the means i S.E.M. and is expressed as mmole/mg protein. ChAc activity is given as the means ± S.E.M. and is expressed as nmoles ACh synthesized/rag protein/h. The number of determinations in each case is indicated in parentheses. C indicates control ; A indicates Alzheimer's diseased brains.
Neurotransmitter receptor binding and enzyme activity in various areas o f control and Alzheimer's diseased brain
TABLE I
O0
479 butyrophenone or a phenothiazine at the time of death whereas control subjects were unmedicated. The diagnosis of AD was made on the basis of microscopical observations of the brain tissue. In all AD cases, histological examinations of the cerebral cortex showed innumerable argyrophilic plaques of the Alzheimer type and widespread neurofibrillary tangles. Control individuals had no known neurological complaints nor was there any evidence of significant histochemical neuropathology. No samples were used when there had been a delay of more than 72 h between death and autQpsy. At autopsy the brains were dissected and the brain regions stored at --70 °C until assay. For binding analysis, cholinergic muscarinic receptor binding was studied using [aH]quinuclidinyl benzilate ([aH]QNB) (13 Ci/mmole) as the ligand15-17, GABA receptor binding was studied using [aH]GABA (35 Ci/mmole) as the ligand4, 5, dopaminergic receptor binding was studied using [aH]spiroperidol (26.4 Ci/mmole)6 and ChAc activity was measured as previously describedll,lL Previous studies have demonstrated that no major postmortem alterations of neurotransmitter receptor binding sites occur in rat brain 4. This suggests that the neurotransmitter receptors in human brain that we have studied are stable to most postmortem changes that might have occurred. Protein content was determined using the method of Lowry et a l l Statistical significance was calculated using a two-tailed Student's t-test and correlations were calculated using a Spearman rank correlation analysis. The results of these preliminary experiments revealed a significant reduction (P < 0.05) in specific [aH]QNB binding in the hippocampus, with no significant changes observed in the caudate nucleus, putamen and frontal cortex (Table I). Saturation studies in the hippocampus indicated that no change in affinity of the receptor for [aH]QNB occurred in AD (data not shown). Receptor binding for [3H]GABA was significantlyreduced--43 ~o in the caudate nucleus and 50 ~ in the frontal cortex of AD brains compared to controls with no significant alterations seen in the putamen and hippocampus. [3H]Spiroperidol binding was significantly reduced, 4 8 ~ in the caudate nucleus. No statistically significant alterations in [aH]spiroperidol binding were found in the frontal cortex, putamen or hippocampus. With regard to enzyme activity, all 4 brain areas studied showed a significant reduction (60-80 ~) in ChAc activity in the AD tissue. The present results demonstrate a significant reduction in ChAc activity in the striatum and substantiate earlier reports 1,~0,~4 of a significant reduction in this enzyme activity in the cerebral cortex and hippocampus in brains of individuals diagnosed as having AD, suggesting that cholinergic cells have partially degenerated in these brain areas in this disorder. The present study also reveals a significant loss of cholinergic muscarinic receptors in the AD hippocampus suggesting that the cell type(s) modulated by the cholinergic system in the hippocampus has also partially degenerated in this disorder. Recently, Davies and Verth 2 have demonstrated that [aH]QNB binding is not altered in AD hippocampus. The differences in our results might be due to variability in tissue dissection, disease diagnosis, severity of the patient's illness as well as different amounts of [3H]QNB and tissue used in the assay.
480 With regard to GABA receptors, the significant decreases observed in the AD caudate nucleus and frontal cortex suggest that there has been a loss of those cell types which are modulated by the GABA system. It is unlikely that the cholinergic cell loss in the AD caudate nucleus is related to the loss of GABA receptors since previous studies have indicated that GABA receptors are not found on cholinergic cells in this brain area 4,z, but it is possible that in the frontal cortex there may be some relationship between the decline in GABA receptor binding and ChAc activity. However, no significant correlation was found between GABA receptor binding and ChAc activity in the frontal cortex, indicating that a GABA-cholinergic link in this brain area is unlikely. The spiroperidol binding alterations in the AD caudate nucleus suggest that there are changes in the dopaminergic system modulating this brain area in AD. The decreases in spiroperidol binding in the caudate nucleus appear to be related to the loss of striatai cholinergic neurons. Similar deficits in dopamine receptor density and ChAc activity have been found in Parkinson's and Huntington's diseased caudate nuclei 11,1'. Recent studies in which rat striatal cell bodies have been destroyed by injection of kainic acid into the corpus striatum have demonstrated that the lesion causes both ChAc activity and spiroperidol binding to be markedly reduced in the striatum 7. This suggests that a large population of striatal dopamine receptors are located on cholinergic cell bodies in the striatum. The lack of change in spiroperidol binding in AD putamen, despite ChAc activity decreases (similar results are found in Parkinson's diseaseU), suggests that a different relationship between dopaminergic receptors and cholinergic neurons may exist in the putamen as compared to the caudate nucleus. Thus, these preliminary results suggest that while the cholinergic system is the most generally affected cell type in AD, other cell types have also degenerated, in particular those which are modulated by the GABA system in the caudate nucleus and frontal cortex, the dopamine system in the caudate nucleus and those which are modulated by the cholinergic system in the hippocampus. It is likely, therefore, that before effective therapy for AD can be designed it will be necessary to identify the neurotransmitter agents utilized by these missing cell types. The authors would like to thank Elaine Mann and Tom McManus for their technical assistance. Supported in part by USPHS Grants and from the Hereditary Disease Foundation. S. J. E. and H. I. Y. are recipients of RSDAs from the NINDS and NIMH.
1 Davies, P. and Maloney, A. J. F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, Dec. 25 (1976) 1403. 2 Davies, P. and Verth, A. H., Regional distribution of muscarinic acetylcholine receptor in normal and Alzheimer's-type dementia brains, Brain Research, 138 (1978) 385-392. 3 DeJong, R. N. and Pope, A., Dementia. In D. B. Tower and T. N. Chase (Eds.), The Nervous System, Raven Press, New York, 1975, pp. 449456. 4 Enna, S..I., Bird, E., Bennett, J., Bylund, D., Yamamura, H. I., Iversen, L. and Snyder, S. H., Huntington's chorea: changes in neurotransmitter receptors in the brain, New Engl. J. Med., 294 (1976) 1305-1309.
481 5 Enna, S. J., Bird, E., Bennett, J., Bylund, D., Iversen, L. and Snyder, S. H., Alterations of brain neurotransmitter receptor binding in Huntington's chorea, Brain Research, 116 (1976) 531-537. 6 Fields, J. Z., Reisine, T. D. and Yamamura, H. I., Biochemical demonstration of the doparninergic receptor in rat and human brain using [aH]spiroperidol, Brain Research, 136 (1977) 578-584. 7 Fields, J. Z., Reisine, T. D. and Yamamura, H. I., Loss of striatal dopaminergic receptor after intrastriatal kainic acid injection, Life Sci., 23 (1978) 569-574. 8 Lloyd, K., Shemer~, L. and Hornykiewicz, O., Distribution of high affinity sodium-independent [aH]gamma-aminobutyric acid ([ZH]GABA) bindingin the human brain: alterations in Parkinson's disease, Brain Research, 127 (1977) 269-278. 9 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, A. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 Perry, E. K., Perry, R., Blessed, G. and Tomlinson, B., Necropsy evidence of central cholinergic deficites in senile dementia, Lancet, Jan. 22 (1977) 189. 11 Reisine, T. D., Fields, J. Z., Yamamura, H. I., Bird, E., Spokes, E., Schreiner, P. and Enna, S. J., Neurotransmitter receptor alterations in Parkinson's disease, Life Sci., 21 (1977) 335-344. 12 Reisine, T. D., Fields, J. Z., Stern, L. Z., Johnson, P. C., Bird, E. and Yamamura, H. I., Alterations in dopaminergic receptors in Huntington's disease, Life ScL, 21 (1977) 1123-1128. 13 Snyder, S. H. and Bennett, J. P., Neurotransmitter receptors in the brain: biochemical identification, Ann. Rev. PhysioL, 38 (1976) 153-175. 14 Spillane, J., White, P., Goodhardt, M., Flack, R., Bowen, D. M. and Davison, A., Selective vulnerability of neurons in organic dementia, Nature (Lond.), 266 (1977) 558-559. 15 Wastek, G., Stern, L., Johnson, P. C. and Yamamura, H. 1., Huntington's disease: regional alterations in muscarinic cholinergic receptor binding in human brain, Life Sci., 19 (1976) 1033-1040. 16 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729. 17 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [aH]quinuclidinyl benzilate, Molec. Pharmacol., 10 (1974) 861-867.
477
Pre- and postsynaptic neurochemical alterations in Alzheimer's disease
TERRY D. REISINE, HENRY |. YAMAMURA, EDWARD D. BIRD, ERNEST SPOKES and S. J. ENNA
Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Ariz. 85724; ( E.D.B. and E.S.) Department of Neurological Surgery and Neurology, Addenbrookes Hospital, Cambridge (Great Britain) and (S.J,E.) Departments of Pharmacology, Neurobiology and Anatomy, University of Texas Medical School at Houston, P.O. Box 20708, Houston Texas 77025 (U.S.A.) (Accepted September 7th, 1978)
Although Alzheimer's disease (AD) is the most common form of senile dementia there is as yet no effective therapy for this disorder. The most profound neuropathological changes observed in this disease are gross atrophy of the cerebral cortical sulci, degeneration of neurons in the hippocampus and the presence of senile plaques, neurofibrillary tangles and argyrophilic inclusion bodies 3. Neurochemically the most significant alteration observed is a marked decrease in choline acetyltransferase (ChAc) activity within the hippocampus, caudate nucleus, amygdala, and cerebral cortex suggesting a selective loss of cholinergic cells in these brain areas 1,1°,14. Interestingly however, as opposed to some other neurological disorders, many of the neuropathological alterations observed in AD are seen, though to a much lesser extent, in a significant number of non-demented elderly individuals, suggesting that these changes are part of the normal aging process. Thus, studies on AD, in addition to better defining this disorder, may yield more general insights into the neurobiology of aging. With the advent of biochemical techniques to measure the presence of synaptic receptor sites for neurotransmitters in brain la it has been possible to obtain new information about the neuropathology of disorders such as Huntington's disease 4,5, 12,12 and Parkinson's diseaseS, 11. This information may be useful in designing rational therapeutic treatments for these disorders and, coupled with other neurochemical studies, has contributed significantly to the understanding of the interneuronal relationships which exist in the normal human brain. In the present investigation, these neurotransmitter binding techniques are used to study possible receptor alterations in selected brain areas obtained at autospy from individuals diagnosed as having had AD. In addition, the activity of ChAc was determined in an attempt to correlate receptor changes with neuronal cell type. Postmortem brain tissue was obtained from 6 individuals with AD having a mean age of 68 years (59-79) and 5 control individuals with a mean age of 69 years (52-82). No control tissue was used from individuals who suffered from an infection or malignancy of the central nervous system. All AD subjects were receiving either a
1454 (6) 267 (4) 24 (6) 67 (6) 2_ 12"
± 12"
± 37*
± 340
1901 (3) 463 (3) 82 (3) 148 (3) ± 10
:i: 12
~ 66
4- 368
1428 (5) 357 (4) 30 (5) 150 (5)
A
~: 23
2_ 8*
± 50
± 214
832 (3) 1800 (3) 5 f3) 24 (3)
C
± 5
± I
:[: 79
-4- 33
Frontal cortex
789 (4) 913 (4) 2 (4) 17 (4)
A
± 4
± 0.3*
2_ 86*
± 34
353 (5) 765 (5) 5 (5) 7 (5)
C
129
± 2
± 1
i
2_ 61
Hippocampus
153 (4) 453 (5) 1 (4) 7 (4)
A
~ 2
:i: 0.1"
± 77
± 41"
* For the neurotransmitter receptor binding assays, the final concentrations of [aH]ligand was 140 p M for [3H]QNB, 25 n M for [3H]GABA and 80 p M for [3H]spiroperidol. ** P < 0.05.
± 12
± 5
± 11
± 112
C
C
A
Putamen
Caudate nucleus
1631 (4) [ZH]GABA 468 (4) Choline acetyltransferase 68 (4) [ZH]spiroperidol 128 (4)
[3H]QNB
Receptor or enzyme*
The receptor binding data are expressed as the means i S.E.M. and is expressed as mmole/mg protein. ChAc activity is given as the means ± S.E.M. and is expressed as nmoles ACh synthesized/rag protein/h. The number of determinations in each case is indicated in parentheses. C indicates control ; A indicates Alzheimer's diseased brains.
Neurotransmitter receptor binding and enzyme activity in various areas o f control and Alzheimer's diseased brain
TABLE I
O0
479 butyrophenone or a phenothiazine at the time of death whereas control subjects were unmedicated. The diagnosis of AD was made on the basis of microscopical observations of the brain tissue. In all AD cases, histological examinations of the cerebral cortex showed innumerable argyrophilic plaques of the Alzheimer type and widespread neurofibrillary tangles. Control individuals had no known neurological complaints nor was there any evidence of significant histochemical neuropathology. No samples were used when there had been a delay of more than 72 h between death and autQpsy. At autopsy the brains were dissected and the brain regions stored at --70 °C until assay. For binding analysis, cholinergic muscarinic receptor binding was studied using [aH]quinuclidinyl benzilate ([aH]QNB) (13 Ci/mmole) as the ligand15-17, GABA receptor binding was studied using [aH]GABA (35 Ci/mmole) as the ligand4, 5, dopaminergic receptor binding was studied using [aH]spiroperidol (26.4 Ci/mmole)6 and ChAc activity was measured as previously describedll,lL Previous studies have demonstrated that no major postmortem alterations of neurotransmitter receptor binding sites occur in rat brain 4. This suggests that the neurotransmitter receptors in human brain that we have studied are stable to most postmortem changes that might have occurred. Protein content was determined using the method of Lowry et a l l Statistical significance was calculated using a two-tailed Student's t-test and correlations were calculated using a Spearman rank correlation analysis. The results of these preliminary experiments revealed a significant reduction (P < 0.05) in specific [aH]QNB binding in the hippocampus, with no significant changes observed in the caudate nucleus, putamen and frontal cortex (Table I). Saturation studies in the hippocampus indicated that no change in affinity of the receptor for [aH]QNB occurred in AD (data not shown). Receptor binding for [3H]GABA was significantlyreduced--43 ~o in the caudate nucleus and 50 ~ in the frontal cortex of AD brains compared to controls with no significant alterations seen in the putamen and hippocampus. [3H]Spiroperidol binding was significantly reduced, 4 8 ~ in the caudate nucleus. No statistically significant alterations in [aH]spiroperidol binding were found in the frontal cortex, putamen or hippocampus. With regard to enzyme activity, all 4 brain areas studied showed a significant reduction (60-80 ~) in ChAc activity in the AD tissue. The present results demonstrate a significant reduction in ChAc activity in the striatum and substantiate earlier reports 1,~0,~4 of a significant reduction in this enzyme activity in the cerebral cortex and hippocampus in brains of individuals diagnosed as having AD, suggesting that cholinergic cells have partially degenerated in these brain areas in this disorder. The present study also reveals a significant loss of cholinergic muscarinic receptors in the AD hippocampus suggesting that the cell type(s) modulated by the cholinergic system in the hippocampus has also partially degenerated in this disorder. Recently, Davies and Verth 2 have demonstrated that [aH]QNB binding is not altered in AD hippocampus. The differences in our results might be due to variability in tissue dissection, disease diagnosis, severity of the patient's illness as well as different amounts of [3H]QNB and tissue used in the assay.
480 With regard to GABA receptors, the significant decreases observed in the AD caudate nucleus and frontal cortex suggest that there has been a loss of those cell types which are modulated by the GABA system. It is unlikely that the cholinergic cell loss in the AD caudate nucleus is related to the loss of GABA receptors since previous studies have indicated that GABA receptors are not found on cholinergic cells in this brain area 4,z, but it is possible that in the frontal cortex there may be some relationship between the decline in GABA receptor binding and ChAc activity. However, no significant correlation was found between GABA receptor binding and ChAc activity in the frontal cortex, indicating that a GABA-cholinergic link in this brain area is unlikely. The spiroperidol binding alterations in the AD caudate nucleus suggest that there are changes in the dopaminergic system modulating this brain area in AD. The decreases in spiroperidol binding in the caudate nucleus appear to be related to the loss of striatai cholinergic neurons. Similar deficits in dopamine receptor density and ChAc activity have been found in Parkinson's and Huntington's diseased caudate nuclei 11,1'. Recent studies in which rat striatal cell bodies have been destroyed by injection of kainic acid into the corpus striatum have demonstrated that the lesion causes both ChAc activity and spiroperidol binding to be markedly reduced in the striatum 7. This suggests that a large population of striatal dopamine receptors are located on cholinergic cell bodies in the striatum. The lack of change in spiroperidol binding in AD putamen, despite ChAc activity decreases (similar results are found in Parkinson's diseaseU), suggests that a different relationship between dopaminergic receptors and cholinergic neurons may exist in the putamen as compared to the caudate nucleus. Thus, these preliminary results suggest that while the cholinergic system is the most generally affected cell type in AD, other cell types have also degenerated, in particular those which are modulated by the GABA system in the caudate nucleus and frontal cortex, the dopamine system in the caudate nucleus and those which are modulated by the cholinergic system in the hippocampus. It is likely, therefore, that before effective therapy for AD can be designed it will be necessary to identify the neurotransmitter agents utilized by these missing cell types. The authors would like to thank Elaine Mann and Tom McManus for their technical assistance. Supported in part by USPHS Grants and from the Hereditary Disease Foundation. S. J. E. and H. I. Y. are recipients of RSDAs from the NINDS and NIMH.
1 Davies, P. and Maloney, A. J. F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, Dec. 25 (1976) 1403. 2 Davies, P. and Verth, A. H., Regional distribution of muscarinic acetylcholine receptor in normal and Alzheimer's-type dementia brains, Brain Research, 138 (1978) 385-392. 3 DeJong, R. N. and Pope, A., Dementia. In D. B. Tower and T. N. Chase (Eds.), The Nervous System, Raven Press, New York, 1975, pp. 449456. 4 Enna, S..I., Bird, E., Bennett, J., Bylund, D., Yamamura, H. I., Iversen, L. and Snyder, S. H., Huntington's chorea: changes in neurotransmitter receptors in the brain, New Engl. J. Med., 294 (1976) 1305-1309.
481 5 Enna, S. J., Bird, E., Bennett, J., Bylund, D., Iversen, L. and Snyder, S. H., Alterations of brain neurotransmitter receptor binding in Huntington's chorea, Brain Research, 116 (1976) 531-537. 6 Fields, J. Z., Reisine, T. D. and Yamamura, H. I., Biochemical demonstration of the doparninergic receptor in rat and human brain using [aH]spiroperidol, Brain Research, 136 (1977) 578-584. 7 Fields, J. Z., Reisine, T. D. and Yamamura, H. I., Loss of striatal dopaminergic receptor after intrastriatal kainic acid injection, Life Sci., 23 (1978) 569-574. 8 Lloyd, K., Shemer~, L. and Hornykiewicz, O., Distribution of high affinity sodium-independent [aH]gamma-aminobutyric acid ([ZH]GABA) bindingin the human brain: alterations in Parkinson's disease, Brain Research, 127 (1977) 269-278. 9 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, A. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 Perry, E. K., Perry, R., Blessed, G. and Tomlinson, B., Necropsy evidence of central cholinergic deficites in senile dementia, Lancet, Jan. 22 (1977) 189. 11 Reisine, T. D., Fields, J. Z., Yamamura, H. I., Bird, E., Spokes, E., Schreiner, P. and Enna, S. J., Neurotransmitter receptor alterations in Parkinson's disease, Life Sci., 21 (1977) 335-344. 12 Reisine, T. D., Fields, J. Z., Stern, L. Z., Johnson, P. C., Bird, E. and Yamamura, H. I., Alterations in dopaminergic receptors in Huntington's disease, Life ScL, 21 (1977) 1123-1128. 13 Snyder, S. H. and Bennett, J. P., Neurotransmitter receptors in the brain: biochemical identification, Ann. Rev. PhysioL, 38 (1976) 153-175. 14 Spillane, J., White, P., Goodhardt, M., Flack, R., Bowen, D. M. and Davison, A., Selective vulnerability of neurons in organic dementia, Nature (Lond.), 266 (1977) 558-559. 15 Wastek, G., Stern, L., Johnson, P. C. and Yamamura, H. 1., Huntington's disease: regional alterations in muscarinic cholinergic receptor binding in human brain, Life Sci., 19 (1976) 1033-1040. 16 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729. 17 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [aH]quinuclidinyl benzilate, Molec. Pharmacol., 10 (1974) 861-867.