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Immunohistochemical distribution of β-protein kinase C in rat hippocampus determined with an antibody against a synthetic peptide sequence
Brain Research
Bulletin,
Vol. 22, pp. 893-897.
0361.9230189 $3.00 + .OO
Maxwell Pergamon Macmillan plc, 1989. Printed in the U.S.A.
Immunohistochemical Distribution of P-Protein Kinase C in Rat Hippocampus Determined With an Antibody Against a Synthetic Peptide Sequence BRYAN L. ROTH,*’ MICHAEL J. IADAROLA,? JOHN P. MEHEGAN* AND DAVID M. JACOBOWITZS *Naval Medical Research Institute, Bethesda, MD 20814 _iLaboratory of Neurobiology and Anesthesiology, National Institute of Dental Research, Bethesda, MD 20892 fLaboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 Received 3 January
1989
ROTH, B. L., M. J. IADAROLA, J. P. MEHEGAN AND D. M. JACOBOWITZ. Immunohisfochemical distribution of P-protein kinase C in rat hippocampus determined with an antibody against a synthetic peptide sequence. BRAIN RES BULL 22(5) 893-897, 1989. -An antibody directed against a synthetic peptide sequence specific for the P-subtype of protein kinase C (PKC) was used to determine the distribution of p-PKC in rat hippocampus by immunocytochemistry. PKC was distributed primarily in the stratum oriens and radiatum of the CA1 region. Positive staining cell bodies were only observed after colchicine treatment in pyramidal cells (CA2-CA4) and granule cells of the dentate gyrus. The discrete localization of various subtypes of PKC should provide clues to their functions. P-Protein kinase C
Hippocampus
Immunohistochemistry
previously detailed (10). A search of the protein database disclosed that the sequence was specific for the P-isozyme of PKC. The conjugated peptide was dissolved in saline (1 mg/ml), mixed with an equal volume of complete Freund’s adjuvant and 1 ml of the emulsion was injected in multiple subcutaneous locations on the backs of two New Zealand White rabbits. The rabbits were boosted every 10 days and bled every 4-6 weeks via the ear artery. For these experiments, the third, fourth and fifth bleeds were used. Protein kinase C was purified to homogeneity by a modification of previously published procedures (17,20) to a final specific activity of 3,400 nmole/min/mg histone III-S phosphorylating activity and 10,000 pmole/mg [3H]-phorbol-12, 13-dibutyrate binding activity; these results are similar to previously published findings (17,20). Ten percent SDS gels were performed as described by Laemmli (9); Western blots were performed as described by Towbin et al. (19) overnight at 20 volts. Nitrocellulose membranes were blocked with 3% gelatin and TBS (500 mM NaCl, 10 mM Tris-Cl, pH 7.40) for 2 hr, washed in TTBS (TBS containing 0.05% Tween-20) then incubated with a 1500 dilution of antibody in a solution of TTBS containing 1% gelatin/l% bovine serum albumin for 2 hr, then incubated with TTBS
PROTEIN kinase C (PKC) is a calcium and phospholipid-dependent enzyme which is activated by phorbol esters and diacylglycerol (DAG) (6) and has been found to be highly enriched in nervous tissue (22). PKC was shown by receptor autoradiography to be concentrated in hippocampus with the highest amounts found in the CA1 region (22). Many neurotransmitters and neuromodulators may activate hippocampal PKC by stimulating hydrolysis of phosphoinositides to release DAG (13,16). In the hippocampus, PKC activation is important for induction of long-term potentiation, feedback inhibition of PI metabolism and modulation of ion-channel activity (1, 3, 16). Recently several isozymes of PKC were identified by cloning and sequencing of cDNAs (7, 8, 14). The present study represents the beginning of a systematic mapping of these subtypes of PKC within the hippocampus. For this report, we used an antibody we prepared to a peptide specific for the P-isozyme of PKC. METHOD
A peptide corresponding to residues 307-327 of the P-PKC (8) (RAKIGQGTKAPEEKTANTISK) was synthesized and purified using an automated peptide synthesizer (Model 430A, Applied Biosystems, Inc.) and coupled to bovine serum albumin as
‘Requests for reprints should be addressed to Bryan L. Roth, M.D., Ph.D., Department University Medical Center, Stanford, CA 94305.
893
of Psychiatry
and Behavioral
Sciences, Room S-253, Stanford
XYJ
K07’H
D
ET Al..
E
FIG. I. Western blot analysis of purified PKC and hippocampal
homogenates. Shown are typical results obtained with antibody to (j-PKC 28. Lane A shows Coomasaie Blue stained molecular weight standards (AMyosin heavy chain, 200 kDa; P-galactosidase. II6 kDa; phosphorylase b. 97 kDa: bovine serum albumin, 66.2 kDa; and ovalbumin 43 kDa). Lane B shows Coomasie Blue stained PKC as a doublet CM,=80 and 78 kDa); lane C show5 a hippocampal homogenate. Lane D shows PKC visualized with the antibody while lane E shows hippocampal PKC (corresponding to lane C) visualized with the antibody.
containing [‘251]-protein A (76 &i/kg; 0. I @/ml) for 2 hr. dried and autoradiographed with Kodak X-OMAT film. For immunocytochemistry two procedures were followed: I ) rats were anesthetized with chloropent and perfused via the ascending aorta with 50-75 ml of cold phosphate-buffered saline (PBS) containing 0.5% sodium nitrate followed by 300-400 ml of 10% formalin (4°C) in 65 mM sodium phosphate buffer (pH 7.0). Brains were removed, cut into slabs 3 mm thick and placed in fixative for 2-3 hr and sectioned on a vibratome as previously described (5). The antiserum was diluted 1: 1000 in PBS containing 0.3% Triton X- 100 and 1% goat serum. 2) Nonperfused normal and colchicine-treated (intraventricular, 100 kg. 2 days) rats were decapitated and brains were removed, cut into 3 mm thick slices and placed in cold 10% formalin for 2 hr, followed by cold 20% sucrose in PBS (pH 7.4) for 3 days. The brain slices were frozen on dry ice and cut in a cryostat ( - 18°C) at 20 pm sections. These sections were incubated in affinity purified antisera diluted 150 in PBS containing 0.3% Triton X-100 and 1% goat serum. Sections were incubated for 2-4 days at 4°C and then washed 3 times with PBS containing 0.2% Triton X-100 for 5 min each and further incubated in fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (I:300 in PBS containing 0.3% Triton X-100) for 30 min. The sections were then washed 3 times with PBS containing 0.2% Triton X-100 for 10 min each, rinsed in PBS alone and mounted in glycerol-PBS mounting medium containing p-phenylenediamine. Fluorescence was monitored under a Leitz Orthoplan fluorescence microscope equipped with a Ploem illuminator. Immunohistochemical controls were 1) preimmune serum and 2) antiserum preabsorbed with purified PKC. Preabsorption was performed by incubating slides with solution consisting of diluted antibody and 1 (LM PKC for 2 days. Both preimmune serum and the preabsorption controls failed to stain tissue (not shown).
RESULTS
Figure 1 shows typical result3 obtained with our methods. Lane B shows the purified PKC p@aration we used as an authentic standard; a doublet corresponding to 78 and 80 kDa is evident. Lane C shows a silver stained gel of hippocampus; lane D shows the Western blot with antigen of lane B with PKC visualized as above, while lane E is the Western blot corresponding to lane C. It is clear that under the conditions of our assay, antibody 2B visualizes only PKC in the hippocampus. This antibody was then used for subsequent histochemical studies. Complete biochemical characterization of PKC and our antibody has been previously reported ( 17). Coronal sections through the brain at the hippocampal level revealed a dense array of fluorescent fibers in the stratum oriens and radiatum which appeared to course in a perpendicular direction to and through the pyramidal cell layer in the CA1 region (Fig. 2A, C). A greater density of fibers was frequently observed in close proximity to the pyramidal cell layer (Fig. 2C). The remaining portion of the hippocampus (CA2-CA4 and dentate gyrus) contained a comparatively sparse number of nerve processes (Fig. 2A). Following colchicine treatment fluorescence was only observed in the pyramidal cells of the CA2ZCA4 region (Figs. 2 and 3) and the granule cells of the dentate gyrus (Fig. 3A, B). Short processes were observed to emanate from these cells. No immunofluorescence could be observed in the pyramidal cells of the CA1 area. These cells were in proximity to fibers which appeared thicker and more intensely fluorescent than those processes in the remaining oriens and radiatum layers (Fig. 2C). DISCUSSION
These results are the first to demonstrate
the distribution of the
P-PROTEIN KINASE C IN HIPPOCAMPUS
895
FIG. 2. Cryostat section of the dorsal hippocampus. Colchicine treatment. (A) Low power view of CAI-CA2 region (X 50). (B) Enlarged view of the CA3 region seen in Fig. 2A. Note cytoplasmic staining of the pyramidal cells of the CA3 area ( X 120). (C) Enlarged view of the CA1 and CA2 area. Dense fibers are seen in the stratum oriens (SO) and stratum radiatum (SR) ( x 75).
p-PKC in rat hippocampus. The immunocytochemical results show that P-PKC is distributed in a heterogenous fashion within the hippocampus. Intense staining was evident throughout CA1 with a dramatic decrease in intensity in the remaining areas. Within the CA 1, an abundant network of fibers was present in the
regions of stratum oriens and radiatum adjacent to the pyramidal cell layer. No fluorescent cells were observed in the CA1 pyramidal layer in rats with or without colchicine treatment, although the other regions (CA2-CA4) did reveal immunoreactivity only after colchicine treatment. The significance of this is not
FIG. 3. Cryostat section of the dentate gyms. Colchicine treatment. (A) Low power view of the granule cells (GC) and the pyramidal cells of the CA4 area ( X 55). (B) Enlarged view CA4 pyramidal cells ( x IIO) and (C) Granule cells ( X 180).
immediately apparent although it would be of interest if this is a reflection of translocation of protein kinase C activity. This phenomenon has been suggested to be responsible for the mediation of hippocampal long-term potentiation (I). Furthermore in situ hybridization studies suggest that PKC is synthesized in pyramidal cell bodies since the P-PKC mRNA is found in pyramidal cells (4). Thus, it would appear that the storage capacity of P-PKC in the perikaryon is minimal (especially in the CA1 area) and that transport into dendrites and axons occurs immediately after synthesis. The enzyme positive fibers in proximity to the pyramidal cells suggests PKC may be involved in feedforward or feedback functioning of pyramidal cells.
Previous studies by Worley et al. (22) examined the distribution of phorbol ester binding sites within the hippocampus. In general, the distribution of the l3-isozyme of PKC parallels the location of phorbol ester binding sites. Interestingly, Worley et al. also noticed few phorbol ester binding sites in the pyramidal cells (22). Other investigators have studied the localization of PKC using monoclonal and polyclonal antibodies. In none of these previous studies was it known which of the various isozymes of PKC were labelled. In general, these studies have found dense staining of pyramidal cells and pyramidal cell processes ( 12,2 1). The patterns of staining previously reported were quite distinct from those of our study. Mochly-Rosen et al. (12). for example. report that the
897
B-PROTEIN KINASE C IN HIPPOCAMPUS
apical dendrites of pyramidal cells were labelled with their monoclonal antibody CK 1.12, while other workers demonstrated staining of pyramidal cell nuclear membranes (21). By contrast, our antibody showed almost no staining of pyramidal cell bodies in the CA1 area and intense staining of both the stratum radiatum and stratum oriens. The localization of dense processes above and below the pyramidal cell layer suggests the possibility that much of the immunoreactivity observed in the CA 1 layer is contained in basket cells which are in the stratum oriens immediately adjacent to the pyr~idal cells (I I, 15, 18). Our inability to microscopically visualize cell bodies in this layer may be due to the large density of processes in the stratum oriens thereby in effect serving to camouflage the cell soma. On the other hand it could be argued that in situ hybridization studies revealed PKC mRNA in the pyramidal cells of CA 1 (31. However, the oligonucleotide probes that Brandt er al. (4) employed to detect the BI and BII forms of PKC are not completely specific for either subtype. Thus, both probes for instance, appear to preferentially recognize the common unspliced precursor mRNA (4), and show a much smaller degree of hybridization to the differentially spliced mature mRNA forms. In situ hybridization studies might, therefore, preferentially recognize cells which synthesize precursor mRNA’s and not mature, spliced mRNA forms. Also, there is no a priori reason to suspect that there is a direct relationship between cellular levels of a particular mRNA and its corresponding protein. For instance, the Brandt ef al. (4) study suggests the BI and BII forms of PKC are equally distributed in hippocampus while a recent study by Nishizuka’s group (14) shows the BII/BI>97/1. Finally. Ase et al. (2) employing type-specific antibodies discerned striking dissimilarities between in situ hybridization studies (4) and immunohis-
tochemical localization of PKC. Thus, it is not surprising that our histochemical results suggest a localization of B-PKC in basket cells in the CA1 region. Ono et al. have found that as many as seven distinct isozymes of rat brain PKC may exist (14). Our antibody is directed against a domain (V3) of the molecule which is quite variable among the PKC subtypes. No sequence homology has been found comparing the sequence of our synthetic antigen and the published sequences of the other PKC subtypes or other proteins for which sequences have been published. Thus, the differences we observed in the localization of B-PKC within hipp~ampus probably reflect the fact that we are studying only one of the seven subtypes of PKC. These results also demonstrate the power of the approach of using a synthetic peptide antigen for studying a multigene family. Conventional techniques of protein purification can only resolve, at most, three peaks of PKC activity each of which probably contains multiple isozymes (7). Thus, obtaining antibodies specific for all seven subtypes is nearly impossible utilizing currently available techniques of protein biochemistry. An alternative approach is the use of synthetic peptide antigens specific for individual subtypes. As our results show, antibodies prepared in such a manner are quite specific and sensitive and can useful for both biochemical and histochemical studies. In conclusion, we describe the distribution of B-PKC in rat hippocampus using an antibody directed against a unique peptide sequence. We found PKC distributed in a heterogenous fashion through the hipp~ampus with intense staining in a network of fibers. Further identi~cation of specific patterns of dis~bution for the various subtypes of PKC should give us additional clues to the function of this multigene enzyme family.
REFERENCES 1. Akers, R. F.; Lovinger, D. M.: Colley, P. A.; Linden, D. J.; Routtenberg. A. The translocation of protein kinase C activity may mediate hippocampal long-term potentiation. Science 231:587-589; 1986. M. S.; Kikkawa, Il.; Ono. Y.; 2. Ase, K.; Saito, N.; Shearman, Igarashi, K.; Tanaka, C.; Nishizuka, Y. Distinct cellular expression of BI and BII-subspecies of protein kinase C in rat cerebellum. J. Neurosci. 8:3850-3856; 1988. 3. Baraban, J. M.; Snyder, S. H.; Alger, B. E. Protein kinase C regulated ionic conductance in hippocampal pyramidal neurons: electrophysiological effects of phorbol esters. Proc. Natl. Acad. Sci. USA 82:2538-2542; 1985. 4. Brandt, S. J.: Neidel, J. E.; Bell, R. M.; Young, W. S. Distinct patterns of expression of different protein kinase C mRNAs in rat tissues. Cell 4957-63; 1987. 5. Jacobowitz, D. M. Removal of discrete fresh regions of the rat brain. Brain Res. SO:1 1l-1 15: 1974. 6. Kikkawa, Il.; Takai, Y.; Tanaka, Y.; Miyake, R.; Nishizuka, Y. Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J. Biol. Chem. 258:11452-l 1455; 1983. 7. Kikkawa, U.: Ono, Y.; Ogita, K.; Fujii, T.; Asaoka, Y.; Sekiguchi, K.; Kosuka, Y.: Igarushi, K.; Nishizuka, Y. Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain. FEBS Lett. 217:227-23 1; 1987. x. Knopf, J. L.; Let, M-H.; Sultzman, L. A.; Kriz, R. W.; Loomis, C. R.: Hewick, R. M.: Bell, R. M. Cloning and exoression of multiole I protein kinase C cDNAs. Cell 46:491-502; 1987. 9. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227680685; 1970. 10. Lidner, W.; Robey, F. A. Automated synthesis and use of Nchloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens. Int. J. Pept. Protein Res. 30:794-800; 1987. 11. Lorente de No, R. Studies on the structure of the cerebral cortex. II. continuation of the study of the ammonic system. J. Psychol. Neurol. 46113-177; 1934.
D.; Basbaum, A. 1.; Koshland, D. E., Jr. Distinct 12 Mochley-Rosen, cellular and regional localization of immunoreactive protein kinase C in rat brain. hoc. Natl. Acad. Sci. USA 844660-4664; 1987. 13. Nicoletti. F.; Meek, J. L.; Iadarola, M. J.; Chuang, D-M.; Roth, B. L.; Costa, E. Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus. J. Neurothem. 46:4046; 1986. 14. Ono, Y .; Fujii, T.; Ogita, K.; Kikkawa, U.; Igarashi, K.; Nishizuka, Y. Identification of three additional members of rat protein kinase C family: 6 E and 5. FEBS Lett. 226125-128; 1987. 15, Ramon y Cajal, S. Histologie du systeme nerveux de I’homme et des vertes. Paris: A. Moloine; 1911. 16. Roth, B. L.; Chuang, D. M. Minireview: multiple mechanisms of serotonergic signal transduction. Life Sci. 41:1051-1064; 1987. 17. Roth, B. L.; Mehegan, J. P.; Jacobowitz, D. M.; Robey, F.; Iadaroia, J. Rat brain protein kinase C: Purification, antibody production and quanti~cation in discrete regions of hip~campus. J. Neurochem.; in press. 18. Schwartzkroin, P. A.; Kunkel, D. D. Morphology of identified intemeurons in the CA1 regions of guinea pig hippocampus. J. Comp. Neurol. 232:205-218; 1985. 19. Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from ~lyac~lamide gels to ni~oceIluIo~ sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979. 20. Walton, G. M.; Bertics, P. J.; Hudson, L. G.; Vedvick, T. S.; Gill, G. N. A three-step purification procedure for protein kinase C: characterization of the purified enzyme. Anal. Biochem. 161:425437; 1987. 21. Wood. J. G.; Girard, P. R.; Mazzei, G. J.; Kuo, J. F. Immunocytochemical localization of protein kinase C in identified neuronal compartments of rat brain. J. Neurosci. 6:2571-2577; 1986. 22. Worley, P. F.; Baraban, J. M.; Snyder, S. H. Heterogenous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding. J. Neurasei. 6:199-207; 1986.
Bulletin,
Vol. 22, pp. 893-897.
0361.9230189 $3.00 + .OO
Maxwell Pergamon Macmillan plc, 1989. Printed in the U.S.A.
Immunohistochemical Distribution of P-Protein Kinase C in Rat Hippocampus Determined With an Antibody Against a Synthetic Peptide Sequence BRYAN L. ROTH,*’ MICHAEL J. IADAROLA,? JOHN P. MEHEGAN* AND DAVID M. JACOBOWITZS *Naval Medical Research Institute, Bethesda, MD 20814 _iLaboratory of Neurobiology and Anesthesiology, National Institute of Dental Research, Bethesda, MD 20892 fLaboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 Received 3 January
1989
ROTH, B. L., M. J. IADAROLA, J. P. MEHEGAN AND D. M. JACOBOWITZ. Immunohisfochemical distribution of P-protein kinase C in rat hippocampus determined with an antibody against a synthetic peptide sequence. BRAIN RES BULL 22(5) 893-897, 1989. -An antibody directed against a synthetic peptide sequence specific for the P-subtype of protein kinase C (PKC) was used to determine the distribution of p-PKC in rat hippocampus by immunocytochemistry. PKC was distributed primarily in the stratum oriens and radiatum of the CA1 region. Positive staining cell bodies were only observed after colchicine treatment in pyramidal cells (CA2-CA4) and granule cells of the dentate gyrus. The discrete localization of various subtypes of PKC should provide clues to their functions. P-Protein kinase C
Hippocampus
Immunohistochemistry
previously detailed (10). A search of the protein database disclosed that the sequence was specific for the P-isozyme of PKC. The conjugated peptide was dissolved in saline (1 mg/ml), mixed with an equal volume of complete Freund’s adjuvant and 1 ml of the emulsion was injected in multiple subcutaneous locations on the backs of two New Zealand White rabbits. The rabbits were boosted every 10 days and bled every 4-6 weeks via the ear artery. For these experiments, the third, fourth and fifth bleeds were used. Protein kinase C was purified to homogeneity by a modification of previously published procedures (17,20) to a final specific activity of 3,400 nmole/min/mg histone III-S phosphorylating activity and 10,000 pmole/mg [3H]-phorbol-12, 13-dibutyrate binding activity; these results are similar to previously published findings (17,20). Ten percent SDS gels were performed as described by Laemmli (9); Western blots were performed as described by Towbin et al. (19) overnight at 20 volts. Nitrocellulose membranes were blocked with 3% gelatin and TBS (500 mM NaCl, 10 mM Tris-Cl, pH 7.40) for 2 hr, washed in TTBS (TBS containing 0.05% Tween-20) then incubated with a 1500 dilution of antibody in a solution of TTBS containing 1% gelatin/l% bovine serum albumin for 2 hr, then incubated with TTBS
PROTEIN kinase C (PKC) is a calcium and phospholipid-dependent enzyme which is activated by phorbol esters and diacylglycerol (DAG) (6) and has been found to be highly enriched in nervous tissue (22). PKC was shown by receptor autoradiography to be concentrated in hippocampus with the highest amounts found in the CA1 region (22). Many neurotransmitters and neuromodulators may activate hippocampal PKC by stimulating hydrolysis of phosphoinositides to release DAG (13,16). In the hippocampus, PKC activation is important for induction of long-term potentiation, feedback inhibition of PI metabolism and modulation of ion-channel activity (1, 3, 16). Recently several isozymes of PKC were identified by cloning and sequencing of cDNAs (7, 8, 14). The present study represents the beginning of a systematic mapping of these subtypes of PKC within the hippocampus. For this report, we used an antibody we prepared to a peptide specific for the P-isozyme of PKC. METHOD
A peptide corresponding to residues 307-327 of the P-PKC (8) (RAKIGQGTKAPEEKTANTISK) was synthesized and purified using an automated peptide synthesizer (Model 430A, Applied Biosystems, Inc.) and coupled to bovine serum albumin as
‘Requests for reprints should be addressed to Bryan L. Roth, M.D., Ph.D., Department University Medical Center, Stanford, CA 94305.
893
of Psychiatry
and Behavioral
Sciences, Room S-253, Stanford
XYJ
K07’H
D
ET Al..
E
FIG. I. Western blot analysis of purified PKC and hippocampal
homogenates. Shown are typical results obtained with antibody to (j-PKC 28. Lane A shows Coomasaie Blue stained molecular weight standards (AMyosin heavy chain, 200 kDa; P-galactosidase. II6 kDa; phosphorylase b. 97 kDa: bovine serum albumin, 66.2 kDa; and ovalbumin 43 kDa). Lane B shows Coomasie Blue stained PKC as a doublet CM,=80 and 78 kDa); lane C show5 a hippocampal homogenate. Lane D shows PKC visualized with the antibody while lane E shows hippocampal PKC (corresponding to lane C) visualized with the antibody.
containing [‘251]-protein A (76 &i/kg; 0. I @/ml) for 2 hr. dried and autoradiographed with Kodak X-OMAT film. For immunocytochemistry two procedures were followed: I ) rats were anesthetized with chloropent and perfused via the ascending aorta with 50-75 ml of cold phosphate-buffered saline (PBS) containing 0.5% sodium nitrate followed by 300-400 ml of 10% formalin (4°C) in 65 mM sodium phosphate buffer (pH 7.0). Brains were removed, cut into slabs 3 mm thick and placed in fixative for 2-3 hr and sectioned on a vibratome as previously described (5). The antiserum was diluted 1: 1000 in PBS containing 0.3% Triton X- 100 and 1% goat serum. 2) Nonperfused normal and colchicine-treated (intraventricular, 100 kg. 2 days) rats were decapitated and brains were removed, cut into 3 mm thick slices and placed in cold 10% formalin for 2 hr, followed by cold 20% sucrose in PBS (pH 7.4) for 3 days. The brain slices were frozen on dry ice and cut in a cryostat ( - 18°C) at 20 pm sections. These sections were incubated in affinity purified antisera diluted 150 in PBS containing 0.3% Triton X-100 and 1% goat serum. Sections were incubated for 2-4 days at 4°C and then washed 3 times with PBS containing 0.2% Triton X-100 for 5 min each and further incubated in fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (I:300 in PBS containing 0.3% Triton X-100) for 30 min. The sections were then washed 3 times with PBS containing 0.2% Triton X-100 for 10 min each, rinsed in PBS alone and mounted in glycerol-PBS mounting medium containing p-phenylenediamine. Fluorescence was monitored under a Leitz Orthoplan fluorescence microscope equipped with a Ploem illuminator. Immunohistochemical controls were 1) preimmune serum and 2) antiserum preabsorbed with purified PKC. Preabsorption was performed by incubating slides with solution consisting of diluted antibody and 1 (LM PKC for 2 days. Both preimmune serum and the preabsorption controls failed to stain tissue (not shown).
RESULTS
Figure 1 shows typical result3 obtained with our methods. Lane B shows the purified PKC p@aration we used as an authentic standard; a doublet corresponding to 78 and 80 kDa is evident. Lane C shows a silver stained gel of hippocampus; lane D shows the Western blot with antigen of lane B with PKC visualized as above, while lane E is the Western blot corresponding to lane C. It is clear that under the conditions of our assay, antibody 2B visualizes only PKC in the hippocampus. This antibody was then used for subsequent histochemical studies. Complete biochemical characterization of PKC and our antibody has been previously reported ( 17). Coronal sections through the brain at the hippocampal level revealed a dense array of fluorescent fibers in the stratum oriens and radiatum which appeared to course in a perpendicular direction to and through the pyramidal cell layer in the CA1 region (Fig. 2A, C). A greater density of fibers was frequently observed in close proximity to the pyramidal cell layer (Fig. 2C). The remaining portion of the hippocampus (CA2-CA4 and dentate gyrus) contained a comparatively sparse number of nerve processes (Fig. 2A). Following colchicine treatment fluorescence was only observed in the pyramidal cells of the CA2ZCA4 region (Figs. 2 and 3) and the granule cells of the dentate gyrus (Fig. 3A, B). Short processes were observed to emanate from these cells. No immunofluorescence could be observed in the pyramidal cells of the CA1 area. These cells were in proximity to fibers which appeared thicker and more intensely fluorescent than those processes in the remaining oriens and radiatum layers (Fig. 2C). DISCUSSION
These results are the first to demonstrate
the distribution of the
P-PROTEIN KINASE C IN HIPPOCAMPUS
895
FIG. 2. Cryostat section of the dorsal hippocampus. Colchicine treatment. (A) Low power view of CAI-CA2 region (X 50). (B) Enlarged view of the CA3 region seen in Fig. 2A. Note cytoplasmic staining of the pyramidal cells of the CA3 area ( X 120). (C) Enlarged view of the CA1 and CA2 area. Dense fibers are seen in the stratum oriens (SO) and stratum radiatum (SR) ( x 75).
p-PKC in rat hippocampus. The immunocytochemical results show that P-PKC is distributed in a heterogenous fashion within the hippocampus. Intense staining was evident throughout CA1 with a dramatic decrease in intensity in the remaining areas. Within the CA 1, an abundant network of fibers was present in the
regions of stratum oriens and radiatum adjacent to the pyramidal cell layer. No fluorescent cells were observed in the CA1 pyramidal layer in rats with or without colchicine treatment, although the other regions (CA2-CA4) did reveal immunoreactivity only after colchicine treatment. The significance of this is not
FIG. 3. Cryostat section of the dentate gyms. Colchicine treatment. (A) Low power view of the granule cells (GC) and the pyramidal cells of the CA4 area ( X 55). (B) Enlarged view CA4 pyramidal cells ( x IIO) and (C) Granule cells ( X 180).
immediately apparent although it would be of interest if this is a reflection of translocation of protein kinase C activity. This phenomenon has been suggested to be responsible for the mediation of hippocampal long-term potentiation (I). Furthermore in situ hybridization studies suggest that PKC is synthesized in pyramidal cell bodies since the P-PKC mRNA is found in pyramidal cells (4). Thus, it would appear that the storage capacity of P-PKC in the perikaryon is minimal (especially in the CA1 area) and that transport into dendrites and axons occurs immediately after synthesis. The enzyme positive fibers in proximity to the pyramidal cells suggests PKC may be involved in feedforward or feedback functioning of pyramidal cells.
Previous studies by Worley et al. (22) examined the distribution of phorbol ester binding sites within the hippocampus. In general, the distribution of the l3-isozyme of PKC parallels the location of phorbol ester binding sites. Interestingly, Worley et al. also noticed few phorbol ester binding sites in the pyramidal cells (22). Other investigators have studied the localization of PKC using monoclonal and polyclonal antibodies. In none of these previous studies was it known which of the various isozymes of PKC were labelled. In general, these studies have found dense staining of pyramidal cells and pyramidal cell processes ( 12,2 1). The patterns of staining previously reported were quite distinct from those of our study. Mochly-Rosen et al. (12). for example. report that the
897
B-PROTEIN KINASE C IN HIPPOCAMPUS
apical dendrites of pyramidal cells were labelled with their monoclonal antibody CK 1.12, while other workers demonstrated staining of pyramidal cell nuclear membranes (21). By contrast, our antibody showed almost no staining of pyramidal cell bodies in the CA1 area and intense staining of both the stratum radiatum and stratum oriens. The localization of dense processes above and below the pyramidal cell layer suggests the possibility that much of the immunoreactivity observed in the CA 1 layer is contained in basket cells which are in the stratum oriens immediately adjacent to the pyr~idal cells (I I, 15, 18). Our inability to microscopically visualize cell bodies in this layer may be due to the large density of processes in the stratum oriens thereby in effect serving to camouflage the cell soma. On the other hand it could be argued that in situ hybridization studies revealed PKC mRNA in the pyramidal cells of CA 1 (31. However, the oligonucleotide probes that Brandt er al. (4) employed to detect the BI and BII forms of PKC are not completely specific for either subtype. Thus, both probes for instance, appear to preferentially recognize the common unspliced precursor mRNA (4), and show a much smaller degree of hybridization to the differentially spliced mature mRNA forms. In situ hybridization studies might, therefore, preferentially recognize cells which synthesize precursor mRNA’s and not mature, spliced mRNA forms. Also, there is no a priori reason to suspect that there is a direct relationship between cellular levels of a particular mRNA and its corresponding protein. For instance, the Brandt ef al. (4) study suggests the BI and BII forms of PKC are equally distributed in hippocampus while a recent study by Nishizuka’s group (14) shows the BII/BI>97/1. Finally. Ase et al. (2) employing type-specific antibodies discerned striking dissimilarities between in situ hybridization studies (4) and immunohis-
tochemical localization of PKC. Thus, it is not surprising that our histochemical results suggest a localization of B-PKC in basket cells in the CA1 region. Ono et al. have found that as many as seven distinct isozymes of rat brain PKC may exist (14). Our antibody is directed against a domain (V3) of the molecule which is quite variable among the PKC subtypes. No sequence homology has been found comparing the sequence of our synthetic antigen and the published sequences of the other PKC subtypes or other proteins for which sequences have been published. Thus, the differences we observed in the localization of B-PKC within hipp~ampus probably reflect the fact that we are studying only one of the seven subtypes of PKC. These results also demonstrate the power of the approach of using a synthetic peptide antigen for studying a multigene family. Conventional techniques of protein purification can only resolve, at most, three peaks of PKC activity each of which probably contains multiple isozymes (7). Thus, obtaining antibodies specific for all seven subtypes is nearly impossible utilizing currently available techniques of protein biochemistry. An alternative approach is the use of synthetic peptide antigens specific for individual subtypes. As our results show, antibodies prepared in such a manner are quite specific and sensitive and can useful for both biochemical and histochemical studies. In conclusion, we describe the distribution of B-PKC in rat hippocampus using an antibody directed against a unique peptide sequence. We found PKC distributed in a heterogenous fashion through the hipp~ampus with intense staining in a network of fibers. Further identi~cation of specific patterns of dis~bution for the various subtypes of PKC should give us additional clues to the function of this multigene enzyme family.
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