Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3195-3199, April 1990 Neurobiology Immunocytocheniical localization of the a subspecies of protein kinase C in rat brain (in situ hybridization histochemistry) ATSUKO ITO*, NAOAKI SAITO*, MIDORI HIRATA*, AKIKO KOSE*, TAKESHI TsUJINO*, CHIKA YOSHIHARA*, Kouji OGITAt, AKIRA KISHIMOTOt, YASUTOMI NISHIZUKAt, AND CHIKAKO TANAKA** Departments of *Pharmacology and tBiochemistry, Kobe University School of Medicine, Kobe 650, Japan Contributed by Yasutomi Nishizuka, January 2, 1990 ABSTRACT The distribution ofthe a subspecies ofprotein types, with limited intracellular localization. The present kinase C (PKC) in rat brain was demonstrated immunocy- studies were undertaken to identify a-PKC in the rat brain by tochemically by using polyclonal antibodies raised against a using immunocytochemistry, and the results show that this synthetic oligopeptide corresponding to the carboxyl-terminal PKC subspecies is enriched in particular cell types. sequence of a-PKC. The a-PKC-specific immunoreactivity was widely but discretely distributed in both gray and white MATERIAL AND matter. The immunoreactivity was associated predominantly METHODS with neurons, particularly with perikaryon, dendrite, or axon, Preparation of Antibodies Against a-PKC. The carboxyl- but little was seen in the nucleus. Glial cells expressed this PKC terminal portion of a-PKC (residues 662-672; Gln-Phe-Val- subspecies poorly, if at all. The highest density of immuno- His-Pro-Ile-Leu-Gln-Ser-Ala-Val) was selected as a se- reactivity was seen in the olfactory bulb, septohippocampal quence specific to a-PKC. The oligopeptide was coupled to nucleus, indusium griseum, islands of Calleja, intermediate keyhole limpet with m-maleimidobenzoic acid N-hydrox- part of the lateral septal nucleus, and Ammon's horn. A ysuccinimide ester. Rabbits were immunized with the immu- moderately high density of the immunoreactivity was seen in nogen by the method described (23, 24) and were bled 1 week the anterior olfactory nucleus, anterior commissure, cingulate after the third booster administration. The IgG fraction was cortex, dentate gyrus, compact part of the substantia nigra, obtained from the antisera by affinity chromatography on interpeduncular nucleus, inferior olive, and olivocerebeilar Sepharose CL-4B coupled to goat anti-rabbit IgG, and the tract. This distribution pattern was consistent with that ob- fraction was used as a-PKC-specific antibodies. tained by in situ hybridization histochemistry. The distribution Immunoblotting Analysis. Specificity of the antibodies was of a-PKC immunoreactivity was different from that of P(,- examined by immunoblotting analysis using the three sub- (,B-I and y-PKC immunoreactivity. These findings suggest that types of rat brain PKC (types I, II, and III) and four a-PKC is involved heavily in the control ofspecific functions of subspecies of PKC (a-, 13-, 1I-, and -PKC). These subspe- some restricted neurons. cies were purified from COS-7 cells transfected with the respective cDNA-containing plasmids as described (3, 4). Protein kinase C (PKC) was once thought to be a single The enzyme samples were subjected to NaDodSO4/7.5% entity, but molecular cloning and enzymatic studies have polyacrylamide slab gel electrophoresis as described by revealed that the enzyme is a large family with multiple Laemmli (25) and transferred to nitrocellulose paper. The subspecies. At least seven subspecies of PKC (a, 81, 813, y, paper was incubated with the a-PKC-specific antibodies, and 8, E, and D have been identified from analysis ofthe rat brain immunoreactive bands were visualized by the peroxidase- cDNA library (reviewed in ref. 1). Early enzymatic studies antiperoxidase method. have shown that rat brain PKC can be separated into three Immunocytochemical Staining. Frontal sections of the rat distinct fractions, types I, II, and III, by hydroxyapatite brain were prepared as described (21) by using a periodate/ column chromatography (2), which correspond to y-, ,1- and lysine/paraformaldehyde fixative containing 0.05% picric 813,-, and a-PKC, respectively (3, 4). These PKC subspecies acid. The sections were preincubated for 20 min with 0.3% are subtly different from one another in their kinetic prop- H202 and 5% normal goat serum in phosphate-buffered saline erties, mode of activation, and most likely substrate speci- (pH 7.4) containing 0.03% Triton X-100 (PBS-T), then incu- ficity (1, 5-8). Early analysis of the mRNA levels has bated for 18 hr at 4°C with the purified antibodies diluted indicated that a-, 1I-, and 81,-PKC are expressed in a variety 1:500 in PBS-T, washed, incubated for an additional 4 hr with of tissues, whereas y-PKC is expressed only in the central goat anti-rabbit IgG (MBL) diluted 1:500, washed, and incu- nervous system (9-12). Enzymatic and immunochemical bated for an additional 90 min with rabbit peroxidase-anti- analysis has also shown that a-PKC (type III) is most peroxidase complex (ICN) diluted 1:500. After three rinses, commonly distributed in many tissues and cell types (13, 14). the sections were developed with 0.02% 3, 3'-diaminobenzi- On the basis of these studies, it has been suggested that dine (Sigma) and 0.2% nickel ammonium sulfate in 50 mM a-PKC plays a role of crucial importance in the control of Tris-HCI (pH 7.4) with 0.005% H202. All sections were common processes in cell functions (1). observed and photographed under a Zeiss microscope. The Several laboratories have carried out immunocyto- pattern ofimmunostaining was analyzed on an IBAS II image chemical studies using antibodies specific to each type of analyzer (Zeiss). PKC (15-19). In earlier reports (20-24), with subspecies- In Situ Hybridization Histochemistry. A synthetic oligode- specific antibodies, it was shown that in the rat brain 1,-, 1I-, oxynucleotide complementary to the rat a-PKC sequence and y-PKC are differentially distributed in particular cell described by Ono et al. (26) was prepared with an Applied Biosystems DNA sythesizer (model 381A). The probe was The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PKC, protein kinase C. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3195 Downloaded by guest on September 26, 2021 31% Neurobiology: Ito et al. Proc. Natl. Acad. Sci. USA 87 (1990) buffer (pH 7.4), and the brains were dissected. Frozen sections 15 pim thick were thaw-mounted on poly-L-lysine- 204.9 * coated glass slides. The sections were treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9%o NaCl (pH 8.0) and transferred through graded alcohols (70-100%) and 103.2w chloroform. After a 3-hr prehybridization in buffer containing 40% (vol/vol) formamide, 10% (wt/vol) dextran, 4x SSC (1x 67.5 SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7), 0.02 M Tris HCI (pH 7.4), 1 x Denhardt's solution (0.02% 41.8 x polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum al- bumin), salmon sperm DNA (20 mg/ml), and yeast tRNA (0.25 mg/ml), hybridization was carried out at 440C for 24 hr 17.8 )m- in the same buffer containing 106 cpm of labeled probe. The section was washed three times in 1 x SSC/20% formamide a b C d e f g h at 460C and exposed to x-ray film for 3-7 days. FIG. 1. Immunoblot analysis with antibodies against a-PKC. Samples were subjected to NaDodSO4/7.5% polyacrylamide slab gel RESULTS electrophoresis. The proteins were transferred to nitrocellulose Characterization of Antibodies. Purified antibodies against paper, incubated with antibodies against the a-PKC-specific oligo- peptide (1:500 dilution), and visualized by the peroxidase- the oligopeptide specific to a-PKC detected a protein in the anti-peroxidase method. Lanes: a, marker proteins; b, crude soluble rat brain soluble fraction that corresponded to PKC with a fraction ofthe whole rat brain; c, type I PKC; d, type II PKC; e, type molecular mass of =80 kDa (Fig. 1, lane b). It was also shown III PKC; f, a-PKC; g, ,/3-PKC; h, ,/11-PKC; i, y-PKC. Molecular sizes that the antibodies detected the 80-kDa band only in the (in kDa) of marker proteins are indicated on the left. fraction of type III but not of type I or II PKC (Fig. 1, lanes c-e). The antibodies stained only a-PKC and not other complementary to bases 2257-2301, present in the 3' non- subspecies encoded by sequence f31, 1I,, or y (Fig. 1, lanes coding region of a-PKC cDNA. The probe was labeled at the f-i). 3' end by using terminal deoxynucleotidyl transferase and Immunocytochemical Localization of a-PKC. General dis- [a-35S]dATP. Anesthetized rats were perfused with ice-cold tribution of a-PKC immunoreactivity. Fig. 2 shows the fixative containing 4% paraformaldehyde in 0.1 M phosphate patterns of immunostaining of a-PKC that were analyzed FIG. 2. Color images of the distribution of a-PKC immunoreactivity in frontal sections of rat brain [rostral (A) to caudal (G)]. The a-PKC immunoreactivity was localized by peroxidase-anti-peroxidase immunocytochemistry. Relative density of the immunoreactivity is shown according to the color scale (highest density, red; lowest density, black). The highest density of immunoreactivity (red) was seen in the external plexiform layer of the olfactory bulb (A), indusium griseum (B), islands of Calleja (B), intermediate part of the lateral septal nucleus (B), and Ammon's horn (C). A moderately high density of immunoreactivity (yellow) was seen in the anterior olfactory nucleus (A), anterior commissure (B), dentate gyrus (C), cingulate cortex (C and D), compact part of the substantia nigra (D), interpeduncular nucleus (D), inferior olive (F), and olivocerebellar tract (F).