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Somatostatin is not co-localized in cholinergic neurons innervating the rat cerebral cortex-hippocampal formation
Brain Research, 243 (1982) 169-172 Elsevier Biomedical Press
169
Somatostatin is not co-localized in cholinergic neurons innervating the rat cerebral cortex-hippocampal formation MICHAEL McKINNEY, PETER DAVIES and JOSEPH T. COYLE* ( M.M. and J.T.C.) Departments of Neuroscience, Psychiatry and the Behavioral Sciences, and Pharmacology and Experimental Therapeutics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 and (P.D.) Departments of Pathology and Neuroscience, Albert Einstein College of Medicine, The Bronx, N Y 10461 (U.S.A.)
(Accepted March 18th, 1982) Key words: somatostatin - - choline acetyltransferase - - rat brain - - lesions - - excitotoxins
The somatostatin content and choline acetyltransferase activity of the rat cerebral cortex and hippocampus were examined after lesions to the nucleus basalis, the fornix and the dorsal hippocampus. Lesions of the nucleus basalis caused reductions in cortical choline acetyltransfera~e activity but had no effect on the concentration of somatostatin. Fornix transection caused a reduction in choline acetyltransferase activity in the dorsal hippocampus, but again was without effect on the somatostatin content. Excitotoxin lesions of the dorsal hippocampus caused a 69 ~o reduction in somatostatin concentration in this structure, with no reduction in choline acetyltransferase activity. The results indicate that somatostatin and choline acetyltransferase are in separate populations of neurons in the rat cerebral cortex and hippocampus. Considerable evidence has accrued that Alzheimer's Disease and senile dementia of the Alzheimer's type (AD/SDAT) are associated with profound and relatively selective reductions in the presynaptic markers for cholinergic neurons in the cerebral cortex and hippocampal formation1, 3,5,17. A major source of cholinergic innervation to the telencephalon are the large acetylcholinesterase positive neurons contained in the nucleus basalis of the basal forebrain which extends anteriorly to include the diagonal band of Broca and the medial septal nucleusa-11,16. Excitotoxin lesions of this complex in rats result in marked and selective reductions in the presynaptic cholinergic markers, depending upon the localization of the lesion within the complex, in the cerebral cortex and hippocampal formationg, ~s. Neuropathologic studies have now demonstrated that the nucleus basalis undergoes degeneration in A D / S D A T , thus providing histopathologic basis for the reduction in cortical cholinergic markers in this disorder 22. Recently, two laboratories independently repor-
ted that the concentration of somatostatin is markedly reduced in the cortex and hippocampus of patients dying with AD/SDAT4,8, 20. Since other neuropeptides such as cholecystokinin and vasoactive intestinal peptide are not consistently decreased in these areas 19,21, this reduction in somatostatin appears to be relatively selective and parallels, to some extent, the decrement in cortical cholinergic markers. Although immunocytochemical studies have demonstrated somatostatin-containing perikarya in the cerebral cortex 7,12, it is unclear at present whether these intrinsic neurons are the major or exclusive source of cortical somatostatin innervation. In the light of the growing evidence of coexistence of neuropeptides in aminergic neurons 13, it seemed possible that the parallel reductions in cholinergic markers and somatostatin in A D / S D A T might result from their co-localization in the basal forebrain neurons that project to the cerebral cortex. To evaluate this hypothesis, we have examined the effects of excitotoxin and ablative lesions of the basal forebrain projections to the rat cerebral cortex
* To whom all correspondence should be addressed at: Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
170 and of the hippocampal formation on the activity of choline acetyltransferase, a specific marker for the cholinergic neurons, and on the levels of somatostatin. Adult male Sprague-Dawley rats, (180-200 g) purchased from Harlan-Sprague-Dawley, were used for the experiments. To lesion the nucleus basalis in the ventral globus pallidus, the rats were anesthetized with Equithesin and placed in a David K o p f stereotaxic apparatus; through a burr hole placed in the calvarium, a Hamilton cannula (0.3 m m in diameter) was positioned in the ventral globus pallidus (P, 0.3 m m ; V, 7.2 m m ; L, 3.0 m m with the intra-auricular line, the midline and pial surface equal to zero and with the incisor bar at 0 ° to the ear bars) and 15 /tg of ibotenic acid (Regis Chemicals) dissolved in 1 #1 of artificial CSF was infused over a period of 2 min 10. To lesion neurons intrinsic to the hippocampal formation, 1.0/tg of kainic acid (Sigma Chemicals, St. Louis, MO; lot 105C4479) was infused into the dorsal hippocampal formation at the following coordinates: P, 2.3 m m ; V, 3.0 m m ; L, 2.0 mm. In some animals, the fornix was transected with a knife cut according to the procedure of Zaczek et al. 24. One week after surgery or excitotoxin lesion, the animals were sacrificed by decapitation and the cerebral cortex and hippocampus ipsilateral and contralateral to the lesion was dissected at 5 °C. The cortical sample consisted of the fronto-parietal cortex superior to the rhinal fissure and medial to the cingulate; in the case of the hippocampal kainate lesion, only the dorsal hippocampus was used. A 10 mg portion of tissue was dissected from the cortex and the dorsal hippocampal formation and immediately frozen upon dry ice for measurement of somatostatin levels. For the somatostatin assay, the frozen tissue was dropped into boiling 2 M acetic acid, and left for 10 min. The mixture was homogenized and an aliquot was removed for protein determination by the method of Lowry et a!.14. After centrifugation at 15,000 g for 30 min, aliquots of the supernatant were freeze-dried and used for somatostatin assay as previously described 4,6. The remaining tissue was immediately homogenized in 50 m M Tris-HC1, p H 7.4, containing 0.2 ~ Triton X-100; and after centrifugation, the supernatant was assayed for the activity of choline acetyltransferase by the method of
Bull and Oderfeld-Nowak 2 and the activity of glutamate decarboxylase by the method of Wilson et al. 23. Protein content was determined by the method of Lowry et al. 14, with bovine serum albumin as standard. Ibotenate lesion of the ventral globus pallidus caused a 6 4 ~ reduction in the specific activity of choline acetyltransferase in the ipsilateral cerebral cortex but did not significantly affect the activity of the enzyme in the hippocampal formation (Table I). In contrast, the concentration of somatostatin was not altered either in the neocortex or in the hippocampal formation. This result indicated that somatostatin was not localized in the cholinergic neuronal perikarya in the ventral globus pallidus that provide innervation to the fronto-parietal cortex but did not rule out the possibility that hippocampal somatostatin might be primarily localized in extrinsic neurons. To evaluate this latter possibility, we examined the effects of transection of the fornix, the primary route of entry of afferent input to the hippocampal formation. Prior transection of the fornix
TABLE 1 Effects o f lesions o f the ventral pallidum or fornix on somatostatin levels
The ventral globus pallidus was lesioned with 15 ~g of ibotenate or the fornix was transected as described in the text. Seven days after lesion, the rats were sacrificed; and specific activity of choline acetyltransferase and the content of somatostatin were measured in the same samples. The results are expressed as the mean ± S.E.M. of the number of animals indicated in parenthesis. Choline acetyltransferase (nrnol/ mg protein/h )
Somatostatin (ng/mgprotein)
(A) Ventral GP lesion Cortex Lesion (5) 13.7 ± 0.8* Control (7) 38.5 ± 4.0 Hippocampus Lesion (5) 40.2 ± 1.6 Control (7) 42.8 ± 1.7
3.15 ~ 0.54 3.24 ± 0.4
(B) Fornix transection Hippocampus Lesion (6) 14.5 ± 1.3" Control (5) 39.2 ± 1.5
3.92 i 0.34 3.57 ± 0.39
3.86 ± 0.38 3.77 ± 0.64
* P < 0.001 versus control by Student's t-test.
171 TABLE II Effects of hippocampal kainate lesion on somatostatin levels
Ether anesthetized rats received a 1.0 jug injection of kainic acid in the dorsal hippocampus one week prior to sacrifice. The specificactivities of glutamate decarboxylaseand choline acetyltransferase and the content of somatostatin were measured in the same dorsal hippocampal samples. The results are expressed as the mean i S.E.M. of 7 lesioned and control preparations. Control
Lesion
Glutamate decarboxylase (nmol/mgprotein/h)
23.6 ± 1.6
Choline acetyltransferase (nmol/mgprotein/h)
38.7 4- 1.2
35.5 4- 1.3
Somatostatin (ng/mg protein)
4.14 ± 0.36
1.27 ± 0.12"
6.3 4- 1.0"
* P < 0.001 versus control by Student's t-test.
caused a 63 ~ reduction in the specific activity of choline acetyltransferase in the ipsilateral hippocampal formation but did not significantly affect the concentration of somatostatin. To establish that somatostatin was indeed associated with neurons intrinsic to the hippocampal formation, we examined the effects of kainic acid lesion of the dorsal hippocampal formation, which causes a selective degeneration of neurons with cell bodies in the region 1°. The kainate lesion resulted in a profound reduction in the specific activity of glutamate decarboxylase and a comparable 69 ~ reduction in the concentration of somatostatin in the dorsal hippocampus (Table II); the selectivity of the lesion was confirmed by the finding that the specific activity of choline acetyltransferase in the dorsal hippocampus
1 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) 459-495. 2 Bull, G. and Oderfeld-Nowak, B., Standardization of a radiochemical assay of choline acetyltransferase and a study of the activation of the enzyme in rabbit brain, J. Neurochem., 18 (1971) 935-947. 3 Davies, P., Neurotransmitter-related enzymes in senile dementia of the Alzheimer type, Brain Research, 171 (1979) 319-327. 4 Davies, P., Katzman, R. and Terry, R. D., Reduced somatostatin-like immunoreactivity in cerebral cortex
was not significantly reduced. These studies provide compelling evidence that somatostatin is localized in a distinctly different population of neurons than those that serve as the major source of cholinergic innervation to the cortex and hippocampal formation. The results from transection of the fornix and kainate lesion of hippocampal intrinsic neurons indicate that somatostatin is contained in neurons intrinsic to the hippocampal formation. Thus, it would appear that the bipolar neurons in the cerebral cortex and hippocampal formation, that have been shown to contain somatostatin by immunocytochemical methods, are the primary, if not the exclusive, source of somatostatin innervation of the cortex and hippocampus. These findings suggest that the decrements in somatostatin levels in the cerebral cortex and hippocampal formation in A D / S D A T reflect degeneration of intrinsic neurons. Thus, the pathology of AD/ SDAT involves not only the degeneration of the aminergic inputs from the brainstem including the basal forebrain cholinergic projection and, in some cases, the locus ceruleus noradrenergic pathway TM but also the selective loss of certain neurons intrinsic to the cerebral cortex. This observation raises intriguing questions about the properties shared in common by these three neurochemicaUy and neuroanatomically dissimilar systems that render them particularly vulnerable to the neurodegenerative process responsible for AD/SDAT. This work was supported in part by N I H Grants A G 01066, A G / N S 02478, NS 13584, M H 26654 and M H 00125 and by the McKnight Foundation. We thank Carol Kenyon and Aurora Thompson for technical assistance.
from cases of Alzheimer disease and Alzheimer senile dementia, Nature (Lond.), 288 (1980) 279-280. 5 Davies, P. and Maloney, A. J. F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, II (1976) 1403. 6 Davies, P. and Terry, R. D., Cortical somatostatin-like immunoreactivity in cases of Alzheimer's disease and senile dementia of the Alzheimer type, NeurobioL Aging, 2 (1981) 9-14. 7 Emson, P. C. and LindvaU, P., Distribution of putative neurotransmitters in the neocortex, Neuroscience, 4 (1979) 1-30.
8 Jones, E. G., Burton, H., Saper, C. B. and Swanson, L.
172
9
10
11 12
13 14 15
16
W., Midbrain diencephalic and cortical relationships of the basal nucleus of Meynert and associated structures in primates, J. comp. NeuroL, 167 (1976) 385-420. Johnston, M. V., McKinney, M. and Coyle, J. T., Evidence for a cholinergic projection to neocortex from neurons in basal forebrain, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5392-5396. Johnston, M. V., McKinney, M. and Coyle, J. T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Brain Res., 43 (1981) 159-172. Kelly, P. H. and Moore, K. E., Decrease of neocortical choline acetyltransferase after lesion of the globus pallidus in the rat, Exper. NeuroL, 61 (1978) 479-484. HSkfelt, T., Elde, R., Johansson, O., Ljungdahl, A., Schultzberg, M., Fuxe, K., Goldstein, M., Nilsson, G., Pernow, B., Terenius, L., Ganten, D., Jeffcoate, S. L., Rehfeld, H. and Said, S., Distribution of peptide-containing neurons. In M. A. Lipton, A. DiMascio and K. F. Killam (Eds.), Psychopharmacology: a Generation of Progress, Raven Press, New York, 1978, pp. 39-66. H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J. M. and Schultzberg, M., Peptidergic neurones, Nature (Lond.), 284 (1980) 515-521. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. S., Protein measurement with the Folin phenol method, J. biol. Chem., 193 (1951) 265-275. Lehman, J., Nagy, J. I., Atmadja, S. and Fibiger, H. C., The nucleus basalis magnocellularis. The origin of a cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. Mesulam, M.-M. and Van Hoesen, G. W., Acetylcholinesterase-rich projections from the basal forebrain of the rhesus monkey to neocortex, Brain Research, 109 (1976) 152-157.
17 Perry, E. K., Gibson, P. H., Blessed, G., Perry, R. H. and Tomlinson, B. E., Neurotransmitter enzyme abnormalities in senile dementia. Choline acetyltransferase and glutamic acid decarboxylase activities in necropsy brain tissue, J. Neurol. Sci., 34 (1977) 247-265. 18 Perry, E. K., Tomlinson, B. E., Blessed, G., Perry, R. H., Cross, A. J. and Crow, T. J., Neuropathological and biochemical observations on the noradrenergic system in Alzheimer's Disease, J. NeuroL Sci., 51 (1981) 279-287. 19 Perry, R. H., Dockray, G. J., Dimaline, R., Perry, E. K., Blessed, G. and Tomlinson, B. E., Neuropeptides in Alzheimer's Disease, depression and schizophrenia: a post mortem analysis of vasoactive intestinal peptide and cholecystokinin in cerebral cortex, J. Neurol. Sci., 51 (1981) 465-472. 20 Rossor, M. N., Emson, P. C., Mountjoy, C. Q., R.oth, M., and lversen, L. L., Reduced amounts of immunoreactive somatostatin in the temporal cortex in senile dementia of Alzheimer type, Neurosci. Lett., 20 (1980) 373-377. 21 Rossor, M., Fahrenkrug, J., Emson, P., Mountjoy, C., Iversen, L. and Roth, M., Reduced cortical choline acetyltransferase activity in senile dementia of Alzheimer type is not accompanied by changes in vasoactive intestinal polypeptide, Brain Research, 201 (1980) 249-253. 22 Whitehouse, P. J., Price, D. L., Clark, A. W., Coyle, J. T. and DeLong, M. R., Alzheimer's disease: evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol., 10 (1981) 122-126. 23 Wilson, S. H., Schrier, B. K., Farber, J. L., Thompson, E. J., Rosenberg, R. W., Blume, A. J. and Nirenberg, M. W., J. biol. Chem., 147 (1972) 3159-3169. 24 Zaczek, R., Hedeen, J. C. and Coyle, J. T., Evidence for a hippocampal-septal glutaminergic pathway in the rat, J. Exp. NeuroL, 65 (1979) 145-156.
169
Somatostatin is not co-localized in cholinergic neurons innervating the rat cerebral cortex-hippocampal formation MICHAEL McKINNEY, PETER DAVIES and JOSEPH T. COYLE* ( M.M. and J.T.C.) Departments of Neuroscience, Psychiatry and the Behavioral Sciences, and Pharmacology and Experimental Therapeutics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 and (P.D.) Departments of Pathology and Neuroscience, Albert Einstein College of Medicine, The Bronx, N Y 10461 (U.S.A.)
(Accepted March 18th, 1982) Key words: somatostatin - - choline acetyltransferase - - rat brain - - lesions - - excitotoxins
The somatostatin content and choline acetyltransferase activity of the rat cerebral cortex and hippocampus were examined after lesions to the nucleus basalis, the fornix and the dorsal hippocampus. Lesions of the nucleus basalis caused reductions in cortical choline acetyltransfera~e activity but had no effect on the concentration of somatostatin. Fornix transection caused a reduction in choline acetyltransferase activity in the dorsal hippocampus, but again was without effect on the somatostatin content. Excitotoxin lesions of the dorsal hippocampus caused a 69 ~o reduction in somatostatin concentration in this structure, with no reduction in choline acetyltransferase activity. The results indicate that somatostatin and choline acetyltransferase are in separate populations of neurons in the rat cerebral cortex and hippocampus. Considerable evidence has accrued that Alzheimer's Disease and senile dementia of the Alzheimer's type (AD/SDAT) are associated with profound and relatively selective reductions in the presynaptic markers for cholinergic neurons in the cerebral cortex and hippocampal formation1, 3,5,17. A major source of cholinergic innervation to the telencephalon are the large acetylcholinesterase positive neurons contained in the nucleus basalis of the basal forebrain which extends anteriorly to include the diagonal band of Broca and the medial septal nucleusa-11,16. Excitotoxin lesions of this complex in rats result in marked and selective reductions in the presynaptic cholinergic markers, depending upon the localization of the lesion within the complex, in the cerebral cortex and hippocampal formationg, ~s. Neuropathologic studies have now demonstrated that the nucleus basalis undergoes degeneration in A D / S D A T , thus providing histopathologic basis for the reduction in cortical cholinergic markers in this disorder 22. Recently, two laboratories independently repor-
ted that the concentration of somatostatin is markedly reduced in the cortex and hippocampus of patients dying with AD/SDAT4,8, 20. Since other neuropeptides such as cholecystokinin and vasoactive intestinal peptide are not consistently decreased in these areas 19,21, this reduction in somatostatin appears to be relatively selective and parallels, to some extent, the decrement in cortical cholinergic markers. Although immunocytochemical studies have demonstrated somatostatin-containing perikarya in the cerebral cortex 7,12, it is unclear at present whether these intrinsic neurons are the major or exclusive source of cortical somatostatin innervation. In the light of the growing evidence of coexistence of neuropeptides in aminergic neurons 13, it seemed possible that the parallel reductions in cholinergic markers and somatostatin in A D / S D A T might result from their co-localization in the basal forebrain neurons that project to the cerebral cortex. To evaluate this hypothesis, we have examined the effects of excitotoxin and ablative lesions of the basal forebrain projections to the rat cerebral cortex
* To whom all correspondence should be addressed at: Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
170 and of the hippocampal formation on the activity of choline acetyltransferase, a specific marker for the cholinergic neurons, and on the levels of somatostatin. Adult male Sprague-Dawley rats, (180-200 g) purchased from Harlan-Sprague-Dawley, were used for the experiments. To lesion the nucleus basalis in the ventral globus pallidus, the rats were anesthetized with Equithesin and placed in a David K o p f stereotaxic apparatus; through a burr hole placed in the calvarium, a Hamilton cannula (0.3 m m in diameter) was positioned in the ventral globus pallidus (P, 0.3 m m ; V, 7.2 m m ; L, 3.0 m m with the intra-auricular line, the midline and pial surface equal to zero and with the incisor bar at 0 ° to the ear bars) and 15 /tg of ibotenic acid (Regis Chemicals) dissolved in 1 #1 of artificial CSF was infused over a period of 2 min 10. To lesion neurons intrinsic to the hippocampal formation, 1.0/tg of kainic acid (Sigma Chemicals, St. Louis, MO; lot 105C4479) was infused into the dorsal hippocampal formation at the following coordinates: P, 2.3 m m ; V, 3.0 m m ; L, 2.0 mm. In some animals, the fornix was transected with a knife cut according to the procedure of Zaczek et al. 24. One week after surgery or excitotoxin lesion, the animals were sacrificed by decapitation and the cerebral cortex and hippocampus ipsilateral and contralateral to the lesion was dissected at 5 °C. The cortical sample consisted of the fronto-parietal cortex superior to the rhinal fissure and medial to the cingulate; in the case of the hippocampal kainate lesion, only the dorsal hippocampus was used. A 10 mg portion of tissue was dissected from the cortex and the dorsal hippocampal formation and immediately frozen upon dry ice for measurement of somatostatin levels. For the somatostatin assay, the frozen tissue was dropped into boiling 2 M acetic acid, and left for 10 min. The mixture was homogenized and an aliquot was removed for protein determination by the method of Lowry et a!.14. After centrifugation at 15,000 g for 30 min, aliquots of the supernatant were freeze-dried and used for somatostatin assay as previously described 4,6. The remaining tissue was immediately homogenized in 50 m M Tris-HC1, p H 7.4, containing 0.2 ~ Triton X-100; and after centrifugation, the supernatant was assayed for the activity of choline acetyltransferase by the method of
Bull and Oderfeld-Nowak 2 and the activity of glutamate decarboxylase by the method of Wilson et al. 23. Protein content was determined by the method of Lowry et al. 14, with bovine serum albumin as standard. Ibotenate lesion of the ventral globus pallidus caused a 6 4 ~ reduction in the specific activity of choline acetyltransferase in the ipsilateral cerebral cortex but did not significantly affect the activity of the enzyme in the hippocampal formation (Table I). In contrast, the concentration of somatostatin was not altered either in the neocortex or in the hippocampal formation. This result indicated that somatostatin was not localized in the cholinergic neuronal perikarya in the ventral globus pallidus that provide innervation to the fronto-parietal cortex but did not rule out the possibility that hippocampal somatostatin might be primarily localized in extrinsic neurons. To evaluate this latter possibility, we examined the effects of transection of the fornix, the primary route of entry of afferent input to the hippocampal formation. Prior transection of the fornix
TABLE 1 Effects o f lesions o f the ventral pallidum or fornix on somatostatin levels
The ventral globus pallidus was lesioned with 15 ~g of ibotenate or the fornix was transected as described in the text. Seven days after lesion, the rats were sacrificed; and specific activity of choline acetyltransferase and the content of somatostatin were measured in the same samples. The results are expressed as the mean ± S.E.M. of the number of animals indicated in parenthesis. Choline acetyltransferase (nrnol/ mg protein/h )
Somatostatin (ng/mgprotein)
(A) Ventral GP lesion Cortex Lesion (5) 13.7 ± 0.8* Control (7) 38.5 ± 4.0 Hippocampus Lesion (5) 40.2 ± 1.6 Control (7) 42.8 ± 1.7
3.15 ~ 0.54 3.24 ± 0.4
(B) Fornix transection Hippocampus Lesion (6) 14.5 ± 1.3" Control (5) 39.2 ± 1.5
3.92 i 0.34 3.57 ± 0.39
3.86 ± 0.38 3.77 ± 0.64
* P < 0.001 versus control by Student's t-test.
171 TABLE II Effects of hippocampal kainate lesion on somatostatin levels
Ether anesthetized rats received a 1.0 jug injection of kainic acid in the dorsal hippocampus one week prior to sacrifice. The specificactivities of glutamate decarboxylaseand choline acetyltransferase and the content of somatostatin were measured in the same dorsal hippocampal samples. The results are expressed as the mean i S.E.M. of 7 lesioned and control preparations. Control
Lesion
Glutamate decarboxylase (nmol/mgprotein/h)
23.6 ± 1.6
Choline acetyltransferase (nmol/mgprotein/h)
38.7 4- 1.2
35.5 4- 1.3
Somatostatin (ng/mg protein)
4.14 ± 0.36
1.27 ± 0.12"
6.3 4- 1.0"
* P < 0.001 versus control by Student's t-test.
caused a 63 ~ reduction in the specific activity of choline acetyltransferase in the ipsilateral hippocampal formation but did not significantly affect the concentration of somatostatin. To establish that somatostatin was indeed associated with neurons intrinsic to the hippocampal formation, we examined the effects of kainic acid lesion of the dorsal hippocampal formation, which causes a selective degeneration of neurons with cell bodies in the region 1°. The kainate lesion resulted in a profound reduction in the specific activity of glutamate decarboxylase and a comparable 69 ~ reduction in the concentration of somatostatin in the dorsal hippocampus (Table II); the selectivity of the lesion was confirmed by the finding that the specific activity of choline acetyltransferase in the dorsal hippocampus
1 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) 459-495. 2 Bull, G. and Oderfeld-Nowak, B., Standardization of a radiochemical assay of choline acetyltransferase and a study of the activation of the enzyme in rabbit brain, J. Neurochem., 18 (1971) 935-947. 3 Davies, P., Neurotransmitter-related enzymes in senile dementia of the Alzheimer type, Brain Research, 171 (1979) 319-327. 4 Davies, P., Katzman, R. and Terry, R. D., Reduced somatostatin-like immunoreactivity in cerebral cortex
was not significantly reduced. These studies provide compelling evidence that somatostatin is localized in a distinctly different population of neurons than those that serve as the major source of cholinergic innervation to the cortex and hippocampal formation. The results from transection of the fornix and kainate lesion of hippocampal intrinsic neurons indicate that somatostatin is contained in neurons intrinsic to the hippocampal formation. Thus, it would appear that the bipolar neurons in the cerebral cortex and hippocampal formation, that have been shown to contain somatostatin by immunocytochemical methods, are the primary, if not the exclusive, source of somatostatin innervation of the cortex and hippocampus. These findings suggest that the decrements in somatostatin levels in the cerebral cortex and hippocampal formation in A D / S D A T reflect degeneration of intrinsic neurons. Thus, the pathology of AD/ SDAT involves not only the degeneration of the aminergic inputs from the brainstem including the basal forebrain cholinergic projection and, in some cases, the locus ceruleus noradrenergic pathway TM but also the selective loss of certain neurons intrinsic to the cerebral cortex. This observation raises intriguing questions about the properties shared in common by these three neurochemicaUy and neuroanatomically dissimilar systems that render them particularly vulnerable to the neurodegenerative process responsible for AD/SDAT. This work was supported in part by N I H Grants A G 01066, A G / N S 02478, NS 13584, M H 26654 and M H 00125 and by the McKnight Foundation. We thank Carol Kenyon and Aurora Thompson for technical assistance.
from cases of Alzheimer disease and Alzheimer senile dementia, Nature (Lond.), 288 (1980) 279-280. 5 Davies, P. and Maloney, A. J. F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, II (1976) 1403. 6 Davies, P. and Terry, R. D., Cortical somatostatin-like immunoreactivity in cases of Alzheimer's disease and senile dementia of the Alzheimer type, NeurobioL Aging, 2 (1981) 9-14. 7 Emson, P. C. and LindvaU, P., Distribution of putative neurotransmitters in the neocortex, Neuroscience, 4 (1979) 1-30.
8 Jones, E. G., Burton, H., Saper, C. B. and Swanson, L.
172
9
10
11 12
13 14 15
16
W., Midbrain diencephalic and cortical relationships of the basal nucleus of Meynert and associated structures in primates, J. comp. NeuroL, 167 (1976) 385-420. Johnston, M. V., McKinney, M. and Coyle, J. T., Evidence for a cholinergic projection to neocortex from neurons in basal forebrain, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5392-5396. Johnston, M. V., McKinney, M. and Coyle, J. T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Brain Res., 43 (1981) 159-172. Kelly, P. H. and Moore, K. E., Decrease of neocortical choline acetyltransferase after lesion of the globus pallidus in the rat, Exper. NeuroL, 61 (1978) 479-484. HSkfelt, T., Elde, R., Johansson, O., Ljungdahl, A., Schultzberg, M., Fuxe, K., Goldstein, M., Nilsson, G., Pernow, B., Terenius, L., Ganten, D., Jeffcoate, S. L., Rehfeld, H. and Said, S., Distribution of peptide-containing neurons. In M. A. Lipton, A. DiMascio and K. F. Killam (Eds.), Psychopharmacology: a Generation of Progress, Raven Press, New York, 1978, pp. 39-66. H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J. M. and Schultzberg, M., Peptidergic neurones, Nature (Lond.), 284 (1980) 515-521. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. S., Protein measurement with the Folin phenol method, J. biol. Chem., 193 (1951) 265-275. Lehman, J., Nagy, J. I., Atmadja, S. and Fibiger, H. C., The nucleus basalis magnocellularis. The origin of a cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. Mesulam, M.-M. and Van Hoesen, G. W., Acetylcholinesterase-rich projections from the basal forebrain of the rhesus monkey to neocortex, Brain Research, 109 (1976) 152-157.
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