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Protein kinase C gamma-like immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat
Neuroscience Letters 311 (2001) 185–188 www.elsevier.com/locate/neulet
Protein kinase C gamma-like immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat Jin-Lian Li a, Yun-Qing Li a, Sakashi Nomura b, Takeshi Kaneko c, Noboru Mizuno d,* a
Department of Anatomy and K. K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, People’s Republic of China b College of Medical Technology, Kyoto University, Kyoto 606-8507, Japan c Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan d Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan Received 20 July 2001; received in revised form 27 July 2001; accepted 30 July 2001
Abstract We examined protein kinase C gamma-immunoreactivity (PKCg-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCg-IR in the MDH was most intense in the SG. The number of neurons with PKCg-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCg-IR, indicating that SG neurons with PKCg might receive nociceptive primary afferent fibers. The results support the notion that PKCg in the MDH may contribute to the regulation of the nociception. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Protein kinase C gamma; Medullary dorsal horn; Substantia gelatinosa; Substance P; I-B4; Immunohistochemistry; Rat
Protein kinase C (PKC) family is involved as a second messenger in signal transduction in various cellular processes [14] (for further review, see Ref. [17]). An isoenzyme of PKC, PKC gamma (PKCg), has been indicated to have a role in sensitization of dorsal horn neurons in certain pain states [11], especially in neuropathic pain [9]. PKCgimmunoreactive (PKCg-ir) neurons were distributed mainly in the substantia gelatinosa (SG) and additionally in the marginal layer and lamina III of the spinal dorsal horn (SDH) of the rat; some of the PKCg-ir neurons in the marginal layer and lamina III showed immunoreactivity for neurokinin 1 receptor (NK1R: substance P (SP) receptor), indicating that SP-containing, nociceptive primary afferent fibers might be in synaptic contact with the PKCg-ir neurons [15]. In the medullary dorsal horn (MDH: spinal trigeminal nucleus caudalis) of the rat, we found that the number of PKCg-ir neurons was much larger in the SG than in the other layers [7]. Thus, in the present study, we investigated whether or not neurons in the SG of the rat MDH might * Corresponding author. Office of the Director, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan. Tel.: 181-325-42-3881; fax: 181-42-321-8678. E-mail address: [email protected] (N. Mizuno).
receive nociceptive primary afferent fibers; double-immunohistochemical experiments were performed to demonstrate light and electron microscopically that SPcontaining fibers and I-B4 (isolectin from the seeds of Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCg-immunoreactivity (PKCg-IR) in the rat MDH. All procedures for the experiments were approved by the Animal Care and Use Committees at the Fourth Military Medical University and at the Graduate School of Medicine, Kyoto University. A total of nine Wistar male rats weighing 200–300 g (Japan SLC; Shizuoka, Japan) were used. The rats anesthetized with chloral hydrate (70 mg/100 g body weight) were perfused transcardially for light or electron microscopic experiments, as described elsewhere [2,4,7]. For light microscopic experiments, the medulla oblongata was cut transversely 20 mm thick into serial transverse sections on a freezing microtome; four series of every fourth serial frozen section were prepared, as described elsewhere [7]. Immunohistochemistry for PKCg was done, as described elsewhere [7]. Briefly, the sections were incubated at room temperature sequentially with: (1) 0.3 mg/ml rabbit antiPKCg IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight; (2) 10 mg/ml of donkey biotinylated anti-rabbit
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IgG antibody (Jackson, West Grove, PA) for 3 h; and (3) avidin-biotinylated peroxidase complex (1:50 dilution; Vector Laboratories, Burlingame, CA) for 3 h. The incubation media were prepared by using 0.05 M PBS containing 0.5% (v/v) Triton X-100, 0.25% (w/v) carrageenan, 0.05% (w/v) NaN3, and 5% (v/v) normal donkey serum (PBS-NDS) in step (1) and step (2), and by using 0.05M PBS containing 0.3% (v/v) Triton X-100 (PBS-T) in step (3). Subsequently, the sections were reacted with 0.02% (w/v) 3.3 0 -diaminobenzidine tetrahydrochloride and 0.002% (v/v) H2O2 in 0.05 M Tris–HCl buffer (pH 7.6). The control experiment was also done. When the primary antibody for PKCg was omitted or replaced with normal IgG, no PKCg-IR was found in the sections. Double immunofluorescence histochemistry for PKCg and SP was performed. Briefly, the sections were incubated at room temperature sequentially with: (1) PBS-NDS containing 0.5 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 2 mg/ml rat anti-SP IgG (Chemicon, Temecula, CA) overnight; (2) PBS-NDS containing 10 mg/ml of donkey biotinylated anti-rat IgG antibody (Jackson) for 3 h; and (3) PBS-T containing 10% (v/v) normal rat serum, 10 mg/ml donkey dichlorotriazinyl aminofluorescein (DTAF)-labeled anti-rabbit IgG antibody (Chemicon) and 5 M mg/ml Texas Red-labeled avidin D (Vector Laboratories, Burlingame, CA) for 4 h. Double immunofluorescence histochemistry for PKCg and I-B4 was also performed. Briefly, the sections were
incubated at room temperature sequentially with: (1) PBSNDS containing 0.5 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 1 mg/ml biotinylated isolectin IB4 (Sigma, St. Louis, MO) overnight; (2) PBS-T containing 5 mg/ml of TR-labeled avidin D (Vector Laboratories) and 10 mg/ml donkey DTAF-labeled anti-rabbit IgG (Chemicon) for 6 h. After the immunofluorescence histochemical staining, the sections were observed with an epifluorescence microscope (Axiophoto; Zeiss, Oberkochen, Germany) under appropriate filters, as described elsewhere [4]. For immunoelectron microscope study, the medulla oblongata was cut serially into frontal sections 50 mm thick on a vibratome (Microslicer DTK-100; Dosaka, Kyoto, Japan); two series of alternate serial sections were prepared, as described elsewhere [4]. For double-immunocytochemistry for PKCg and SP, the immunogold-silver method for PKCg was combined with the immunoperoxidase method for SP. Briefly, the sections were incubated at room temperature for 24 h with a mixture of 0.75 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 2 mg/ml rat anti-SP IgG (Chemicon) in TBS containing 2% (v/v) normal goat serum (TBS-G). Then, the sections were incubated at room temperature overnight with TBS-G containing 10 mg/ml donkey biotinylated anti-rat IgG antibody (Jackson) and 10 mg/ml goat anti-rabbit IgG antibody conjugated to 1.4 nm gold particles (Nanoprobes, Stony Brook, NY).
Fig. 1. Digital images of two sections (a,b) and (c,d) through the MDH of a rat, taken with a confocal laser-scanning microscope. In one section (a,b), PKCg- and SP-ir neuronal components are visualized with DTAF (green) and Texas Red (red), respectively. In another section (c,d), PKCg-ir and I-B4-labeled neuronal components are visualized with DTAF and Texas Red, respectively. The border between the marginal layer (I) and the SG (II) and that between the SG (II) and the magnocellular layer of the MDH are roughly indicated by the dotted lines in (a) and (c). Arrows in (b) and (d) indicate SP-ir (b) and I-B4-labeled (d) axon terminals which are in close contact with PKCgir neurons in the SG (II) of the MDH. Scale bars: 20 mm in (a) and (c); 5 mm in (b) and (d).
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Fig. 2. Electron micrographs showing a SP-ir axon terminal (asterisk) which is in asymmetric synaptic contact with a PKCgir dendritic profile (Den) in the SG of the MDH. The axon terminal and the dendritic profile are labeled by the immunoperoxidase method and by the immunogold-silver labeling method, respectively. Arrowheads indicate the postsynaptic specialization. Scale bar, 0.15 mm.
Electron microscopic immunocytochemistry for PKCg was also combined with I-B4 labeling. Briefly, the sections were incubated at room temperature for 24 h with TBS-G containing rabbit anti-PKC 0.75 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 1 mg/ml biotinylated isolectin I-B4 (Sigma). Then, the sections were incubated at room temperature overnight with TBS-G containing10 mg/ ml goat anti-rabbit IgG antibody conjugated to 1.4 nm gold particles (Nanoprobes). After PKCg/SP and PKCg/I-B4 incubations, the sections were processed for: (1) postfixation with 1% glutaraldehyde in 0.1 M PB for 10 min; (2) silver enhancement with HQ Silver Kit (Nanoprobes); (3) incubation with ABC Kit (Vector) diluted at 1:50 in 50 mM TBS for 3 h at room temperature; (4) visualization of SP-IR by incubation with 0.05 M Tris–HCl (pH 7.6) containing 0.02% diaminobenzidine tetrahydrochloride and 0.003% H2O2, for 20–30 min at room temperature; (5) osmification with 1% OsO4 in 0.1 M PB for 1 h; (6) counterstaining with 1% (w/v) urany1 acetate; and (7) flat-embedding in Durcupan (Fluka, Buchs, Switzerland). Ultrathin sections were prepared and examined as described elsewhere [2,4]. Immunohistochemical findings concerning the distribution of PKCg-IR in the MDH were substantially the same as reported previously [7]. Briefly, PKCg-IR was most intense in the SG; it was localized in perikarya and dendrites of neuronal cells. The number of PKCg-ir neurons were much larger in the SG than in the other MDH layers. The density of SP-ir axonal components in the MDH was high in
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the SG, especially in its outer part, as well as in the marginal layer, although a number of SP-ir axonal components were also distributed in the deeper layers, Epifluorescence and confocal laser-scanning microscopy also confirmed these findings (Fig. 1a,b). I-B4 binding in the MDH was observed mainly in the marginal layer and the SG, as reported previously in the rat SDH [2]; the distribution density of I-B4-labeled axonal components was higher in the outer part of the SG than in the marginal layer and the inner part of the SG (Fig. 1c). Most of the PKCg-ir neurons in the SG were small in size, and were oval, fusiform, triangular, or polygonal in shape; their cytoplasm was rather scanty as compared with their nuclei (Fig. 1b,d). The dendrites and cell bodies of many PKCg-ir neurons in the SG were in contact with SP-ir and IB4-labeled axon terminals (Fig. 1b,d). A few PKCg-ir neurons in the marginal layer were also observed to be in contact with SP-ir and I-B4-labeled axonal components (data not shown). SP-ir and I-B4-labeled axon terminals were further observed electron microscopically to make asymmetric synaptic contact with somatic and axonal profiles showing PKCg-IR (labeled with immunogold) (Figs. 2 and 3).
Fig. 3. Electron micrographs showing a I-B4-labeled axon terminal (asterisk) which is in asymmetric synaptic contact with a PKCg-ir dendritic profile (Den) in the SD of the MDH. The dendritic profile is labeled by the immunogold-silver labeling method. Arrowheads indicate the postsynaptic specialization. Scale bar, 0.15 mm.
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In the present study, the distribution pattern of neurons with PKCg-IR was in accordance with that reported previously in the rat SDH [15] and MDH [7]. In the rat SDH, the great majority (92%) of cells with PKCg-IR were GABA-immunonegative, possibly excitatory interneurons [15]. In the rat MDH, over 90% of trigeminothalamic neurons in the marginal layer showed PKCg-IR, whereas no trigeminothalamic neurons in the SG displayed PKCg-IR [7]. Thus, we have assumed that the PKCg-ir neurons in the SG are intrinsic neurons, although the SG of the MDH contain a number of projection neurons [3,5,6]. The SG of the MDH receives many thin-myelinated and unmyelinated primary afferent fibers and is implicated in the processing of nociceptive information (for review, see Ref. [16]). SP is contained in primary afferent fibers of small diameter and is implicated in transmission of noxious information (for review, see Ref. [1,18]). The isolectin I-B4 binds selectively to unmyelinated primary afferent fibers (for review, see Ref. [4]). Thus, the present results have indicated that SP-containing and I-B4-labled axon terminals of nociceptive primary afferent fibers make synapses on many PKCg-expressing neurons in the SG of the MDH. In summary, the present results, with those of our previous study [7], indicate that PKCg is expressed in both projection and intrinsic neurons in the superficial layers of the MDH, and support the notion that PKC is involved in the regulation of nociceptive activity of MDH neurons [8– 13,19,20]. This work was supported in part by Grants-in-Aid from the National Natural Science Foundation of China (39800044, 39870262, 39970239), from the Foundation for University Key Teacher of the Ministry of Education of China, and from the Ministry of Education, Science, Sports and Culture of Japan (12680743). [1] Levine, J.D., Fields, H.L. and Basbaum, A.I., Peptides and the primary afferent nociceptor, J. Neurosci., 13 (1993) 2273–2286. [2] Li, H., Ohishi, H., Kinoshita, A., Shigemoto, R., Nomura, S. and Mizuno, N., Localization of a metabotropic glutamate receptor, mGluR7, in axon terminals of presumed nociceptive, primary afferent fibers in he superficial layers of the spinal dorsal horn: an electron microscope study in the rat, Neurosci. Lett., 223 (1997) 153–156. [3] Li, J.-L., Ding, Y.-Q., Shigemoto, R. and Mizuno, N., Distribution of trigeminothalamic and spinothalamic-tract neurons showing substance P receptor-like immunoreactivity in the rat, Brain Res., 719 (1996) 207–212. [4] Li, J.-L., Kaneko, T., Nomura, S., Li, Y.-Q. and Mizuno, N., Association of serotonin-like immunoreactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat, J. Comp. Neurol., 384 (1997) 127– 141.
[5] Li, J.-L., Li, Y.-Q., Kaneko, T. and Mizuno, N., Preprodynorphin-like immunoreactivity in medullary dorsal horn neurons projecting to the thalamic regions in the rat, Neurosci. Lett., 264 (1999) 13–16. [6] Li, Y.-Q., Li, H., Kaneko, T. and Mizuno, N., Substantia gelatinosa neurons in the medullary dorsal horn: an intracellular labeling study in the rat, J. Comp. Neurol., 411 (1999) 399–412. [7] Li, Y.-Q., Li, J.-L., Li, H., Kaneko, T. and Mizuno, N., Protein Kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat, Brain Res., 913 (2001) 159–164. [8] Lin, Q., Peng, Y.B. and Willis, W.D., Possible role of protein kinase C in the sensitization of primate spinothalamic tract neurons, J. Neurosci., 16 (1996) 3026–3034. [9] Malmberg, A.B., Chen, C., Tonegawa, S. and Basbaum, A.I., Preserved acute pain and reduced neuropathic pain in mice lacking PKC, Science, 278 (1997) 279–283. [10] Mao, J., Price, D.D., Phillips, L.I., Lu, J. and Mayer, D.J., Increases in protein kinase C gamma immunoreactivity in the spinal cord dorsal horn of rats with painful mononeuropathy, Neurosci. Lett., 198 (1995) 75–78. [11] Martin, W.J., Lui, H., Wang, H., Malmberg, A.B. and Basbaum, A.I., Inflammation-induced up-regulation of protein kinase Cg immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing, Neuroscience, 88 (1999) 1267–1274. [12] Miletic, V., Bowen, K.K. and Miletic, G., Loose ligation of the rat sciatic nerve is accompanied by changes in the subcellular content of protein kinase C beta II and gamma in the spinal dorsal horn, Neurosci. Lett., 288 (2000) 199–202. [13] Munro, F.E., Fleetwood-Walker, S.M. and Mitchell, R., Evidence for a role of protein kinase C in the sustained activation of rat dorsal horn neurons evoked by cutaneous mustard oil application, Neurosci. Lett., 170 (1994) 199– 202. [14] Nishizuka, Y., Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 (1992) 607–614. [15] Polga´ r, E., Fowler, J.H., McGill, M.M. and Todd, A.J., The types of neuron which contain protein kinase C gamma in rat spinal cord, Brain Res., 833 (1999) 71–80. [16] Sessle, B.J., Mechanisms of trigeminal and occipital pain, Pain Rev., 3 (1996) 91–116. [17] Tanaka, C. and Nishizuka, Y., The protein kinase C family for neuronal signaling, Annu. Rev. Neurosci., 17 (1994) 551– 567. [18] Todd, A.J. and Spike, R.C., The localization of classical transmitters and neuropeptides within neurons in laminae I-III of the mammalian spinal dorsal horn, Prog. Neurobiol., 41 (1993) 609–645. [19] Wajima, Z., Hua, X.-Y. and Yaksh, T.L., Inhibition of spinal protein kinase C blocks substance P-mediated hyperalgesia, Brain Res., 877 (2000) 314–321. [20] Yashpal, K., Pitcher, G.M., Parent, A., Quirion, R. and Coderre, T.J., Noxious thermal and chemical stimulation induce increases in 3H-phorbol 12,13-dibutyrate binding in spinal cord dorsal horn as well as persistent pain and hyperalgsia, which is reduced by inhibition of protein kinase C, J. Neurosci., 15 (1995) 3263–3272.
Protein kinase C gamma-like immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat Jin-Lian Li a, Yun-Qing Li a, Sakashi Nomura b, Takeshi Kaneko c, Noboru Mizuno d,* a
Department of Anatomy and K. K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, People’s Republic of China b College of Medical Technology, Kyoto University, Kyoto 606-8507, Japan c Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan d Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan Received 20 July 2001; received in revised form 27 July 2001; accepted 30 July 2001
Abstract We examined protein kinase C gamma-immunoreactivity (PKCg-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCg-IR in the MDH was most intense in the SG. The number of neurons with PKCg-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCg-IR, indicating that SG neurons with PKCg might receive nociceptive primary afferent fibers. The results support the notion that PKCg in the MDH may contribute to the regulation of the nociception. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Protein kinase C gamma; Medullary dorsal horn; Substantia gelatinosa; Substance P; I-B4; Immunohistochemistry; Rat
Protein kinase C (PKC) family is involved as a second messenger in signal transduction in various cellular processes [14] (for further review, see Ref. [17]). An isoenzyme of PKC, PKC gamma (PKCg), has been indicated to have a role in sensitization of dorsal horn neurons in certain pain states [11], especially in neuropathic pain [9]. PKCgimmunoreactive (PKCg-ir) neurons were distributed mainly in the substantia gelatinosa (SG) and additionally in the marginal layer and lamina III of the spinal dorsal horn (SDH) of the rat; some of the PKCg-ir neurons in the marginal layer and lamina III showed immunoreactivity for neurokinin 1 receptor (NK1R: substance P (SP) receptor), indicating that SP-containing, nociceptive primary afferent fibers might be in synaptic contact with the PKCg-ir neurons [15]. In the medullary dorsal horn (MDH: spinal trigeminal nucleus caudalis) of the rat, we found that the number of PKCg-ir neurons was much larger in the SG than in the other layers [7]. Thus, in the present study, we investigated whether or not neurons in the SG of the rat MDH might * Corresponding author. Office of the Director, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan. Tel.: 181-325-42-3881; fax: 181-42-321-8678. E-mail address: [email protected] (N. Mizuno).
receive nociceptive primary afferent fibers; double-immunohistochemical experiments were performed to demonstrate light and electron microscopically that SPcontaining fibers and I-B4 (isolectin from the seeds of Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCg-immunoreactivity (PKCg-IR) in the rat MDH. All procedures for the experiments were approved by the Animal Care and Use Committees at the Fourth Military Medical University and at the Graduate School of Medicine, Kyoto University. A total of nine Wistar male rats weighing 200–300 g (Japan SLC; Shizuoka, Japan) were used. The rats anesthetized with chloral hydrate (70 mg/100 g body weight) were perfused transcardially for light or electron microscopic experiments, as described elsewhere [2,4,7]. For light microscopic experiments, the medulla oblongata was cut transversely 20 mm thick into serial transverse sections on a freezing microtome; four series of every fourth serial frozen section were prepared, as described elsewhere [7]. Immunohistochemistry for PKCg was done, as described elsewhere [7]. Briefly, the sections were incubated at room temperature sequentially with: (1) 0.3 mg/ml rabbit antiPKCg IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight; (2) 10 mg/ml of donkey biotinylated anti-rabbit
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 17 1- 1
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IgG antibody (Jackson, West Grove, PA) for 3 h; and (3) avidin-biotinylated peroxidase complex (1:50 dilution; Vector Laboratories, Burlingame, CA) for 3 h. The incubation media were prepared by using 0.05 M PBS containing 0.5% (v/v) Triton X-100, 0.25% (w/v) carrageenan, 0.05% (w/v) NaN3, and 5% (v/v) normal donkey serum (PBS-NDS) in step (1) and step (2), and by using 0.05M PBS containing 0.3% (v/v) Triton X-100 (PBS-T) in step (3). Subsequently, the sections were reacted with 0.02% (w/v) 3.3 0 -diaminobenzidine tetrahydrochloride and 0.002% (v/v) H2O2 in 0.05 M Tris–HCl buffer (pH 7.6). The control experiment was also done. When the primary antibody for PKCg was omitted or replaced with normal IgG, no PKCg-IR was found in the sections. Double immunofluorescence histochemistry for PKCg and SP was performed. Briefly, the sections were incubated at room temperature sequentially with: (1) PBS-NDS containing 0.5 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 2 mg/ml rat anti-SP IgG (Chemicon, Temecula, CA) overnight; (2) PBS-NDS containing 10 mg/ml of donkey biotinylated anti-rat IgG antibody (Jackson) for 3 h; and (3) PBS-T containing 10% (v/v) normal rat serum, 10 mg/ml donkey dichlorotriazinyl aminofluorescein (DTAF)-labeled anti-rabbit IgG antibody (Chemicon) and 5 M mg/ml Texas Red-labeled avidin D (Vector Laboratories, Burlingame, CA) for 4 h. Double immunofluorescence histochemistry for PKCg and I-B4 was also performed. Briefly, the sections were
incubated at room temperature sequentially with: (1) PBSNDS containing 0.5 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 1 mg/ml biotinylated isolectin IB4 (Sigma, St. Louis, MO) overnight; (2) PBS-T containing 5 mg/ml of TR-labeled avidin D (Vector Laboratories) and 10 mg/ml donkey DTAF-labeled anti-rabbit IgG (Chemicon) for 6 h. After the immunofluorescence histochemical staining, the sections were observed with an epifluorescence microscope (Axiophoto; Zeiss, Oberkochen, Germany) under appropriate filters, as described elsewhere [4]. For immunoelectron microscope study, the medulla oblongata was cut serially into frontal sections 50 mm thick on a vibratome (Microslicer DTK-100; Dosaka, Kyoto, Japan); two series of alternate serial sections were prepared, as described elsewhere [4]. For double-immunocytochemistry for PKCg and SP, the immunogold-silver method for PKCg was combined with the immunoperoxidase method for SP. Briefly, the sections were incubated at room temperature for 24 h with a mixture of 0.75 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 2 mg/ml rat anti-SP IgG (Chemicon) in TBS containing 2% (v/v) normal goat serum (TBS-G). Then, the sections were incubated at room temperature overnight with TBS-G containing 10 mg/ml donkey biotinylated anti-rat IgG antibody (Jackson) and 10 mg/ml goat anti-rabbit IgG antibody conjugated to 1.4 nm gold particles (Nanoprobes, Stony Brook, NY).
Fig. 1. Digital images of two sections (a,b) and (c,d) through the MDH of a rat, taken with a confocal laser-scanning microscope. In one section (a,b), PKCg- and SP-ir neuronal components are visualized with DTAF (green) and Texas Red (red), respectively. In another section (c,d), PKCg-ir and I-B4-labeled neuronal components are visualized with DTAF and Texas Red, respectively. The border between the marginal layer (I) and the SG (II) and that between the SG (II) and the magnocellular layer of the MDH are roughly indicated by the dotted lines in (a) and (c). Arrows in (b) and (d) indicate SP-ir (b) and I-B4-labeled (d) axon terminals which are in close contact with PKCgir neurons in the SG (II) of the MDH. Scale bars: 20 mm in (a) and (c); 5 mm in (b) and (d).
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Fig. 2. Electron micrographs showing a SP-ir axon terminal (asterisk) which is in asymmetric synaptic contact with a PKCgir dendritic profile (Den) in the SG of the MDH. The axon terminal and the dendritic profile are labeled by the immunoperoxidase method and by the immunogold-silver labeling method, respectively. Arrowheads indicate the postsynaptic specialization. Scale bar, 0.15 mm.
Electron microscopic immunocytochemistry for PKCg was also combined with I-B4 labeling. Briefly, the sections were incubated at room temperature for 24 h with TBS-G containing rabbit anti-PKC 0.75 mg/ml rabbit anti-PKCg IgG (Santa Cruz Biotechnology) and 1 mg/ml biotinylated isolectin I-B4 (Sigma). Then, the sections were incubated at room temperature overnight with TBS-G containing10 mg/ ml goat anti-rabbit IgG antibody conjugated to 1.4 nm gold particles (Nanoprobes). After PKCg/SP and PKCg/I-B4 incubations, the sections were processed for: (1) postfixation with 1% glutaraldehyde in 0.1 M PB for 10 min; (2) silver enhancement with HQ Silver Kit (Nanoprobes); (3) incubation with ABC Kit (Vector) diluted at 1:50 in 50 mM TBS for 3 h at room temperature; (4) visualization of SP-IR by incubation with 0.05 M Tris–HCl (pH 7.6) containing 0.02% diaminobenzidine tetrahydrochloride and 0.003% H2O2, for 20–30 min at room temperature; (5) osmification with 1% OsO4 in 0.1 M PB for 1 h; (6) counterstaining with 1% (w/v) urany1 acetate; and (7) flat-embedding in Durcupan (Fluka, Buchs, Switzerland). Ultrathin sections were prepared and examined as described elsewhere [2,4]. Immunohistochemical findings concerning the distribution of PKCg-IR in the MDH were substantially the same as reported previously [7]. Briefly, PKCg-IR was most intense in the SG; it was localized in perikarya and dendrites of neuronal cells. The number of PKCg-ir neurons were much larger in the SG than in the other MDH layers. The density of SP-ir axonal components in the MDH was high in
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the SG, especially in its outer part, as well as in the marginal layer, although a number of SP-ir axonal components were also distributed in the deeper layers, Epifluorescence and confocal laser-scanning microscopy also confirmed these findings (Fig. 1a,b). I-B4 binding in the MDH was observed mainly in the marginal layer and the SG, as reported previously in the rat SDH [2]; the distribution density of I-B4-labeled axonal components was higher in the outer part of the SG than in the marginal layer and the inner part of the SG (Fig. 1c). Most of the PKCg-ir neurons in the SG were small in size, and were oval, fusiform, triangular, or polygonal in shape; their cytoplasm was rather scanty as compared with their nuclei (Fig. 1b,d). The dendrites and cell bodies of many PKCg-ir neurons in the SG were in contact with SP-ir and IB4-labeled axon terminals (Fig. 1b,d). A few PKCg-ir neurons in the marginal layer were also observed to be in contact with SP-ir and I-B4-labeled axonal components (data not shown). SP-ir and I-B4-labeled axon terminals were further observed electron microscopically to make asymmetric synaptic contact with somatic and axonal profiles showing PKCg-IR (labeled with immunogold) (Figs. 2 and 3).
Fig. 3. Electron micrographs showing a I-B4-labeled axon terminal (asterisk) which is in asymmetric synaptic contact with a PKCg-ir dendritic profile (Den) in the SD of the MDH. The dendritic profile is labeled by the immunogold-silver labeling method. Arrowheads indicate the postsynaptic specialization. Scale bar, 0.15 mm.
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In the present study, the distribution pattern of neurons with PKCg-IR was in accordance with that reported previously in the rat SDH [15] and MDH [7]. In the rat SDH, the great majority (92%) of cells with PKCg-IR were GABA-immunonegative, possibly excitatory interneurons [15]. In the rat MDH, over 90% of trigeminothalamic neurons in the marginal layer showed PKCg-IR, whereas no trigeminothalamic neurons in the SG displayed PKCg-IR [7]. Thus, we have assumed that the PKCg-ir neurons in the SG are intrinsic neurons, although the SG of the MDH contain a number of projection neurons [3,5,6]. The SG of the MDH receives many thin-myelinated and unmyelinated primary afferent fibers and is implicated in the processing of nociceptive information (for review, see Ref. [16]). SP is contained in primary afferent fibers of small diameter and is implicated in transmission of noxious information (for review, see Ref. [1,18]). The isolectin I-B4 binds selectively to unmyelinated primary afferent fibers (for review, see Ref. [4]). Thus, the present results have indicated that SP-containing and I-B4-labled axon terminals of nociceptive primary afferent fibers make synapses on many PKCg-expressing neurons in the SG of the MDH. In summary, the present results, with those of our previous study [7], indicate that PKCg is expressed in both projection and intrinsic neurons in the superficial layers of the MDH, and support the notion that PKC is involved in the regulation of nociceptive activity of MDH neurons [8– 13,19,20]. This work was supported in part by Grants-in-Aid from the National Natural Science Foundation of China (39800044, 39870262, 39970239), from the Foundation for University Key Teacher of the Ministry of Education of China, and from the Ministry of Education, Science, Sports and Culture of Japan (12680743). [1] Levine, J.D., Fields, H.L. and Basbaum, A.I., Peptides and the primary afferent nociceptor, J. Neurosci., 13 (1993) 2273–2286. [2] Li, H., Ohishi, H., Kinoshita, A., Shigemoto, R., Nomura, S. and Mizuno, N., Localization of a metabotropic glutamate receptor, mGluR7, in axon terminals of presumed nociceptive, primary afferent fibers in he superficial layers of the spinal dorsal horn: an electron microscope study in the rat, Neurosci. Lett., 223 (1997) 153–156. [3] Li, J.-L., Ding, Y.-Q., Shigemoto, R. and Mizuno, N., Distribution of trigeminothalamic and spinothalamic-tract neurons showing substance P receptor-like immunoreactivity in the rat, Brain Res., 719 (1996) 207–212. [4] Li, J.-L., Kaneko, T., Nomura, S., Li, Y.-Q. and Mizuno, N., Association of serotonin-like immunoreactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat, J. Comp. Neurol., 384 (1997) 127– 141.
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