174
Brain Research, 549 (1991) 174-177 © 1991 Elsevier Science Publishers B.V. 0006-8993/911503.50 ADONIS 000689939124664U
BRES 24664
Localization of Mn-superoxide dismutase (Mn D) in cholinergic and sornatostatin-containlng neurons in the rat neostriatum Shinobu Inagaki 1, Keichiro Suzuki 2, Naoyuki Taniguchi 2 and Hiroshi Takagi 1 1Department of Anatomy, Osaka City University Medical School, Abeno-ku, Osaka (Japan) and 2Department of Biochemistry, Osaka University Medical School, Kita-ku, Osaka (Japan)
(Accepted 12 February 1991) Key words: Mn-superoxide dismutase; Choline acetyltransferase; Somatostatin; Neostriatum; Rat
We used rabbit antisera against manganese (Mn)-superoxide dismutase for immunohistochemical studies of localization in the rat neostriatum. Immunostaining was intense in large-sized neurons and several medium-sized neurons, but it was moderate to weak in other cells. Double immunostaining with monoclonal antibody to choline acetyltransferase or somatostatin demonstrated large-sized, Mn-SOD immunoreactive neurons to be cholinergic, and some medium-sized neurons which were intensely immunoreactive for Mn-SOD to contain somatostatinergic.
Changes in neurotransmitters and neuropeptides in such neurodegenerative disorders as Parkinson's and Huntington's disease, have been reported 7. Although the chemical nature of individual striatal neurons has been established 7, our understanding of degenerative mechanisms in the basal ganglia is still poor. Free radicals may play a role in the degeneration of neurons in Parkinson's disease z'4. Superoxide dismutase (SOD) catalyzes the conversion of superoxide radicals to hydrogen peroxide and molecular oxygen, and probably protects cells against the potential toxicity of reactive free radicals produced from oxygen 12. Mammalian tissues contain two main forms of SOD: Mn-SOD and Cu/Zn-SOD. Mn-SOD is a manganese-containing enzyme present in mitochondria, whereas Cu/Zn-SOD is a copper- and zinc-containing enzyme localized in the cytoplasm of various cells 1'6'1°. In the striatum of Huntington's disease, medium-sized spiny neurons are the first and most severely affected neurons, whereas large and medium aspiny neurons, including cholinergic and somatostatin-containing intrinsic neurons are selectively preserved 7'11. However, little is known regarding the immunohistochemicai localization of this enzyme and its relationships with neurotransmitters in the neostriatum. The aim of this study was to localize Mn-SOD immunoreactivity in the neostriatum and study the relationship between the localization of Mn-SOD and acetylcholine- and somatostatin-containing neuron systems. Five adult male Wistar rats weighing 100-150 g were
used. The animals were anesthetized with pentobarbital (50 mg/kg) and perfused with 200 ml of a formaldehyde fixative (2% paraformaldehyde-0.2% picric acid in 0.1 M phosphate buffer). The brain was postfixed in this solution overnight, followed by immersion in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Frontal sections with a thickness of 30/~m were cut on a cryostat and processed for double immunostaining 15. Forebrain sections were first incubated overnight at 4 °C in a mixture containing rabbit polyclonal antibody to rat Mn-SOD 9 and rat monoclonal antibody to choline acetyltransferase (CHAT) 5, or mouse monoclonal antibody to somatostatin ~6. The sections were then incubated overnight at 4 °C with a mixture of the second antibody, fluorescent isothiocyanate (FITC)-labeled goat antirabbit IgG (Miles) to detect SOD, and Texas red-labeled sheep anti-rat IgG (Amersham) to detect ChAT or Texas red-labeled sheep anti-mouse IgG (Amersham) to detect somatostatin. Following 3 rinses in buffer, the sections were mounted on glass slides and examined with an epifluorescence microscope (Olympus) equipped with a B-dichronic filter system for FITC fluorescence and a G-dichronic filter system for Texas-red fluorescence. The preparation and specificities of the antisera to Mn-SOD, CHAT, and somatostatin have been previously described 5' 8 10,16. To test the specificity, control experiments were performed by: (1) omission of the primary antisera: (2) replacement of the primary antisera with normal rabbit,
Correspondence: S. Inagaki, Department of Anatomy 1st Division, Osaka City University Medical School, Asahimachi 1-4-54, Abeno-ku, Osaka, 545 Japan.
175 rat and mouse sera: and (3) use of primary Mn-SOD antisera following its absorption with purified Mn-SOD (at 10-6 M). No immunostaining was observed in the tissue sections prepared under these control conditions. Mn-SOD immunoreactivity was concentrated in neuronal cells, while immunoreactivity in glial cells was very weak, except around blood vessels. Although the neostriatum showed moderate immunoreactivity (Fig. 1A), several groups of neurons were intensely immunoreactive. These appeared to be often large-sized neurons. Some of the medium-sized neurons also demonstrated intense Mn-SOD immunostaining, but most were only moderately immunoreactive (Fig. 1A). Double staining
for Mn-SOD and ChAT showed the concurrent localization of these substances within large-sized neurons throughout the neostriatum (Fig. 1). Furthermore, some medium-sized neurons intensely immunostained for MnSOD were also immunoreactive for somatostatin, while others were not (Fig. 2). Many somatostatin-immunoreactive neurons were moderately immunoreactive for Mn-SOD, while a few were only very weakly Mn-SOD immunoreactive. The present study demonstrated intense Mn-SOD immunoreactivity within almost all large-sized, cholinergic neurons and several groups of medium-sized neurons containing somatostatin in the neostriatum. There was
Fig. 1. Fluorescent photomicrographs showing immunoreactivity for Mn-SOD (A) and ChAT (B) in the caudate-putamen in a single section. Arrows indicate intensely Mn-SOD immunoreactive neurons that are also immunostained for CHAT. These are large-sized neurons. Arrowheads indicate medium-sized neurons which are intensely immunoreactive for Mn-SOD, but not for CHAT. Scale (A and B): 50 #m.
176
Fig. 2. Fluorescent photomicrigraphs showing immunoreactivity for Mn-SOD (A) and somatostatin (B) in the caudate-putamen of a single section. Arrows indicate medium-sized neurons which are simultaneously immunostained for Mn-SOD and somatostatin, while small arrowheads indicate examples of medium-sized neurons which are intensely immunoreaetive for Mn,SOD but not somatostatin. The large arrowhead (A) indicates a large-sized neuron which is strongly immunostained for Mn-SOD but not for somatostatin. Scale (A and B): 50 /zm.
also a population of medium-sized neurons which were intensely immunostained for M n - S O D but not immunoreactive for somatostatin. O n e possible transmitter in these medium-sized neurons is G A B A , as most mediumsized neurons are G A B A e r g i c 7. We previously demonstrated prominent topographic distribution in the hippocampal subfields 1. CA1 pyramidal cells, which are particularly vulnerable to transient cerebral ischemia 14, were weakly immunostained for M n - S O D , whereas CA3 pyramidal cells, which are resistant to cerebral ischemia, were intensely immunore-
active. Antiperoxidative enzymes such as M n - S O D and Cu/Zn-SOD may be involved in defense against reactive oxygen free radicals formed as a result of ischemia and subsequent reoxygenation 3'14. However, magnocellular basal forebrain neurons, which undergo selective degeneration in Alzheimer's disease, demonstrated very intense immunoreactivity 9. Increased S O D activity has been reported in fibroblast cell lines derived from familial Alzheimer's patients 17 and in the basal ganglia in Parkinson's disease 13. Mitochondrial S O D activity was higher in Parkinson's disease; however, there was no
177 difference b e t w e e n cytosol S O D activity in the parkinsonian substantia nigra and control tissues. Increased S O D activity in the mitochondrial fraction may be a protective response to elevated levels of toxic free radicals in the parkinsonian basal ganglia; alternatively, it m a y induce cell death through the accumulation of h y d r o g e n peroxide. T h e r e f o r e , high levels of M n - S O D in the cholinergic neuronal system and some somatostatinergic n e u r o n systems in the neostriatum may be factors in the p a t h o l o g y and t r e a t m e n t of human neurodegen-
erative disorders such as P a r k i n s o n ' s and H u n t i n g t o n ' s diseases 11,13.
1 Akai, E, Maeda, M., Suzuki, K., Inagaki, S., Takagi, H. and Taniguchi, N., Immunocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat, Neurosci. Lett., 115 (1990) 19-23. 2 Ambani, L.M., Van Woert, M.H. and Murphy, S., Brain peroxidase and catalase in Parkinson's disease, Arch. Neurol., 32 (1975) 114-118. 3 Chan, P.H., Chu, L. and Fishman, R.A., Reduction of activities of superoxide dismutase but not of glutathione peroxidase in rat brain regions following decapitation ischemia, Brain Research, 439 (1988) 388-390. 4 Cohen, G., Oxyradical toxicity in catecholamine neurons, Neurotoxicology, 5 (1984) 77-82. 5 Eckenstein, E and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363-368. 6 Fridovich, I., Superoxide dismutase, Annu. Rev. Biochem., 44 (1975) 147-159. 7 Graybiel, A.M. and Ragsdale Jr., C.W., Biochemical anatomy of the striatum. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 427-503. 8 Iizuka, S., Taniguchi, N. and Makita, A., Enzyme-like immunosorbent assay for human manganese-containing superoxide dismutase and its content in lung cancer, J. Natl. Cancer Inst., 72 (1984) 1043-1049. 9 Inagaki, S., Takagi,H., Suzuki, K., Akai, F. and Taniguchi, N., Intense immunoreactivity for Mn-superoxide dismutase (MnSOD) in cholinergic and non-cholinergic neurons in the rat basal
forebrain, Brain Research, 541 (1991) 354-357. 10 Kawaguchi, T., Noji, S., Uda, T., Nakashima, Y., Takeyasu, A., Kawai, Y., Takagi, H., Tohyama, M. and Taniguchi, N., A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase, J. Biol. Chem., 264 (1989) 5762-5767. 11 Kowall, N.W., Ferrante, R.J. and Martin, J.B., Patterns of cell loss in Huntington's disease, Trends Neurosci., 10 (1987) 24-29. 12 McCord, J.M., Keele Jr., B.B. and Fridovich, I., An enzymebased theory of obligate anaerobiosis: the physiological function of superoxide dismutase, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 1024-1027. 13 Saggu, H., Cooksey, J., Dexter, D., Wells, ER., Lees, A., Jenner, P. and Marsden, C.D., A selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra, J. Neurochem., 53 (1989) 692-697. 14 Siesj6, B.K., Cell damage in the brain: a speculative synthesis, J. Cereb. Blood Flow Metab., 1 (1981) 155-185. 15 Shimada, S., Inagaki, S., Kubota, Y., Kito, S., Shiotani, Y. and Tohyama, M., Coexistence of substance P and enkephalin-like peptides in single neurons of the rat hypothalamus, Brain Research, 425 (1987) 256--262. 16 Vincent, S.R., Mclntosh, C.H.S., Buchan, A.N.J. and Brown, J.C., Central somatostatin systems revealed with monoclonal antibodies, J. Comp. Neurol., 238 (1985) 169-186. 17 Zemlan, EP., Thienhaus, O.J. and Bosmann, H.B., Superoxide dismutase activity in Alzheimer's disease: possible mechanism for paired helical filament formation, Brain Research, 476 (1989) 160-162.
We wish to thank Dr. F. Eckenstein for providing antibody to CHAT, Dr. J.C. Brown for a gift of somatostatin antibody and Dr. S.R. Vincent for improving the manuscript and helpful suggestions. This work was supported by the Grant-in-Aid for Scientific Research on Priority Areas No. 02240106 from the Ministry of Education, Science and Culture, Japan, and a grant provided by the Ichiro Kanehara Foundation.
Brain Research, 549 (1991) 174-177 © 1991 Elsevier Science Publishers B.V. 0006-8993/911503.50 ADONIS 000689939124664U
BRES 24664
Localization of Mn-superoxide dismutase (Mn D) in cholinergic and sornatostatin-containlng neurons in the rat neostriatum Shinobu Inagaki 1, Keichiro Suzuki 2, Naoyuki Taniguchi 2 and Hiroshi Takagi 1 1Department of Anatomy, Osaka City University Medical School, Abeno-ku, Osaka (Japan) and 2Department of Biochemistry, Osaka University Medical School, Kita-ku, Osaka (Japan)
(Accepted 12 February 1991) Key words: Mn-superoxide dismutase; Choline acetyltransferase; Somatostatin; Neostriatum; Rat
We used rabbit antisera against manganese (Mn)-superoxide dismutase for immunohistochemical studies of localization in the rat neostriatum. Immunostaining was intense in large-sized neurons and several medium-sized neurons, but it was moderate to weak in other cells. Double immunostaining with monoclonal antibody to choline acetyltransferase or somatostatin demonstrated large-sized, Mn-SOD immunoreactive neurons to be cholinergic, and some medium-sized neurons which were intensely immunoreactive for Mn-SOD to contain somatostatinergic.
Changes in neurotransmitters and neuropeptides in such neurodegenerative disorders as Parkinson's and Huntington's disease, have been reported 7. Although the chemical nature of individual striatal neurons has been established 7, our understanding of degenerative mechanisms in the basal ganglia is still poor. Free radicals may play a role in the degeneration of neurons in Parkinson's disease z'4. Superoxide dismutase (SOD) catalyzes the conversion of superoxide radicals to hydrogen peroxide and molecular oxygen, and probably protects cells against the potential toxicity of reactive free radicals produced from oxygen 12. Mammalian tissues contain two main forms of SOD: Mn-SOD and Cu/Zn-SOD. Mn-SOD is a manganese-containing enzyme present in mitochondria, whereas Cu/Zn-SOD is a copper- and zinc-containing enzyme localized in the cytoplasm of various cells 1'6'1°. In the striatum of Huntington's disease, medium-sized spiny neurons are the first and most severely affected neurons, whereas large and medium aspiny neurons, including cholinergic and somatostatin-containing intrinsic neurons are selectively preserved 7'11. However, little is known regarding the immunohistochemicai localization of this enzyme and its relationships with neurotransmitters in the neostriatum. The aim of this study was to localize Mn-SOD immunoreactivity in the neostriatum and study the relationship between the localization of Mn-SOD and acetylcholine- and somatostatin-containing neuron systems. Five adult male Wistar rats weighing 100-150 g were
used. The animals were anesthetized with pentobarbital (50 mg/kg) and perfused with 200 ml of a formaldehyde fixative (2% paraformaldehyde-0.2% picric acid in 0.1 M phosphate buffer). The brain was postfixed in this solution overnight, followed by immersion in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Frontal sections with a thickness of 30/~m were cut on a cryostat and processed for double immunostaining 15. Forebrain sections were first incubated overnight at 4 °C in a mixture containing rabbit polyclonal antibody to rat Mn-SOD 9 and rat monoclonal antibody to choline acetyltransferase (CHAT) 5, or mouse monoclonal antibody to somatostatin ~6. The sections were then incubated overnight at 4 °C with a mixture of the second antibody, fluorescent isothiocyanate (FITC)-labeled goat antirabbit IgG (Miles) to detect SOD, and Texas red-labeled sheep anti-rat IgG (Amersham) to detect ChAT or Texas red-labeled sheep anti-mouse IgG (Amersham) to detect somatostatin. Following 3 rinses in buffer, the sections were mounted on glass slides and examined with an epifluorescence microscope (Olympus) equipped with a B-dichronic filter system for FITC fluorescence and a G-dichronic filter system for Texas-red fluorescence. The preparation and specificities of the antisera to Mn-SOD, CHAT, and somatostatin have been previously described 5' 8 10,16. To test the specificity, control experiments were performed by: (1) omission of the primary antisera: (2) replacement of the primary antisera with normal rabbit,
Correspondence: S. Inagaki, Department of Anatomy 1st Division, Osaka City University Medical School, Asahimachi 1-4-54, Abeno-ku, Osaka, 545 Japan.
175 rat and mouse sera: and (3) use of primary Mn-SOD antisera following its absorption with purified Mn-SOD (at 10-6 M). No immunostaining was observed in the tissue sections prepared under these control conditions. Mn-SOD immunoreactivity was concentrated in neuronal cells, while immunoreactivity in glial cells was very weak, except around blood vessels. Although the neostriatum showed moderate immunoreactivity (Fig. 1A), several groups of neurons were intensely immunoreactive. These appeared to be often large-sized neurons. Some of the medium-sized neurons also demonstrated intense Mn-SOD immunostaining, but most were only moderately immunoreactive (Fig. 1A). Double staining
for Mn-SOD and ChAT showed the concurrent localization of these substances within large-sized neurons throughout the neostriatum (Fig. 1). Furthermore, some medium-sized neurons intensely immunostained for MnSOD were also immunoreactive for somatostatin, while others were not (Fig. 2). Many somatostatin-immunoreactive neurons were moderately immunoreactive for Mn-SOD, while a few were only very weakly Mn-SOD immunoreactive. The present study demonstrated intense Mn-SOD immunoreactivity within almost all large-sized, cholinergic neurons and several groups of medium-sized neurons containing somatostatin in the neostriatum. There was
Fig. 1. Fluorescent photomicrographs showing immunoreactivity for Mn-SOD (A) and ChAT (B) in the caudate-putamen in a single section. Arrows indicate intensely Mn-SOD immunoreactive neurons that are also immunostained for CHAT. These are large-sized neurons. Arrowheads indicate medium-sized neurons which are intensely immunoreactive for Mn-SOD, but not for CHAT. Scale (A and B): 50 #m.
176
Fig. 2. Fluorescent photomicrigraphs showing immunoreactivity for Mn-SOD (A) and somatostatin (B) in the caudate-putamen of a single section. Arrows indicate medium-sized neurons which are simultaneously immunostained for Mn-SOD and somatostatin, while small arrowheads indicate examples of medium-sized neurons which are intensely immunoreaetive for Mn,SOD but not somatostatin. The large arrowhead (A) indicates a large-sized neuron which is strongly immunostained for Mn-SOD but not for somatostatin. Scale (A and B): 50 /zm.
also a population of medium-sized neurons which were intensely immunostained for M n - S O D but not immunoreactive for somatostatin. O n e possible transmitter in these medium-sized neurons is G A B A , as most mediumsized neurons are G A B A e r g i c 7. We previously demonstrated prominent topographic distribution in the hippocampal subfields 1. CA1 pyramidal cells, which are particularly vulnerable to transient cerebral ischemia 14, were weakly immunostained for M n - S O D , whereas CA3 pyramidal cells, which are resistant to cerebral ischemia, were intensely immunore-
active. Antiperoxidative enzymes such as M n - S O D and Cu/Zn-SOD may be involved in defense against reactive oxygen free radicals formed as a result of ischemia and subsequent reoxygenation 3'14. However, magnocellular basal forebrain neurons, which undergo selective degeneration in Alzheimer's disease, demonstrated very intense immunoreactivity 9. Increased S O D activity has been reported in fibroblast cell lines derived from familial Alzheimer's patients 17 and in the basal ganglia in Parkinson's disease 13. Mitochondrial S O D activity was higher in Parkinson's disease; however, there was no
177 difference b e t w e e n cytosol S O D activity in the parkinsonian substantia nigra and control tissues. Increased S O D activity in the mitochondrial fraction may be a protective response to elevated levels of toxic free radicals in the parkinsonian basal ganglia; alternatively, it m a y induce cell death through the accumulation of h y d r o g e n peroxide. T h e r e f o r e , high levels of M n - S O D in the cholinergic neuronal system and some somatostatinergic n e u r o n systems in the neostriatum may be factors in the p a t h o l o g y and t r e a t m e n t of human neurodegen-
erative disorders such as P a r k i n s o n ' s and H u n t i n g t o n ' s diseases 11,13.
1 Akai, E, Maeda, M., Suzuki, K., Inagaki, S., Takagi, H. and Taniguchi, N., Immunocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat, Neurosci. Lett., 115 (1990) 19-23. 2 Ambani, L.M., Van Woert, M.H. and Murphy, S., Brain peroxidase and catalase in Parkinson's disease, Arch. Neurol., 32 (1975) 114-118. 3 Chan, P.H., Chu, L. and Fishman, R.A., Reduction of activities of superoxide dismutase but not of glutathione peroxidase in rat brain regions following decapitation ischemia, Brain Research, 439 (1988) 388-390. 4 Cohen, G., Oxyradical toxicity in catecholamine neurons, Neurotoxicology, 5 (1984) 77-82. 5 Eckenstein, E and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363-368. 6 Fridovich, I., Superoxide dismutase, Annu. Rev. Biochem., 44 (1975) 147-159. 7 Graybiel, A.M. and Ragsdale Jr., C.W., Biochemical anatomy of the striatum. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 427-503. 8 Iizuka, S., Taniguchi, N. and Makita, A., Enzyme-like immunosorbent assay for human manganese-containing superoxide dismutase and its content in lung cancer, J. Natl. Cancer Inst., 72 (1984) 1043-1049. 9 Inagaki, S., Takagi,H., Suzuki, K., Akai, F. and Taniguchi, N., Intense immunoreactivity for Mn-superoxide dismutase (MnSOD) in cholinergic and non-cholinergic neurons in the rat basal
forebrain, Brain Research, 541 (1991) 354-357. 10 Kawaguchi, T., Noji, S., Uda, T., Nakashima, Y., Takeyasu, A., Kawai, Y., Takagi, H., Tohyama, M. and Taniguchi, N., A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase, J. Biol. Chem., 264 (1989) 5762-5767. 11 Kowall, N.W., Ferrante, R.J. and Martin, J.B., Patterns of cell loss in Huntington's disease, Trends Neurosci., 10 (1987) 24-29. 12 McCord, J.M., Keele Jr., B.B. and Fridovich, I., An enzymebased theory of obligate anaerobiosis: the physiological function of superoxide dismutase, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 1024-1027. 13 Saggu, H., Cooksey, J., Dexter, D., Wells, ER., Lees, A., Jenner, P. and Marsden, C.D., A selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra, J. Neurochem., 53 (1989) 692-697. 14 Siesj6, B.K., Cell damage in the brain: a speculative synthesis, J. Cereb. Blood Flow Metab., 1 (1981) 155-185. 15 Shimada, S., Inagaki, S., Kubota, Y., Kito, S., Shiotani, Y. and Tohyama, M., Coexistence of substance P and enkephalin-like peptides in single neurons of the rat hypothalamus, Brain Research, 425 (1987) 256--262. 16 Vincent, S.R., Mclntosh, C.H.S., Buchan, A.N.J. and Brown, J.C., Central somatostatin systems revealed with monoclonal antibodies, J. Comp. Neurol., 238 (1985) 169-186. 17 Zemlan, EP., Thienhaus, O.J. and Bosmann, H.B., Superoxide dismutase activity in Alzheimer's disease: possible mechanism for paired helical filament formation, Brain Research, 476 (1989) 160-162.
We wish to thank Dr. F. Eckenstein for providing antibody to CHAT, Dr. J.C. Brown for a gift of somatostatin antibody and Dr. S.R. Vincent for improving the manuscript and helpful suggestions. This work was supported by the Grant-in-Aid for Scientific Research on Priority Areas No. 02240106 from the Ministry of Education, Science and Culture, Japan, and a grant provided by the Ichiro Kanehara Foundation.
