354

Brain Research, 541 (1991) 354-357

Elsevier BRES 24542

Intense immunoreactivity for Mn-superoxide dismutase (Mn-SOD) in cholinergic and non-cholinergic neurons in the rat basal forebrain S. Inagakl 1, H Takagl 1, K Suzuki 2, F Akal 3 and N Tamguchl 2 1Department of Anatomy, Osaka City Umverslty Medical School, Abeno-ku, Osaka (Japan), 2Department of Blochemzstry, Osaka Umversay Med,cal School, Ktta-ku, Osaka (Japan) and ~Department of Neurosurgery, Kmkt University School of Medicine, Sayama, Osaka (Japan) (Accepted 13 November 1990)

Key words Mn-superoxlde dlsmutase, Chohne acetyltransferase, Basal forebram, Rat

The lmmunohlstochemlcai localization of manganese (Mn)-superoxlde dtsmutase (Mn-SOD) was studied in the rat basal forebraln using polyclonal antibodies to Mn-SOD Neurons of the basal forebram exhibit a high density of Mn-SOD lmmunoreactwlty Double immunostammg with a monoclonal antibody to choline acetyltransferase demonstrated that both chohnergic and non-cholinerglc neurons in the basal forebraln are intensely Jmmunoreactlve for Mn-SOD

Superoxlde dlsmutases (SOD) catalyze the conversion of superoxlde radicals to O 2 and H 2 0 2 , and It IS very likely that they protect cells against the potential toxicity of free radical produced from oxygen 15 Mammahan tissues contain two mare forms of SOD, Mn-SOD and Cu/Zn-SOD Mn-SOD is a manganese-containing enzyme present in mltochondria, whereas Cu/Zn-SOD is a copper- and zinc-containing enzyme locahzed in the cytoplasm of various cells il i3 Biochemical studies have indicated that these enzymes are abundant In the brain as well as m the hver 3 Withm the brain, a prominent topographical distribution of Mn-SOD lmmunoreactIvity has been noted In the hlppocampal subfields i, however, little is known regarding the immunohlstochemlcal locallzatlon of this enzyme and its relationship with neurotransmitters m other areas of the bram 6 The aim of this study was to localize the Mn-SOD immunoreactlvity in the basal forebraln, and study the relationship of lmmunohlstochemlcal locahzatlon of Mn-SOD and chohnergic neuronal systems m this area Three adult male Wistar rats weighing 100 g were used The animals were anesthetized with pentobarbltal (50 mg/kg) and perfused with 200 ml of a formaldehyde fixative (2% paraformaldehyde-0 2% plcrlc acid in 0 1 M phosphate buffer) The brain was postfixed m 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 m a cryostat and processed for double immunostammg 17

Forebraln sections were first incubated overmght at 4 °C m a mixture containing rabb~t polyclonal antibody to rat Mn-SOD and rat monoclonal antibody to chohne acetyltransferase (CHAT) 7 The sections were then incubated overnight at 4 °C with a mixture of the second antiserum, fluorescent isothlocyanate (FITC)-labeled goat antirabbit IgG (Miles) to detect SOD, and Texas red-labeled sheep anti-rat IgG (Amersham) to detect ChAT immunoreactlvity Following 3 rinses In buffer, the sections were mounted on glass slides and examined with an eplfluorescence microscope (Olympus) equipped with a B-dlchronic filter system for FITC fluorescence and a G-dlchronlc filter system for Texas-red fluorescence The preparation and specIfiClties of the antlsera to Mn-SOD and ChAT have been previously described 1 7 12,13 To test the specificity of the immunostainIng, control experiments were performed by (1) omission of the primary antIsera, (2) replacement of the primary antlsera with normal rabbit and rat sera, and (3) use of primary Mn-SOD antiserum following its absorption with purified Mn-SOD (at 10 -6 M ) NO immunostammg was observed in the tissue sections prepared under these control conditions Sodium dodecyl sulfate-polyacrylamlde gel electrophoresls was performed on a 12% gel with 3% stacking gel, followed by overnight electroelutlon onto nitrocellulose paper (constant current, 100 mA) Immunoblot detection of the enzyme was performed with polyclonal rabbit antibody to rat Mn-SOD, and a second antibody,

Correspondence S Inagakl, Department of Anatomy 1st Division, Osaka City University Medical School, Asahlmachl 1-4-54, Abeno-ku, Osaka, 545 Japan 0006-8993/91/$03 50 ~) 1991 Elsevier Science Publishers B V (Biomedical Division)

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Fig 1 Immunoblot detection of rat Mn-SOD with polyclonal antibody Mltochondnal fraction from rat hver (lane 1), heart (lane 2) and brain (lane 3), and purified Mn-SOD (lane 4)

peroxldase-conlugated anti-rabbit IgG goat antibody ( T A G O ) Peroxldase actwity was then detected with a solution of 1 mg of O-dmmsldlne/0 012% H 2 0 2 m 10 m M Trls-HCl (pH 7 4)

Immunoblot analysis mdlcated that the M n - S O D anUbody only reacts with M n - S O D from mltochondrlal fraction of rat liver, heart and brain, and purified Mn-SOD (Fig 1) M n - S O D ]mmunoreactlvlty was concentrated m neuronal cells m the brain, whereas ~mmunoreactlvlty m ghal cells was absent or only weak, except around vessels The magnocellular nuclei of the rat basal forebram showed very intense M n - S O D lmmunoreactwlty (Fig 2) as compared with the ]mmunoreactwlty in pyramidal cells m the neocortex Neurons demonstrating M n - S O D lmmunoreactwlty appeared to be assooated with nuclei containing the basal forebram chohnerglc system, mcludmg the medial septum and the verttcal and horizontal hmbs of the diagonal band (Fig 2) Double staining for M n - S O D and C h A T showed the concurrent localization of these substances (Fig 3) m the basal forebram Almost all ChAT-lmmunoreactwe p e n k a r y a in the medial septum and vertical and horizontal hmbs of the dmgonal band lmmunostamed for M n - S O D , and Mn-SOD-lmmunoreactlve cells were more extenstvely d]stnbuted m these areas (Fig 3) Within the medial septum and verhcal limb of the diagonal band, most

Fig 2 Fluorescent photomicrographs showing Mn-SOD ]mmunoreactlwty m the basal forebram Intense Mn-SOD lmmunoreactwlty ]s seen m numbers of neurons m the medial septum (MS) and verncal hmb of the diagonal band (A,B), and in the horizontal hmb of the diagonal band (HDB) (C,D) Scales A + C, 100gm, B + D, 50gm

356

Fig 3 Fluorescent photomtcrographs showing the lmmunoreacUvltyfor ChAT (A) and Mn-SOD (B) m the medml septum (MS) and vertical hmb of the dmgonalband of a single section Arrows md~cate neurons lmmunostamed for ChAT and Mn-SOD, and arrowheads indicate several examples of Mn-SOD lmmunoreactwe neurons which lack ChAT lmmunostammg A large arrow indicates the mldhne Chohnerglc neurons are generally located m the lateral portmn of this regmn Scale A + B, 50 ktm

chohnerglc neurons were located laterally m these areas (Fig 3A) Furthermore, non-cholmergtc cells m the basal forebram also lmmunostamed intensely for Mn-SOD m the basal forebram (Fig 3B) Within the mechal septum and verlacal hmb of the dmgonal band, non-cholmerglc neurons, wbach were usually located m the mechal porlaon, displayed intense tmmunostammg for thts enzyme (Fig 3B) Wltban the horizontal hmb of the &agonal band, SOD-lmmunoreactave, cholmerglc neurons were mterrmngled with SODtmmunoreactwe non-cholmergac neurons The present study demonstrated intense SOD tmmunoreact~vlty m chohnerglc and non-chohnerg~c neurons in the basal forebraln, that ~s, the medml septum and the vertical and horizontal hmbs of the dmgonal band One possible transmitter in SOD-lmmunoreactlve non-chohnerg~c neurons may be G A B A GABA-contammg, nonchohnerg~c neurons are also present m the projectmns to the hlppocampus and n e o c o r t e x 9'14 The neocortexh~ppocampus clrcmt is widely beheved to be crucial for memory This circuit is influenced by subcortlcal affe-

rents derwed from neurons m the basal forebram Chohnerglc input to the hlppocampus from the septum is hkely to be revolved m feed-forward dlsmhlbmon 4 Electrophyslologlcal studies indicated that non-chohnerglc afferent neurons from the septum facdltate__responses evoked by st~mulatmn of the major excitatory pathways m the hlppocampus s Non-chohnerglc (probably GABA-medtated) input to the hlppocampus may be revolved m dlsmhlblUon of the pyramidal neurons of the hlppocampus 1° The achvlty of the antloxldatwe enzymes, Mn-SOD and Cu/Zn-SOD, may relate to the defense capabdlty of brain tissues, such as the hlppocampus and cerebral cortex, against reactwe oxygen free radicals formed as the result of lschemla and subsequent reoxygenaUon5A6 Therefore, high levels of Mn-SOD m the chohnerglc and non-chohnerglc basal forebram systems, which extenswely mnervate the neocortex and hlppocampus 2'914, may be slgmficant factors m the pathology and treatment of human neurological disorders such as Alzhelmer's disease TM

357 We wish to thank Dr Eckenstem for providing antiserum to ChAT This work was partly supported by the Grant-m-Aid for Scaentlfic Research on Priority Areas No 02240106 from the

Mlmstry of Education, Soence and Culture, Japan, and a grant prowded by the Ichlro Kanehara Foundation

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Intense immunoreactivity for Mn-superoxide dismutase (Mn-SOD) in cholinergic and non-cholinergic neurons in the rat basal forebrain.

The immunohistochemical localization of manganese (Mn)-superoxide dismutase (Mn-SOD) was studied in the rat basal forebrain using polyclonal antibodie...
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