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Substance P immunoreactivity within neuritic plaques
Neuroscience Letters, 58 (1985) 139-144
139
Elsevier Scientific Publishers Ireland Ltd. NSL 03400 SUBSTANCE P IMMUNOREACTIVITY WITHIN NEURITIC PLAQUES
DAVID M. ARMSTRONG l'* and ROBERT D. TERRY 1.2 Departments o f INeuroscienees and 2pathology, University o f California - San Diego, School o f Medicine, La Jolla, CA 92093 (U.S,A.)
(Received March 15th, 1985; Accepted April 15th, 1985)
Key words." neuropeptide - substance P - immunocytochemistry - Alzheimer's disease - senile plaque -
human
In the present immunocytochemical study we examined brain tissue of patients with Alzheimer's disease in order to determine the relationship of substance P (SP)-labeled processes to neuritic plaques. Swollen neuropeptidergic processes were consistently observed within a relatively small percentage of the plaques. These data provide a morphologic correlate to the biochemical finding that SP levels are reduced in the brain tissue of patients with Alzheimer's disease, and further indicate that Alzheimer's disease affects multiple neurotransmitter and neuropeptide systems. Biochemical analysis o f brain tissue o f patients with Alzheimer's disease reveals that a n u m b e r o f neurotransmitter and neuropeptide systems are involved in this disease. A m o n g these are noradrenalin [16], serotonin [6], acetylcholine [5, 10~ 17], substance P (SP) [8], and somatostatin [11]. Yet it remains to be determined how some o f these biochemical abnormalities relate to the neuropathologic features o f Alzheimer's disease. Recently, several laboratories have begun to employ i m m u n o cytochemical techniques in order to visualize the relationship o f neuropeptide- and neurotransmitter-specific cellular elements to the neuritic plaque. In particular, i m m u n o c y t o c h e m i c a l studies using antibodies against choline acetyltransferase and histochemical studies for acetylcholinesterase demonstrated that cholinergic processes are associated with the simian plaque [13, 21]. Similarly, examination o f selected autopsied specimens from patients with Alzheimer's disease reveal that approximately 2 0 ~ o f all plaques contain the neuropeptide somatostatin [3]. The undecapeptide SP is also reduced in the brain o f patients with Alzheimer's disease [8], yet the relationship o f SP immunoreactive processes to the neuritic plaque is unknown. In the present study, we examined autopsied specimens o f patients with Alzheimer's disease by means o f light microscopic i m m u n o c y t o c h e m i c a l techniques in order to determine this relationship. Autopsied material was obtained from 12 patients with Alzheimer's disease, aged 64--96 years (mean = 78) o f age. The brains were obtained from 4 to 18 h after death *Author for correspondence and reprint requests at: University of California, San Diego, Department of Neurosciences, M-024, School of Medicine, La Jolla, CA 92093, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
140 (mean =9 h). At autopsy, the specimens were divided midsagittally, and the left hemisphere was placed in 10~o buffered formalin for routine histologic examination and morphometric analysis. From the right hemisphere, selected areas were removed for immunocytochemical studies, and the remaining hemi-brain was frozen for subsequent biochemical analysis. The diagnosis of Alzheimer's disease was confirmed in each patient following examination of Thioflavin-S-stained tissue sections from neoand paleocortex and observing numerous senile plaques and neurofibrillary tangles. This stain permits the detection of the two principle components of the plaque: amyloid and degenerating neuritic processes. The arrangement of these two components determines its type: primitive, mature or classical, and end-stage [22]. Similarly, 11 control patients, aged 54-89 years (mean = 66) of age were verified histologically by the absence of neuritic plaques and neurofibrillary tangles. The postmortem interval from this group ranged from 3 to 26 h (mean = 11 h). From both the Alzheimer and control group of patients, tissue was collected from the precentral (motor), superior temporal, and midfrontal gyri; superior parietal lobule, hippocampus, and amygdala of the right hemisphere and was placed in Bouin's fixative as previously described [3]. Vibratome sections 50 /tm thick were collected in phosphate buffer and were immunocytochemically labeled for SP (Accurate Chemicals) using a modification of the peroxidase-anti-peroxidase method [20]. The specificity of this well-characterized monoclonal antibody [9] was confirmed by the absence of immunolabeled profiles in tissue sections incubated with antisera previously absorbed with microgram concentrations of synthetic substance P (4.04 mg SP/3 ml diluted antiserum) (Fig. 1C). Subsequent to the immunocytochemical labeling procedure, the tissue sections were mounted on glass slides and counterstained with Thioflavin-S. The relationship of the neuritic plaques to the SP-labeled profiles was obtained by examining each tissue section alternately with fluorescence and Nomarski optics. By this mean, we were readily able to visualize the brown peroxidase-labeled profiles and the yellowgreen fluorescent neuritic plaques in the same tissue section. The number of plaques per tissue section and the percentage of these plaques that were associated with SPlabeled profiles were each determined by two investigators. In the normal brain, SP immunoreactivity was localized primarily to beaded varicose processes (Fig. I B). This staining pattern was evident throughout each region examined, and the intensity of the reaction product appeared unaffected by the length of the postmortem interval. SP immunoreactivity was also observed within an occasional neuronal perikaryon, but only in regions outside of the cortex, e.g. hypothalamus and medulla (Fig. 1A). Within the tissue of patients with Alzheimer's disease, SP-labeled profiles appeared either as beaded varicose processes or as distended profiles. Occasionally, these distended profiles were observed distinct from any lesion (Fig. 1D), but in most instances they were associated with a plaque (Fig. 2A-F). The plaques were most often of a mature type (i.e., central core of amyloid surrounded by a dense halo of degenerating neurites), yet occasional SP-immunoreactive profiles were associated with plaques that appeared to consist solely of degenerating neurites (i.e., primitive
141
Fig. 1. SP immunoreactivity from selected regions of non-demented control patiens (A-C) and a patient with Alzheimer's disease (D). A: photomicrograph showing a SP-containing neuron within the hypothalamus of a non-demented patient. B: photomicrograph showing a SP-labeled axon within the motor cortex of a non-demented patient. Labeled axons are characterized by periodic varicosities (arrows). C: photomicrograph showing the absence of any specific immunolabeling in a tissue section (motor cortex) incubated with preabsorbed antisera. D: photomicrograph showing an aggragation of SP immunoreactivity within the hippocampus of a patient with Alzheimer's disease. This profile is unusual in that it is not associated with any senile plaque. A-D: x 630. plaques). The SP-containing profiles were observed for the most part within the peripheral portion o f the plaque and varied considerably with respect to the n u m b e r o f labeled profiles in a single plaque. This variability ranged from a single punctate varicosity (Fig. 2A, B) to massive grape-like clusters o f immunolabeled profiles (Fig. 2E, F). The percentage o f plaques that were associated with any a m o u n t o f SP was relatively small and accounted for approximately 5~o o f all plaques. This percentage was consistent for all o f the regions examined and was independent o f the total n u m b e r o f plaques for a given region. The present study is the first published demonstration that SP-labeled processes are associated with neuritic plaques, and it provides a m o r p h o l o g i c correlate to the biochemical investigations that show SP to be reduced in brains o f patients with Alzheimer's disease [8]. In patients with Alzheimer's disease and in age-matched con-
142
Fig. 2. Three examples of SP immunolabeling within senile plaques. A: Nomarski photomicrograph showing several 'dot-like' SP-labeled profiles within the motor cortex of a patient with Alzheimer's disease. B: the corresponding fluorescent image shows that these immunolabeled neurites are within a plaque. The plaque is of mature type (i.e., a dense core of amyloid surrounded by a lighter halo of degenerating nerve processes). The arrows in A correspond in location to the arrows in B. C: photomicrograph showing a small aggregation of SP-immunolabeled profiles in the motor cortex of a patient with Alzheimer's disease. D: corresponding fluorescent image. E: photomicrograph showing a massive cluster of SP-immunoreactive neurites in the motor cortex of a patient with Alzheimer~s disease. F: corresponding fluorescent image. A-F: × 560. trois, S P i m m u n o r e a c t i v i t y w a s d e t e c t e d p r i m a r i l y in b e a d e d v a r i c o s e p r o c e s s e s . Electron microscopic immunocytochemical
studies reveal that these labeled varicose
143 processes consist primarily of synaptic boutons and axon segments [1, 12] and suggests a transmitter role for SP in central nervous system (CNS) [14]. In order to elucidate further the relationship of SP-labeled profiles to neuritic plaques, tissue sections were processed for immunocytochemistry and then counterstained with Thioflavin-S. This method allowed us to visualize SP-labeled processes and fluorescent senile plaques in the same tissue section. The dystrophic SP-labeled profiles were observed within approximately 5~o of all plaques. This can be compared with nearly 20~o of all plaques that were previously found to be associated with processes containing somatostatin immunoreactivity [3]. The greater number of plaques that are associated with somatostatin most likely reflects a greater concentration of this neuropeptide than of SP [7]. Like somatostatin, SP was observed within the peripheral, i.e., the neuritic, portion of the plaque. The labeled profiles of both peptides were usually distended and distinct in appearance when compared to be beaded varicose processes observed in normal brain. The concentration of swollen profiles within each plaque varied considerably and ranged from a simple dot-like structure to a massive clustering of peptidergic swellings. In that these dystrophic profiles are observed within plaques that appear to be primitive (i.e., without significant amyloid) suggests that peptidergic neurites are important to the early development of the plaque. Furthermore, it is likely that these swollen profiles belong to peptidergic neurons that are degenerating and thus collectively their loss would account for the significant reduction in activity that has been reported biochemically [8]. The source of these SP-labeled profiles remains to be determined. It is possible that, like somatostatin, some of the labeled profiles arise from local neurons in the cortex [4]. It is generally believed, however, that most of the cortical SP arises from an extrinsic source, most probably from neurons in the brainstem [18]. Recently, we demonstrated that a group of SP-labeled neurons in the laterodorsal tegmental nucleus projects to the cortex and also contains the cholinergic biosynthetic enzyme choline acetyltransferase [2, 23]. The results of that study provide an anatomical framework with which to suggest that in Alzheimer's disease a single mechanism may act on both cholinergic and SP systems. At the present time, it is unclear whether the effect on these two systems is due to a primary involvement of the cell bodies or whether it may be secondary to a cortical event [15, 19]. Nor is it clear whether cholinergic-, SP- and somatostatin-containing processes are present within the same plaque. The results of this work and previous investigations indicate that neuritic plaques are composed of neurites from several neurotransmitter and neuropeptide systems. This would imply that in Alzheimer's disease several transmitter systems (cholinergic, noradrenergic, glutaminergic, peptidergic and serotonergic, and probably others) as well as several anatomic locations (neocortex, hippocampus/parahippocampus, amygdala, basal forebrain and rostral brainstem) are involved. Not all neurons of any transmitter system are involved, so one must conclude that those cells affected from each of these systems and locations have a particular factor in common which leads to their susceptibility to whatever causes the disorder. This work was supported in part by a grant from the McKnight Foundation and NIH Grants AG-05344 and AG-05386.
144 1 Armstrong, D.M., Pickel, V.M. and Reis, D.J., Electron microscopic immunocytochemical localization of substance P in the area postrema of rat, Brain Res., 243 (1982) 141-146. 2 Armstrong, D.M., Saper, C.B., Levey, A.I., Wainer,B.H. and Terry, R.D., Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol., 216(1983) 53 68. 3 Armstrong, D.M., LeRoy, S., Shields, D. and Terry, R.D., Somatostatin-like immunoreactivity within neuritic plaques, Brain Res., in press. 4 Beach, T.G. and McGeer, E.G., Neocortical substance P neurons in the baboon: an immunohistochemical finding. Neurosci. Lett., 41 (1983) 265 270. 5 Bowen, D.M., Smith, C.B., White, P. and Davidson, A.N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-495. 6 Bowen, D.M., Biochemical assessment of neurotransmitter and metabolic dysfunction and cerebral atrophy in Alzheimer's disease. In R. Katzman (Ed.), Biological Aspects of Alzheimer's Disease, Banbury Report, Vol. 15, Cold Spring Harbor Laboratory, 1983, pp. 219-232. 7 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 Res., 218 (1981) 219-232. 8 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. 9 Cuello, A.C., Galfre, G., and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad. Sci. USA, 76 (1979) 3532-3536. 10 Davies, P. and Maloney, A.J.R., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, ii (1976) 1403. 11 Davies, P., Katzman, R. and Terry, R.D., Reduced somatostatin-like immunoreactivity in cerebralcortex from cases of Alzheimer's disease and Alzheimer senile dementia, Nature (Lond.), 288 (1980) 279-280. 12 DiFiglia, M., Aronin, N. and Leeman, S.E., Light microscopic and ultrastructurat localization of immunoreactive substance P in the dorsal horn of monkey spinal cord, Neuroscience, 7 (1982) 1127 1139. 13 Kitt, C.A., Price, D.L., Struble, R.G., Cork, L.C., Wainer, B.H., Becher, M.W. and Mobley, W.C., Evidence for cholinergic neurites in senile plaques, Science, 226 (1984) 1443-1445. 14 Nicoll, R.A., Schenker, C. and Leeman, S.E., Substance P as a transmitter candidate, Annu. Rev. Neurosci., 3 (1980) 277 68. 15 Pearson, R.C.A., Garter, K.C. and Powell, T.P.S., Retrograde cell degeneration in the basal nucleus in monkey and man, Brain Res., 261 (1983) 321 326. 16 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, .I. Neurol. Sci., 51 (1981) 279 287. 17 Perry, E.K., Perry, R.H., Blessed, G. and Tomlinson, B.E., Necropsy evidence of central cholinergic deficits in senile dementia, Lancet, i (1977) 189. 18 Sakanaka, M., Shiosaka, S., Takatsuki, K. and Tohyama, M., Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex of the rat, Brain Res., 259 (1983) 123-126. 19 Sofroniew, M.V., Pearson, R.C.A., Eckenstein, F., Cuello, A.C. and Powell, T.P.S., Retrograde changes in cholinergic neurons in the basal forebrain of the rat following cortical damage, Brain Res., 289 (1983) 370-374. 20 Sternberger, L.A., Immunocytochemistry, 2nd. Edn., John Wiley and Sons, New York, Chicago, Brisbane, Toronto, 1979. 21 Struble, R.G., Cork, L.C., Whitehouse, P.J. and Price, D.L., Cholinergic innervation in neuritic plaques, Science, 216 (1982) 413-415. 22 Terry, R.D. and Wisniewski, H.M., The ultrastructure of 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. & A. Churchill, London, 1970, pp. 145-168. 23 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., Substance P in the ascending cholinergic reticular system, Nature (Lond.), 306 (1983) 688 691.
139
Elsevier Scientific Publishers Ireland Ltd. NSL 03400 SUBSTANCE P IMMUNOREACTIVITY WITHIN NEURITIC PLAQUES
DAVID M. ARMSTRONG l'* and ROBERT D. TERRY 1.2 Departments o f INeuroscienees and 2pathology, University o f California - San Diego, School o f Medicine, La Jolla, CA 92093 (U.S,A.)
(Received March 15th, 1985; Accepted April 15th, 1985)
Key words." neuropeptide - substance P - immunocytochemistry - Alzheimer's disease - senile plaque -
human
In the present immunocytochemical study we examined brain tissue of patients with Alzheimer's disease in order to determine the relationship of substance P (SP)-labeled processes to neuritic plaques. Swollen neuropeptidergic processes were consistently observed within a relatively small percentage of the plaques. These data provide a morphologic correlate to the biochemical finding that SP levels are reduced in the brain tissue of patients with Alzheimer's disease, and further indicate that Alzheimer's disease affects multiple neurotransmitter and neuropeptide systems. Biochemical analysis o f brain tissue o f patients with Alzheimer's disease reveals that a n u m b e r o f neurotransmitter and neuropeptide systems are involved in this disease. A m o n g these are noradrenalin [16], serotonin [6], acetylcholine [5, 10~ 17], substance P (SP) [8], and somatostatin [11]. Yet it remains to be determined how some o f these biochemical abnormalities relate to the neuropathologic features o f Alzheimer's disease. Recently, several laboratories have begun to employ i m m u n o cytochemical techniques in order to visualize the relationship o f neuropeptide- and neurotransmitter-specific cellular elements to the neuritic plaque. In particular, i m m u n o c y t o c h e m i c a l studies using antibodies against choline acetyltransferase and histochemical studies for acetylcholinesterase demonstrated that cholinergic processes are associated with the simian plaque [13, 21]. Similarly, examination o f selected autopsied specimens from patients with Alzheimer's disease reveal that approximately 2 0 ~ o f all plaques contain the neuropeptide somatostatin [3]. The undecapeptide SP is also reduced in the brain o f patients with Alzheimer's disease [8], yet the relationship o f SP immunoreactive processes to the neuritic plaque is unknown. In the present study, we examined autopsied specimens o f patients with Alzheimer's disease by means o f light microscopic i m m u n o c y t o c h e m i c a l techniques in order to determine this relationship. Autopsied material was obtained from 12 patients with Alzheimer's disease, aged 64--96 years (mean = 78) o f age. The brains were obtained from 4 to 18 h after death *Author for correspondence and reprint requests at: University of California, San Diego, Department of Neurosciences, M-024, School of Medicine, La Jolla, CA 92093, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
140 (mean =9 h). At autopsy, the specimens were divided midsagittally, and the left hemisphere was placed in 10~o buffered formalin for routine histologic examination and morphometric analysis. From the right hemisphere, selected areas were removed for immunocytochemical studies, and the remaining hemi-brain was frozen for subsequent biochemical analysis. The diagnosis of Alzheimer's disease was confirmed in each patient following examination of Thioflavin-S-stained tissue sections from neoand paleocortex and observing numerous senile plaques and neurofibrillary tangles. This stain permits the detection of the two principle components of the plaque: amyloid and degenerating neuritic processes. The arrangement of these two components determines its type: primitive, mature or classical, and end-stage [22]. Similarly, 11 control patients, aged 54-89 years (mean = 66) of age were verified histologically by the absence of neuritic plaques and neurofibrillary tangles. The postmortem interval from this group ranged from 3 to 26 h (mean = 11 h). From both the Alzheimer and control group of patients, tissue was collected from the precentral (motor), superior temporal, and midfrontal gyri; superior parietal lobule, hippocampus, and amygdala of the right hemisphere and was placed in Bouin's fixative as previously described [3]. Vibratome sections 50 /tm thick were collected in phosphate buffer and were immunocytochemically labeled for SP (Accurate Chemicals) using a modification of the peroxidase-anti-peroxidase method [20]. The specificity of this well-characterized monoclonal antibody [9] was confirmed by the absence of immunolabeled profiles in tissue sections incubated with antisera previously absorbed with microgram concentrations of synthetic substance P (4.04 mg SP/3 ml diluted antiserum) (Fig. 1C). Subsequent to the immunocytochemical labeling procedure, the tissue sections were mounted on glass slides and counterstained with Thioflavin-S. The relationship of the neuritic plaques to the SP-labeled profiles was obtained by examining each tissue section alternately with fluorescence and Nomarski optics. By this mean, we were readily able to visualize the brown peroxidase-labeled profiles and the yellowgreen fluorescent neuritic plaques in the same tissue section. The number of plaques per tissue section and the percentage of these plaques that were associated with SPlabeled profiles were each determined by two investigators. In the normal brain, SP immunoreactivity was localized primarily to beaded varicose processes (Fig. I B). This staining pattern was evident throughout each region examined, and the intensity of the reaction product appeared unaffected by the length of the postmortem interval. SP immunoreactivity was also observed within an occasional neuronal perikaryon, but only in regions outside of the cortex, e.g. hypothalamus and medulla (Fig. 1A). Within the tissue of patients with Alzheimer's disease, SP-labeled profiles appeared either as beaded varicose processes or as distended profiles. Occasionally, these distended profiles were observed distinct from any lesion (Fig. 1D), but in most instances they were associated with a plaque (Fig. 2A-F). The plaques were most often of a mature type (i.e., central core of amyloid surrounded by a dense halo of degenerating neurites), yet occasional SP-immunoreactive profiles were associated with plaques that appeared to consist solely of degenerating neurites (i.e., primitive
141
Fig. 1. SP immunoreactivity from selected regions of non-demented control patiens (A-C) and a patient with Alzheimer's disease (D). A: photomicrograph showing a SP-containing neuron within the hypothalamus of a non-demented patient. B: photomicrograph showing a SP-labeled axon within the motor cortex of a non-demented patient. Labeled axons are characterized by periodic varicosities (arrows). C: photomicrograph showing the absence of any specific immunolabeling in a tissue section (motor cortex) incubated with preabsorbed antisera. D: photomicrograph showing an aggragation of SP immunoreactivity within the hippocampus of a patient with Alzheimer's disease. This profile is unusual in that it is not associated with any senile plaque. A-D: x 630. plaques). The SP-containing profiles were observed for the most part within the peripheral portion o f the plaque and varied considerably with respect to the n u m b e r o f labeled profiles in a single plaque. This variability ranged from a single punctate varicosity (Fig. 2A, B) to massive grape-like clusters o f immunolabeled profiles (Fig. 2E, F). The percentage o f plaques that were associated with any a m o u n t o f SP was relatively small and accounted for approximately 5~o o f all plaques. This percentage was consistent for all o f the regions examined and was independent o f the total n u m b e r o f plaques for a given region. The present study is the first published demonstration that SP-labeled processes are associated with neuritic plaques, and it provides a m o r p h o l o g i c correlate to the biochemical investigations that show SP to be reduced in brains o f patients with Alzheimer's disease [8]. In patients with Alzheimer's disease and in age-matched con-
142
Fig. 2. Three examples of SP immunolabeling within senile plaques. A: Nomarski photomicrograph showing several 'dot-like' SP-labeled profiles within the motor cortex of a patient with Alzheimer's disease. B: the corresponding fluorescent image shows that these immunolabeled neurites are within a plaque. The plaque is of mature type (i.e., a dense core of amyloid surrounded by a lighter halo of degenerating nerve processes). The arrows in A correspond in location to the arrows in B. C: photomicrograph showing a small aggregation of SP-immunolabeled profiles in the motor cortex of a patient with Alzheimer's disease. D: corresponding fluorescent image. E: photomicrograph showing a massive cluster of SP-immunoreactive neurites in the motor cortex of a patient with Alzheimer~s disease. F: corresponding fluorescent image. A-F: × 560. trois, S P i m m u n o r e a c t i v i t y w a s d e t e c t e d p r i m a r i l y in b e a d e d v a r i c o s e p r o c e s s e s . Electron microscopic immunocytochemical
studies reveal that these labeled varicose
143 processes consist primarily of synaptic boutons and axon segments [1, 12] and suggests a transmitter role for SP in central nervous system (CNS) [14]. In order to elucidate further the relationship of SP-labeled profiles to neuritic plaques, tissue sections were processed for immunocytochemistry and then counterstained with Thioflavin-S. This method allowed us to visualize SP-labeled processes and fluorescent senile plaques in the same tissue section. The dystrophic SP-labeled profiles were observed within approximately 5~o of all plaques. This can be compared with nearly 20~o of all plaques that were previously found to be associated with processes containing somatostatin immunoreactivity [3]. The greater number of plaques that are associated with somatostatin most likely reflects a greater concentration of this neuropeptide than of SP [7]. Like somatostatin, SP was observed within the peripheral, i.e., the neuritic, portion of the plaque. The labeled profiles of both peptides were usually distended and distinct in appearance when compared to be beaded varicose processes observed in normal brain. The concentration of swollen profiles within each plaque varied considerably and ranged from a simple dot-like structure to a massive clustering of peptidergic swellings. In that these dystrophic profiles are observed within plaques that appear to be primitive (i.e., without significant amyloid) suggests that peptidergic neurites are important to the early development of the plaque. Furthermore, it is likely that these swollen profiles belong to peptidergic neurons that are degenerating and thus collectively their loss would account for the significant reduction in activity that has been reported biochemically [8]. The source of these SP-labeled profiles remains to be determined. It is possible that, like somatostatin, some of the labeled profiles arise from local neurons in the cortex [4]. It is generally believed, however, that most of the cortical SP arises from an extrinsic source, most probably from neurons in the brainstem [18]. Recently, we demonstrated that a group of SP-labeled neurons in the laterodorsal tegmental nucleus projects to the cortex and also contains the cholinergic biosynthetic enzyme choline acetyltransferase [2, 23]. The results of that study provide an anatomical framework with which to suggest that in Alzheimer's disease a single mechanism may act on both cholinergic and SP systems. At the present time, it is unclear whether the effect on these two systems is due to a primary involvement of the cell bodies or whether it may be secondary to a cortical event [15, 19]. Nor is it clear whether cholinergic-, SP- and somatostatin-containing processes are present within the same plaque. The results of this work and previous investigations indicate that neuritic plaques are composed of neurites from several neurotransmitter and neuropeptide systems. This would imply that in Alzheimer's disease several transmitter systems (cholinergic, noradrenergic, glutaminergic, peptidergic and serotonergic, and probably others) as well as several anatomic locations (neocortex, hippocampus/parahippocampus, amygdala, basal forebrain and rostral brainstem) are involved. Not all neurons of any transmitter system are involved, so one must conclude that those cells affected from each of these systems and locations have a particular factor in common which leads to their susceptibility to whatever causes the disorder. This work was supported in part by a grant from the McKnight Foundation and NIH Grants AG-05344 and AG-05386.
144 1 Armstrong, D.M., Pickel, V.M. and Reis, D.J., Electron microscopic immunocytochemical localization of substance P in the area postrema of rat, Brain Res., 243 (1982) 141-146. 2 Armstrong, D.M., Saper, C.B., Levey, A.I., Wainer,B.H. and Terry, R.D., Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol., 216(1983) 53 68. 3 Armstrong, D.M., LeRoy, S., Shields, D. and Terry, R.D., Somatostatin-like immunoreactivity within neuritic plaques, Brain Res., in press. 4 Beach, T.G. and McGeer, E.G., Neocortical substance P neurons in the baboon: an immunohistochemical finding. Neurosci. Lett., 41 (1983) 265 270. 5 Bowen, D.M., Smith, C.B., White, P. and Davidson, A.N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-495. 6 Bowen, D.M., Biochemical assessment of neurotransmitter and metabolic dysfunction and cerebral atrophy in Alzheimer's disease. In R. Katzman (Ed.), Biological Aspects of Alzheimer's Disease, Banbury Report, Vol. 15, Cold Spring Harbor Laboratory, 1983, pp. 219-232. 7 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 Res., 218 (1981) 219-232. 8 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. 9 Cuello, A.C., Galfre, G., and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad. Sci. USA, 76 (1979) 3532-3536. 10 Davies, P. and Maloney, A.J.R., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, ii (1976) 1403. 11 Davies, P., Katzman, R. and Terry, R.D., Reduced somatostatin-like immunoreactivity in cerebralcortex from cases of Alzheimer's disease and Alzheimer senile dementia, Nature (Lond.), 288 (1980) 279-280. 12 DiFiglia, M., Aronin, N. and Leeman, S.E., Light microscopic and ultrastructurat localization of immunoreactive substance P in the dorsal horn of monkey spinal cord, Neuroscience, 7 (1982) 1127 1139. 13 Kitt, C.A., Price, D.L., Struble, R.G., Cork, L.C., Wainer, B.H., Becher, M.W. and Mobley, W.C., Evidence for cholinergic neurites in senile plaques, Science, 226 (1984) 1443-1445. 14 Nicoll, R.A., Schenker, C. and Leeman, S.E., Substance P as a transmitter candidate, Annu. Rev. Neurosci., 3 (1980) 277 68. 15 Pearson, R.C.A., Garter, K.C. and Powell, T.P.S., Retrograde cell degeneration in the basal nucleus in monkey and man, Brain Res., 261 (1983) 321 326. 16 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, .I. Neurol. Sci., 51 (1981) 279 287. 17 Perry, E.K., Perry, R.H., Blessed, G. and Tomlinson, B.E., Necropsy evidence of central cholinergic deficits in senile dementia, Lancet, i (1977) 189. 18 Sakanaka, M., Shiosaka, S., Takatsuki, K. and Tohyama, M., Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex of the rat, Brain Res., 259 (1983) 123-126. 19 Sofroniew, M.V., Pearson, R.C.A., Eckenstein, F., Cuello, A.C. and Powell, T.P.S., Retrograde changes in cholinergic neurons in the basal forebrain of the rat following cortical damage, Brain Res., 289 (1983) 370-374. 20 Sternberger, L.A., Immunocytochemistry, 2nd. Edn., John Wiley and Sons, New York, Chicago, Brisbane, Toronto, 1979. 21 Struble, R.G., Cork, L.C., Whitehouse, P.J. and Price, D.L., Cholinergic innervation in neuritic plaques, Science, 216 (1982) 413-415. 22 Terry, R.D. and Wisniewski, H.M., The ultrastructure of 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. & A. Churchill, London, 1970, pp. 145-168. 23 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., Substance P in the ascending cholinergic reticular system, Nature (Lond.), 306 (1983) 688 691.