Somatostatin-like immunoreactivity within neuritic plaques

Brain Research, 338 (1985) 71-79 Elsevier

71

BRE 10831

Somatostatin-Like Immunoreactivity Within Neuritic Plaques DAVID M. ARMSTRONG 1, SHERYL LeROY I, DENNIS SHIELDS 2 and ROBERT D. TERRY 1

IDepartment of Pathology and 2Department of Anatorny, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) (Accepted October 2nd, 1984)

Key words: Alzheimer's disease - - somatostatin - - neuritic plaques - - immunocytochemistry - - human - - autopsy

Alzheimer's disease or senile dementia of the Alzheimer type (SDAT) is a progressive neurodegenerative disease that is characterized pathologically by two types of microscopic lesions in the neocortex: the neurofibrillary tangle and neuritic plaque. The concentration of neuritic plaques is correlated with significant reductions in the level of specific neurotransmitter and neuropeptide systems in autopsied brains of patients with SDAT, including decreased amounts of the tetradecapeptide, somatostatin. The clinical effects of reduced cortical somatostatin activity in patients with SDAT is unclear, nor is it known whether somatostatinergic neurons participate in either lesion. In the present study we employed ligth microscopic immunocytochemistry to determine whether somatostatin-containing neurons participate in the formation of neuritic plaques. Examination of selected cortical regions from autopsied brains revealed 20-50% of all neuritic plaques contained somatostatin-positive profiles indicating that processes of somatostatinergic neurons are associated with neuritic plaque formation.

INTRODUCTION Alzheimer's disease or senile d e m e n t i a of the Alzheimer type ( S D A T ) is a progressive n e u r o d e g e n e rative disease affecting about 7 or 8% of the population aged 65 years or over 41. Patients with S D A T are characterized clinically by marked deficits in their intellectual functions and pathologically by two types of microscopic lesions within their cerebral cortex: the neuritic plaque and neurofibrillary tangle. The present study investigates the neuritic plaque and its relationship to chemically defined n e u r o n a l elements. Within the neocortex of patients with S D A T three types of neuritic plaques are recognized with the light microscope 42. The first type, or primitive plaque, is made up of degenerating neuritic processes with a few reactive cells. These neurites have been identified as both axons and dendrites 42. The second or classical type of plaque is similar to the first with respect to neurites, but has a central amyloid core which may vary in size. The third or end stage plaque consists of a central core of amyloid, surrounded by few or no neuritic processes. The neurite is believed

to represent the nidus of plaque formation. It has been suggested that the degeneration of these neuronal processes results in the attraction of reactive cells and subsequently the deposition of amyloid 46. Performance on mental status tests in patients with S D A T correlates inversely with the n u m b e r of neuritic plaques in the neocortex6 and positively with decreases in specific neurotransmitter content 31. Reductions in various cholinergic indices from neocortical regions of brains with S D A T remain the most dramatic and consistent neurochemical abnormality in S D A T 7,13,15,3°.3<36,44. Decreased cholinergic activity is thought to result from a degeneration of cholinergic cortical afferents from the basal forebrain m22.24,25,27,28,37,45. In addition, the results of a recent acetylcholinesterase histochemical study suggest that dystrophic cholinergic processes participate in the formation of neuritic plaques ~0. Significant but less dramatic reductions have also been reported for the neuropeptides, substance p~2 and somatostatin 1<2935. It is not known, however, whether these peptide-containing systems are also involved in the formation of plaques. In the present study we have examined autopsied specimens from the neocortex of

Correspondence: D. M. Armstrong, Department of Neurosciences M-024, School of Medicine. University of California at San Diego, CA. U.S.A. 01106-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)

"7,')

patients with S D A T using light microscopic inmmnocytochemistry to d e t e r m i n e if somatostatin-positive processes participate in the formation of neuritic plaques. MATERIALS AND METHODS

Preparation of antiserum. Synthetic somatostatin14 was o b t a i n e d from Boehringer M a n n h e i m Biochemicals and used to p r e p a r e an antiserum in rabbits~. The antiserum, designated RSS-1, recognizes both native somatostatin as well as the somatostatin precursors, p r o s o m a t o s t a t i n and p r e p r o s o m a t o s t a tin 43. Thus the term somatostatin-like i m m u n o r e a c tivity is used to describe the localization of the somatostatin antiserum. R S S - I , however, does not recognize any of the o t h e r pancreatic hormones. lmmunocytochemistry A u t o p s i e d material was selected from i2 patients with S D A T , aged 64 - 96 years (mean = 78 years of age), with p o s t - m o r t e m delays ranging from 4 to 18 h (mean = 9 h). The patients were chosen because each displayed varying concentrations of neuritic plaques within selected brain regions and was without confounding neurological or neuropathological disorders. A n additional group of patients (n = 11) with little or no evidence of neuritic plaques, aged 54 89 years (mean = 66 years of age), with post-mortem delays between 3 and 26 h (mean = 11 h) was used as control patients and a means to d e t e r m i n e acceptable p o s t - m o r t e m delays and optimal fixation schedules. Tissue was collected from the pre-central ( m o t o r ) , superior temporal, mid-frontal gyri, superior parietal lobule, hippocampus and amygdala of the right hemisphere and fixed in Bouin's solution for 48 h. The tissue was sectioned on a vibrating m i c r o t o m e -

( V i b r a t o m e ) to a thickness of 50 ktm. The Vibratome sections were transfered to spot test plates (Fisher) containing 0.1 M PO4-buffer in preparation toJ the immunocytochemieal, peroxidase anti--peroxidase, labeling p r o c e d u r e -w. This labeling p r o c e d u r e consists of the sequential incubation of the tissue sections with: (1) 0.3% H : O 2 in0.1 M Tris-saline (30 rain); (2) 1:1000 dilution of anti-somatostatin antibody or control sera (overnight). Control serum included either p r e i m m u n e serum (i.e. normal rabbit serum collected prior to immunization) or antiserum previously absorbed with 50,ug somatostatin per (), 1 ml undiluted antiserum (Fig. 1E and F). Dilutions of the primary antibody were p r e p a r e d with 1~?c goat serum in 0.1 M Tris-saline containing 0.25% Triton X-100. (3) G o a t anti-rabbit IgG (Cappel) diluted 1:400 with 1% goat serum in Tris-saline (1 h); (4) rabbit peroxidase- a n t i p e r o x i d a s e complex (Cappel) diluted 1:4(]0 with 1% goat serum in Tris-saline (1 h); and (5) 0.05% solution of 3.3'-diaminobenzidine and 0.01 (~i H?O 2 (6 rain) to yield a brown reaction product, Sections were m o u n t e d on gelatin-coated glass slides, oven dried at 60 °C for 30 rain, and then counterstained with Thioflavine-S to visualize neuritic plaques. This staining p r o c e d u r e consists of: (i) 7-rain incubation in 1% aqueous Thioflavine-S; (ii) differentiation in 3 changes of 80~)'~ ethanol; (iii) dehydration through graded ethanol; and (iv) covering with glass coverslips using f l u o r o m o u n t mountant ( B D H Chemicals). Using a fluorescent microscope in combination with standard bright-field or Nomarski optics, we were able to observe the yellow/green fluorescent neuritic plaques and the brown peroxidaselabeled somatostatin profiles m a single section. The number of plaques per tissue section and the percentage of these plaques that were associated with somatostatin-positive profiles were each d e t e r m i n e d by two investigators.

Fig. 1. Somatostatin-like immunoreactivity from selected brain regions of patients with senile dementia of the Alzheimer's type (C and D) and of non-demented control patients (A and B). A: photomicrograph showing somatostatin-like immunoreactivity localized to a beaded varicose process within the parietal cortex of a non-demented patient. B: photomicrograph showing somatostatin-labeled neuronal perikarya within the amygdala of a non-demented patient. C: photomicrograph shows a distended somatostatin-containing profile (arrow) located at one end of a somewhat normal appearing varicose process. The tissue section is from the parietal cortex of a patient with SDAT. D: photomicrograph shows an aggregation of somatostatin-like immunoreactivity within the parietal cortex of a patient with SDAT. Although this profile is plaque-like in appearance it is not associated with fluorescent neurites or amyloid which are characteristic of 'true' plaques. Specificity of the somatostatin labeling procedure is determined by the absence of the immunocytochemical reaction in sections in which the primary antibody is substituted with preimmune serum (El or blocked antiserum to the neuropeptide (F). Both E and F are from the parietal cortex of a patient with SDAT. Bar = 5(11~m,

0

4 J

C

0

/

',,aO

74

75 RESULTS Within the autopsied specimens from patients without pathologic evidence of S D A T , somatostatinlike immunoreactivity was localized to b e a d e d varicose processes (Fig. 1A) and to a lesser extent within neuronal perikarya and proximal processes (Fig. 1B). This labeling p a t t e r n was evident in all specimens regardless of p o s t - m o r t e m intervals, although the peroxidase reaction p r o d u c t a p p e a r e d to be m o r e intense in material with relatively short (under 9 h) autopsy delays. In similar regions of brain from patients with S D A T , somatostatin-like immunoreactivity was also localized to a few neuronal perikarya but mostly to b e a d e d varicose processes. To examine the association of somatostatin-like immunoreactivity with neuritic plaques, tissues were first viewed for Thioflavine-S reactivity. The Thioflavine-S stain permits detection of both amyloid and neuritic processes and allows for the classification of neuritic plaques into their respective types: primitive, classical, or end stage. Somatostatin-positive processes were observed with m o r e or less equal frequency within all three types of plaques. While the concentration of neuritic plaques varies from specimen to specimen, the percentage of these lesions that were associated with somatostatinergic profiles remained constant. Within the neocortex and hippocampus a p p r o x i m a t e l y 20% of the plaques contained somatostatin. This percentage was nearly 50% in the amygdala, but many more somatostatin-positive processes were observed in this region than in the previous areas. The concentration of somatostatin within the plaques was further characterized as severe, intermediate, or mild (Fig. 2A, C and E). The numbers of plaques that contained i n t e r m e d i a t e (Fig. 2D) or mild (Fig. 2F) concentrations of somatostatin were a p p r o x i m a t e l y equal and accounted for over 90% of all s o m a t o s t a t i n - p l a q u e associations.

Less than 10% of all plaques that were associated with somatostatin contained dense concentrations of this n e u r o p e p t i d e . The somatostatin-positive processes typically app e a r e d swollen or distended and often f o r m e d grapelike clusters (Fig. 2A) either within the p e r i p h e r a l (i.e. neuritic) portion of a primitive or classical plaque or i m m e d i a t e l y adjacent to the amyloid core of an end stage plaque. Within these same tissue sections swollen somatostatin-positive processes were also o b s e r v e d which were not associated with neuritic plaques. These swollen processes a p p e a r e d either alone as part of a varicose process (Fig. 1C), or less frequently f o r m e d dense aggregates of somatostatin immunoreactivity (Fig. 1D). Swollen neuritic processes were also seen in control patients but not with the frequency found in brains of patients with S D A T . A u t o p s i e d specimens from elderly control patients also contained a few neuritic plaques. T h e y too were associated with somatostatinergic profiles and app e a r e d similar to the s o m a t o s t a t i n - p l a q u e associations seen in patients with S D A T . Somatostatin-containing neuronal p e r i k a r y a were o b s e r v e d in brains of patients with S D A T and in normal controls. The n u m b e r of somatostatin-containing neurons, however, were too few to assess accurately whether a loss of somatostatin-positive neurons occurs in S D A T c o m p a r e d to aged m a t c h e d controls. H o w e v e r , it is our impression that there is little if any decrease in the n u m b e r of somatostatinergic neurons in S D A T . DISCUSSION The present study is the first d e m o n s t r a t i o n of somatostatin-positive processes within neuritic plaques and provides a morphologic correlate to the biochemical finding that somatosatin levels are r e d u c e d

Fig. 2. Three examples of somatostatin-like immunoreactivity within neuritic plaques. Photomicrographs A, C and E show the peroxidase-labeled somatostatin neurites as seen with bright-field (Nomarski) optics. Photomicrographs B, D and F show the corresponding fluorescent images. Photomicrographs A and B, and C and D, and E and F, respectively, characterize dense, intermediate, and mild concentrations of somatostatin within plaques. The white arrows in photomicrographs B, D and F correspond in location to the black arrows in A, C and E. Notice the distended, swollen appearance of the somatostatin-labeled neurites. In photomicrograph A these labeled neurites form a grape-like cluster. The somatostatin-labeled profiles are found within the periphery of neuritic plaques (see photomicrographs B, D and F). The peripheral portion of the plaque consists mostly of neurites and is seen in photomicrographs B, D and F as the light and wispy fluorescent material in contrast to the dense, deeply fluorescent amyloid core. All three plaques are characteristic of intermediate or classical plaques. Bar - 50 #m.

'76 in the neocortex of patients with SDAT. Our results are in agreement with those from earlier immunocytochemical studies that have shown the presence of somatostatin-positive neuronal perikarya and varicose processes in the cerebral cort e x 5,18-20,23,26,32,3s and with radioimmunological studies that report considerable quantities of somatostatin in the cerebral cortex s,H. In addition, the presence of somatostatin-like immunoreactivity within autopsy material with post-mortem delays up to 26 h is in agreement with biochemical studies that report that somatostatin is not readily susceptible to post mortern degradation 9,~6. The detection of relatively few somatostatin-positive perikarya in the cortex of human autopsy specimens is consistent with the finding of relatively low peptide levels in the perikarya of non-colchicinetreated animals in most regions of the central nervous system3.19-21. The relative paucity of labeled perikarya may also reflect technical limitations such as accessibility of the antibody to the neuropeptide in cell bodies. This possibility is supported by earlier immunocytochemical studies that report widespread distribution of somatostatin-positive cell bodies following proteolytic treatment of the tissue sections with pronase 2°,21. Alternatively, previous immunocytochemical studies have reported the preferential labeling of neuronal processes and terminals or of cell bodies, depending upon the molecular species of the somatostatin being localized 23,26. The RSS-1 antibody employed in the present study recognizes in vitro both native somatostatin as well as the somatostatin precursors, prosomatostatin and preprosomatostatin 4-~. However, the capacity of this antibody to recognize these somatostatin-related peptides may vary, and in turn affect its ability to label terminals, processes, and perikarya with equal intensity. The relatively low concentration of somatostatinlabeled perikarya within cortical and subcortical regions limits our ability to assess quantitatively whether there is a loss in the number of somatostatinergic cell bodies in the brains of patients with SDAT compared with age-matched controls. However, our impression that there is not a significant decrease in the number of somatostatin-positive neurons in patients with SDAT is in agreement with the results of a recent immunocytochemical study by S. Vincent (personal communication) who likewise did not observe

SDAT-related decreases in the number of somatostatinergic neurons. In the present study, somatostatin-like immunore-. activity is preferentially localized to beaded varicose processes. Our previous light and electron microscopic immunocytochemical studies of neuropeptides in the rat brain would suggest that most of these somatostatin-labeled processes were axons :,a. The authors of other immunocytochemical studies, however, have observed that similar appearing varicose swellings may belong to dendrites as well as to axons 17. Additional electron microscopic immunocytochemical studies are luther needed to identify these labeled processes. However, the localization of somatostatin at least in part within axon terminals provides further evidence for the hypothesis that somatostatin functions as a neurotransmitter in certain parts of the nervous system. In the present study neuritic plaques were readily observed in tissue sections incubated with Thioflavine-S. This stain permits visualization of both dystrophic neurites and amyloid. The arrangement of these two components of the neuritic plaque determines its type: primitive, classical or end stage. In addition, Thioflavine-S in combination with the immunocytochemical localization of somatostatin permits visualization of both neuritic plaques and neuropeptidergic processes in a single tissue section, Somatostatin-positive processes were associated more or less equally with all three types of plaques. The labeled profiles were typically swollen and often formed grape-like clusters within the peripheral (i.e. neuritic) region of the plaque. These results are in agreement with the results of a recent Golgi analysis of neuritic plaques in the hippocampus of patients with SDAT in which similar appearing bulbous swellings were described in plaques33. Many of these impregnated profiles were identified as either axons or dendrites and could be traced to nearby pyramidal and non-pyramidal neuron types. The present study provides evidence that some of these local neurons that participate in the formation of neuritic plaques contain somatostatin. However, it is possible that some of these somatostatin-positive dystrophic neurites could originate from neurons lying in structures further away. For example, dystrophic cholinergic neurites are observed within neuritic plaques and originate for the most part from neurons iying in the

77 basal forebrain 40.

of distended somatostatin-containing profiles in the

In the present study approximately 20% of all the plaques that we observed in the neocortex were associated with somatostatin-positive processes. Our methods, however, may lead to an underestimation

neocortex of autopsy specimens from n o n - d e m e n t e d patients, suggest that many of these labeled profiles

of the total n u m b e r of plaques that were associated with this neuropeptide since we did not examine thin

electron microscopic studies of h u m a n biopsy material from patients with S D A T report many distended

represent non-pathologic sites of interaction of somatostatinergic neurons with other cells. Alternatively,

serial sections and because the peroxidase reaction

processes throughout the neuropi142. These processes

product is largely restricted to the surface of the tis-

contained n u m e r o u s mitochondria and might repre-

sue, due to incomplete penetration of the antiserum into the thick Vibratome section. In contrast to the somewhat low n u m b e r of s o m a t o s t a t i n - p l a q u e asso-

sent the earliest stage of plaque formation. This

ciations that were observed in the neocortex, nearly one-half of all plaques in the amygdala contained somatostatin-like immunoreactivity. These data are consistent with our findings of n u m e r o u s somatostatin-positive processes in the amygdala and with radioimmunological studies that report levels of somatostatin in the amygdala 4 - 6 times those of the neocortex s, 11.

would indicate that the swollen somatostatin-positive processes observed in the present study, in addition to representing sites of cellular interaction, may also represent pathologic structures that are part of the early evolution of neuritic plaques. The present study provides evidence that swollen somatostatin-positive processes, probably originat-

In addition to the many associations that were ob-

ing from local cortical neurons, are associated with neuritic plaques. Our data, together with the biochemical finding that somatostatin levels are reduced in the neocortex of patients with S D A T , support the

served between somatostatin and plaques, n u m e r o u s swollen somatostatin-containing processes were also

hypothesis that a dysfunctioning of the somatostatinergic neuropeptide system is an important element

observed in the absence of plaques. These labeled profiles were seen either as part of a varicose process, or less frequently as part of a dense aggregate of somatostatin-like immunoreactivity. Our data are consistent with the results of a previous immunocytochemical study in which similar swellings were ob-

to the development of neuritic plaques.

served at sites where somatostatin-positive processes come into close contact with other n o n - i m m u n o r e a c rive cells 3s. These results, together with our findings

and AM-21860. D.S. holds an Irma T. Hirschl Award and is the recipient of Research Career D e v e l o p m e n t Award 1 K04 A M 01208.

REFERENCES 1 Arimura, A., Sato, H., Coy, D. H. and Schally, A. V., Radioimmunoassay for GH-release inhibiting hormone, Proc. Soc. exp. Biol. Med., 148 (1975) 784-789. 2 Armstrong, D. M., Beaudet, A., Miller, R. J. and Pickel, V. M., Immunocytochemical study of the ultrastructural morphology and capacity of enkephalin-containing neurons to take up [3H]serotonin, Brain Research, 310 (1984) 269-278. 3 Armstrong, D. M., Pickel, V. M., Joh. T. H., Reis, D. J. and Miller, R. J., Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius of rat brain, J. comp, Neurol., 196 (1981) 505-517. 4 Armstrong, D. M., Pickel, V. M. and Reis, D. J., Electron microscopic immunocytochemical localization of substance P in the area postrema of rat, Brain Research, 243 (1982) 141-146.

ACKNOWLEDGEMENTS This work was supported in part by a grant from the McKnight F o u n d a t i o n and NIH grants AG-02478

5 Bennett-Clarke, C.. Romagnano, M. A. and Joseph, S. A., Distribution of somatostatin in the rat brain: Telencephalon and diencephalon, Brain Research, 188 (1980) 473-486. 6 Blessed, G., Tomlinson, B. E. and Roth, M., The association between quantitative measures of dementia and of senile changes in the cerebral grey matter of elderly subjects, Brit. J. Psychiat., 114 (1968) 797-811. 7 Bowen, D. M., Smith, C. B., White, P. and Davison, A. N., Neurotransminer-related enzymes and indices of hypoxia in senile dementia and other abiotrophics, Brain, 99 (1976) 459-495. 8 Brownstein, M.. Arimura, A., Sato, H., Schally, A. V. and Kizer, J. S., The regional distribution of somatostatin in the rat brain, Endocrinology, 96 (1975) 1456-1461. 9 Buck, S. H., Deshmukh, P. P., Burks, T. F. and Yamamura, H. I., A survery of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system, Neurobiol. A~in~, 2 (1981) 257-264.

78 10 Candy, J. M., Perry, R. H., Perry, E. K., Irving, D., Blessed, G., Fairbairn, A. F. and Tomlinson, B. E., Pathological changes in the nucleus of Meynert in Alzheimer's and Parkinson's diseases, J. Neurol. Sci.. 54 (1983) 277-289. l I Cooper, P. E., Fernstrom, M. H., Rorstad, O. P., Leeman, S. E. and Martin, J. B., The regional distribution of somatostatin, substance P and neurotensin in human brain, Brain Research, 218 (19811 219-232. 12 Crystal, H. A. and Davies, P., Cortical substance P-like immunoreactivity in cases of Alzheimer's disease and senile dementia of the Alzheimer type, J. Neurochem., 38 (1982) 1781-1784. 13 Davies, P., Neurotransmitter-related enzymes in senile dementia of the Alzheimer type, Brain Research, 171 (1979) 319-327. 14 Davies, P., Katzman, R. and Terry, R. D., Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer disease and Alzheimer senile dementia, Nature (Lond), 288 (1980) 279-280. 15 Davies, P. and Maloney, A. J. R., Selective Ioss of central cholinergic neurons in Alzheimer's disease, Lancet, II (1976) 1403. 16 Davies, P. and Thompson, A., Postmortem stability of somatostatin-like immunoreactivity in mouse brain under conditions simulating handling of human autopsy material, Neurochem. Res. , 6 (1981)787-791. 17 DiFiglia, M. and Aronin, N., Ultrastructural features ofimmunoreactive somatostatin neurons in the rat caudate nucleus, J. Neurosci., 2 (19821 1267-1274. 18 Feldman, S. C., Lichenstein, E. S. and Silverman, A., Distribution of somatostatin in the mouse brain, Anat. Rec., 193 (19791 535-536. 19 Finley, J. C. W., Grossman, G. H. DiMeo, P. and Petrusz, P., Somatostatin-containing neurons in the rat brain: widespread distribution revealed by immunocytochemistry after pretreatment with pronase, Amer. J. Anat.. 193 (19781 483-488. 20 Finley, J. C. W., Maderdrut, J. L., Roger, L..1. and Petrusz, P., The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system, Neuroscience, 6 (1981) 2173-2192. 21 Hokfelt, T., Elde, R., Johansson, O~, Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the rat nervous system, Neurosci. Lett., 5 (1977) 25-3l. 22 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 (19791 5392-5396. 23 Krisch, B., Differing immunoreactivities of somatostatin in the cortex and the hypothalamus of the rat. A light and electron microscopic study, Cell Tiss. Res., 212 (1980) 457-464. 24 Lehmann, 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. 25 Mesulam, M.-M., Mufson, E. J.; Levey, A. 1. and Wainer, B. H., Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the Rhesus monkey, J. comp. Neurol., 214 (19831 170-197.

26 Morrison, J. H., Benoit, R., Magistretti, P, I and Btoom, F. E., Immunohistochemical distribution of pro-somatostatin-related peptides in cerebral cortex, Brain Research. 262 (1983) 344-351. 27 Nagai, T., McGeer, P. L., Peng, J. H,, Mc(icc~, E. ~.;. and Dolman, C. E., Choline acetyltransferase immunohistochemistry in brains of Alzheimer's disease patients and controls, Neurosci. Lett., 36 (1983) 195-199. 28 Pearson, R. C. A., Gatter, K. C. and Poweli. I'. P. S.. Retrograde cell degeneration m the basal nucleus in monkey and man, Brain Research, 261 (1983) 321-326. 29 Perry, E. K.. Blessed, G., Tomlinson, B. E., Perry, R. H , Crow, T. J., Cross, A. J., Dockray, G. J., Dimaline, R. and Arregui, A., Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes, Neurobiol. Aging, 2 (1981) 251-256~ 30 Perry, E. K., Perry, R. H.. Blessed, G. and Tomlinson, B. E., Necropsy evidence of central cholinergic deficits in senile dementia, Lancet, I (19771 189. 31 Perry, E. K., Tomlinson, B. E.. Blessed, (i., Bergmann, K., Gibson, P. H. and Perry, R. H., Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia, Brit. Med. ,i., 2 (1978) 1457-1459. 32 Petrusz, P., Sar, M., Grossman, G. H. and Kizer, J. S., Synaptic terminals with somatostatin-like immunoreactivity in the rat brain, Brain Research, 137 (1977) 181-187. 33 Probst, A., Basler, V., Bron, B. and Ulrich, J., Neuritic plaques in senile dementia of Alzheimer type: a Golgi analysis in the hippocampal region, Brain Research, 268 (1983) 249-254. 34 Reisine, T. D., Yamamura, H. I., Bird, E. D., Spokes, E. and Enna, S. J., Pre- and postsynaptic neurochemical alterations in Alzheimer's disease, Brain Research, 159 (19781 477-481. 35 Rossor, M. N., Emson, P. C., Mountjoy, C. Q., Roth, M. and Iversen, L. L., Reduced amounts of immunoreactive somatostatin in the temporal cortex in senile dementia of Alzheimer type, Neurosci. Lett., 20 (1980) 373~377. 36 Rossor, M., Fahrenkrug, J., Emson, P., Mount joy, 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. 37 Rossor, M. N., Svendsen, C., Hunt, S. P., Mountjoy, C, Q., Roth, M. and Iversen, L. L., The substantia innominata in A'lzheimer's Disease: An histochemical and biochemical study of cholinergic marker enzymes, Neurosci. Lett,, 28 (1982) 217-222. 38 Sorensen, K. V., Somatostatin: localization and distribution in the cortex and the subcortical white matter of human brain, Neuroscience, 7 (1982) 1227-1232. 39 Sternberger, L. A., lmmunocytochemistrv, 2nd Ed., John Wiley and Sons, New York-Chicago-Brisbane-Toronto, 1979. 40 Struble, R. G., Cork, L. C., Whitehouse, P. J. and L., Cholinergic innervation in neuritic plaques, Science. 216 (19821 413-415. 41 Terry, R. D., Brain disease in aging, especially senile dementia. In R. D. Terry, C. L. Bolis and G. Toffano (Eds.), Neural Aging and its Implications in Human Neurological Pathology, Vol. 18, Raven Press, New York, 1982, pp. 43-52. 42 Terry, R. D. and Wisniewski. H. M., The ultrastructure of

79 the neurofibrillary tangle and the senile plaque. In G. E. W. Wolstenholme and M. O'Connor (Eds.), Ciba Foundation Symposium on Alzheimer Disease and Related Conditions, J. and A. Churchill, London, 1970, pp. 145-168. 43 T. G. Warren and Shields, D., Cell-free biosynthesis of multiple preprosomatostatins: characterization of hybrid selection and amino terminal sequencing, Biochemistry, in press. 44 White, P., Goodhardt, M. J., Keet, J. P., Hiley, C. R., Carasco, L. H., Williams, 1. E. I. and Bowen, D. M., Neocorti-

cal cholinergic neurons in elderly people, Lancet, I (1977) 668-671. 45 Whitehouse, P. J., Clark, A. W., Price, D. L., Struble, R. G., Delong, M. R. and Coyle, J. T., Alzheimer's disease (AD): loss of cholinergic neurons in the nucleus basalis, J. Neuropath. exp. Neurol. , 40 (1981) 323. 46 Wisniewski, H. M. and Terry, R. D., Re-examination of the pathogenesis of the senile plaque. In H. M. Zimmerman (Ed.), Progress in Neuropathology, Vol. 2, Grune and Stratton, New York, 1973, pp. 1-26.