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Protein kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat
Brain Research 913 (2001) 159–164 www.elsevier.com / locate / bres
Short communication
Protein kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat a a a b c, Yun-Qing Li , Jin-Lian Li , Hui Li , Takeshi Kaneko , Noboru Mizuno * a
Department of Anatomy and K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’ an 710032, PR China b Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606 -8501, Japan c Office of the Director, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183 -8526, Japan Accepted 13 June 2001
Abstract We examined protein kinase C gamma-like immunoreactivity (PKCg-LI) of trigeminothalamic neurons in the rat medullary dorsal horn (MDH) after injecting a retrograde tracer, Fluoro-Gold (FG), into the thalamus. Over 90% of FG-labeled neurons in the marginal layer (lamina I) and a few FG-labeled neurons in the superficial part of the magnocellular layer (lamina III) showed PKCg-LI. No PKCg-neurons in the substantia gelatinosa (lamina II) were labeled with FG. PKCg-mediated regulation of trigeminothalamic neurons may contribute to the changes in MDH activity during persistent pain. 2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation, anatomy and physiology Keywords: Protein kinase C gamma; Medullary dorsal horn; Trigeminothalamic tract neuron; Immunohistochemistry; Rat
The protein kinase C (PKC) family, which is activated by 1,2-diacylglycerol in the presence of Ca 21 and phospholipids, is involved as a second messenger in signal transduction in various cellular processes [13–15] (for review, see Ref. [19]). A growing body of evidence indicates that the activation of protein kinase C gamma (PKCg) in the spinal cord is involved in spinal hyperexcitability and / or persistent pain state [7–10,16]. Immunohistochemical studies have shown that PKCg is expressed at a high concentration in neurons of the superficial laminae (laminae I–III) of the spinal dorsal horn (SDH) [7,9–11,16]. On the other hand, it has been reported that in adult normal rats, PKCg-like immunoreactivity (-LI) was observed in 20–30% of neurokinin 1 (NK1) receptor-containing neurons in laminae I and III of the SDH [16]. Since NK1 receptor-LI is expressed in some spinothalamic neurons in the superficial laminae of the rat SDH [5], and since substance P (SP), the main endogenous ligand for *Corresponding author. Tel.: 181-42-325-3881, ext. 4417; fax: 18142-321-8678. E-mail address: [email protected] (N. Mizuno).
NK1 receptor, is implicated in transmission of noxious information from the periphery to the central nervous system [1,2,6] (for reviews, see Refs. [4,21]), it appears to be likely that some PKCg-positive neurons with NK1 receptor may send projection fibers to the thalamic regions and are implicated in nociception. However, there is at present no direct evidence indicating expression of PKCg in pain-tract neurons. The medullary dorsal horn (MDH; spinal trigeminal nucleus caudalis), which is functionally analogous to the SDH [3], is enriched in SP-containing primary afferent fibers [18,20], and some trigeminothalamic neurons in the rat MDH receive synapses from SP-immunoreactive axon terminals [17]. It has also been shown that the rat MDH contains many NK1 receptor-immunoreactive neurons [12], and that some trigeminal thalamic neurons in the rat MDH express NK1 receptor-LI [5]. Thus, in the present study, we examined whether or not the trigeminothalamic neurons in the rat MDH (brainstem homologue of spinothalamic neurons in the rat SDH) might express PKCg-LI; a retrograde tract-tracing method with fluorescent dye, Fluoro-Gold (FG), was combined with immunofluorescence histochemistry for PKCg.
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02777-9
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A total of nine Sprague–Dawley male rats weighing 300–350 g were used for the present study. All procedures for the experiments were approved by the Animal Care and Use Committees at the Fourth Military Medical University (Xi’an, People’s Republic of China) and at the Graduate School of Medicine, Kyoto University (Kyoto, Japan). In the rats anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg body weight), stereotaxic injection of FG (Fluorochrome, Denver, CO) was made unilaterally into the posteromedial ventral thalamic nucleus (VPM), midline-intralaminar thalamic nuclei (ML-IL), or posterior thalamic nuclei (Po); each of the three kinds of injection was performed in three rats. A volume of 0.05– 0.2 ml of 4% FG which was dissolved in distilled water was injected by pressure over a period of 15–20 min through a glass micropipette (tip diameter: 25–30 mm) which was attached to a 1-ml Hamilton microsyringe mounted on a microdriver. After injection, the rats were allowed to survive for 72 h, then were anesthetized deeply with sodium pentobarbital (100 mg / kg body weight, i.p.) and perfused transcardially with 100 ml of 0.01 M phosphate-buffered saline (PBS; pH 7.3), followed with 500 ml of 0.1 M phosphate buffer (PB; pH 7.3) containing 4% (w / v) paraformaldehyde and 75% (v / v)-saturated picric acid. After perfusion, the brains were removed immediately, placed into the same fixative for 4 h at 48C, then immersed in 0.1 M PB containing 30% (w / v) sucrose overnight at 48C. Subsequently, the brainstems were cut transversely 20 mm thick on a freezing microtome. The sections were collected serially into four dishes containing PBS; each dish contained a series of every fourth section. The first series of sections was mounted onto gelatincoated glass slides and stained lightly with 0.5% Neutral Red. The second series of sections was mounted onto clean glass slides, air-dried, and then used for determination of the location and extent of the FG injection sites and for observation of FG-labeled neurons in the MDH. The third series of sections was processed for PKCg immunofluorescence histochemistry. Briefly, the sections were incubated at room temperature sequentially with: (i) rabbit antisera against PKCg (1:3000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight; (ii) biotinylated donkey antirabbit IgG antibody (1:200 dilution; Chemicon, Temecula, CA) for 4–6 h; and (iii) Texas Red-conjugated avidin D (1:1000 dilution; Vector, Burlingame, CA) for 2–3 h. Then the sections were mounted onto clean glass slides, airdried, and cover-slipped with PBS containing 50% (v / v) glycerin and 2.5% (w / v) triethylenediamine (anti-fading agent). Subsequently, the sections were observed with an epifluorescence microscope (Axiophoto; Zeiss, Oberkochen, Germany) under appropriate filters for FG-labeled neurons (excitation 360–370 nm; emission .397 nm) and Texas Red (excitation 530–585 nm; emission >615 nm). In each rat, the numbers of FG-labeled neurons, PKCg-like immunoreactive neurons, and FG-labeled neurons with PKCg-LI in the MDH contralateral to the FG injection
were counted in ten sections by using the third series of the every fourth serial section. The fourth series of sections was used for PKCg immunohistochemistry and for control purposes: some sections were incubated at room temperature sequentially with: (i) rabbit anti-PKCg IgG (1:3000 dilution; Santa Cruz Biotechnology) overnight; (ii) 10 mg / ml of donkey biotinylated anti-rabbit IgG antibody (Jackson, West Grove, PA) for 3 h; and (iii) avidin– biotinylated peroxidase complex (1:50; 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) NaN 3, and 5% (v / v) normal donkey serum in steps (i) and (ii), and by using 0.05 M PBS containing 0.3% (v / v) Triton X-100 in step (iii). Subsequently, the sections were reacted with 0.02% (w / v) 3,39-diaminobenzidine tetrahyrochloride and 0.002% (v / v) H 2 O 2 in 0.05 M Tris–HCl buffer (pH 7.6). In the control experiments, when the
Fig. 1. Photomicrographs of a section through a middle level of the medullary dorsal horn, showing PKCg-like immunoreactivity in low (a) and a higher magnification (b). I, lamina I (marginal layer); II, lamina II (substantia gelatinosa); III, lamina III (magnocellular layer). Scale bar represents 100 mm for (a) and 75 mm for (b).
Y.-Q. Li et al. / Brain Research 913 (2001) 159 – 164
antibody for PKCg was omitted or replaced with normal IgG, no immunoreactivity for PKCg was found. Immunoreactivity for PKCg in the MDH was most marked in lamina II, especially in its outer part (Fig. 1); it was localized within cell bodies and dendrites of MDH neurons. The number of MDH neurons with PKCg-LI was
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much larger in lamina II than in laminae I and III, and was much larger in lamina III than in lamina I (Table 1). In all nine rats injected with FG into the VPM, ML-IL, or Po (Fig. 2), many FG-labeled neurons were seen in the MDH bilaterally with a clear contralateral predominance. FG-labeled neurons in the MDH (Fig. 3a) were encountered more frequently in the marginal layer (lamina I) than in the substantia gelatinosa (lamina II) or the magnocellular layer (lamina III); only a small number of FG-labeled neurons were seen in the substantia gelatinosa (Table 1). Over 90% (92.0–93.0%) of FG-labeled neurons in lamina I showed PKCg-LI (Fig. 3; Table 1); these FGlabeled neurons with PKCg-LI constituted 29.5–44.6% of the total population of PKCg-like immunoreactive neurons in lamina I (Table 1). FG-labeled neurons with PKCg-LI were rarely encountered in lamina III and were not found in lamina II. Thus, in the present study, trigeminothalamic MDH neurons with PKCg-LI were substantially localized within the marginal layer, although MDH neurons with PKCg-L were distributed not only in the marginal layer (lamina I), but also in the substantia gelatinosa (lamina II), and superficial part of the magnocellular layer (MDH part homologous to lamina III of the SDH). The localization of PKCg-LI within cell bodies and dendrites implied a postsynaptic role of PKCg in these neurons. Previous reports of immunocytochemical studies concerning the distribution of PKCg-LI in the spinal cord of the rat have been somewhat variable: Mori et al. [11] and Malmberg et al. [7] observed PKCg-LI only in neurons in lamina II, particularly in lamina IIi, and Martin et al. [9] reported that spinal neurons with PKCg-LI were distributed mainly in laminae II and only occasionally in lamina ´ et al. [16] observed PKCgIII. On the other hand, Polgar like immunoreactive neurons in all superficial laminae (laminae I–III) of the rat SDH, and Miletic et al. [10] also reported that PKCg-LI was concentrated within laminae I and II. The present results in the MDH were consistent ´ et al. [16]. with those in the SDH reported by Polgar Malmberg et al. [7] have suggested that the activity of PKCg in interneurons in the inner part of laminae II was critical for the development of neuropathic pain after nerve injury. On the other hand, it has been assumed that PKCg
Fig. 2. Projection drawings of transverse sections through the thalamus, showing the Fluoro-Gold (FG) injection sites (blackened areas) in nine rats. The FG injections were centered on the posteromedial ventral nucleus (VPM) in R2, R5 and R7, on the posterior nuclei (Po) in R9, R12 and R16, or on the midline-intralaminar nuclear regions of the thalamus in R17, R20 and R24. The drawings of sections are arranged rostrocaudally (from top to bottom) in each rat. 3V, third ventricle; CL, centrolateral nucleus; CM, centromedial nucleus; f, fornix; fr, retroflexus fascicle; Hb, habenula; ic, internal capsule; IMD, intermediodorsal nucleus; MD, mediodorsal nucleus; ml, medial lemniscus; mt, mammillothalamic tract; opt, optic tract; PF, parafascicular nucleus; PV, paraventricular nucleus; Re, reuniens nucleus; Rh, rhomboid nucleus; VPL, posterolateral ventral nucleus.
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Fig. 3. Fluorescent photomicrographs showing FG-labeled (a) and PKCg-immunoreactive (b) neurons in the marginal layer (I), substantia gelatinosa (II) and magnocellular layer (III) of the medullary dorsal horn of a rat (R5), which was injected with FG contralaterally into the VPM (Fig. 1). The field in (a) is the same as that in (b). Double-arrowheads indicate FG-labeled neurons with PKCg-like immunoreactivity. Arrows indicate neurons which were PKCg-like immunoreactive but not labeled with FG Scale bar represents 35 mm.
predominated in excitatory interneurons in the superficial laminae of the rat SDH, although some GABAergic neurons in the superficial laminae also showed PKCg-LI [9,16]. In the present study, we observed PKCg-LI in some projection neurons (trigeminothalamic neurons) in lamina I of the MDH. Some of these neurons may express NK1 receptor [5,16] and be activated by substance P, which is released from primary nociceptive afferent fibers (for review, see Refs. [4,21]). In lamina II (substantia gelatinosa) which has been well known to be deeply involved in
nociception, the number of neurons with PKCg-LI was ten times more than that in lamina I, although no PKCg-like immunoreactive neurons in lamina II were labeled with FG injected into the thalamic regions (Table 1). These possible interneurons in lamina II with PKCg-LI may also be involved in nociception. Thus, it may be assumed that PKCg-mediated regulation of trigeminothalamic neurons and many possible interneurons in lamina II may contribute to the changes in MDH activity during persistent pain in the orofacial regions.
Y.-Q. Li et al. / Brain Research 913 (2001) 159 – 164
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Table 1 Numbers of FG-labeled neurons (FG-cells), neurons with PKCg-like immunoreactivity (PKC-cells) and FG-cells with PKCg-like immunoreactivity (FG / PKC-cells) in the marginal layer (lamina I), substantia gelatinosa (lamina II) and magnocellular layer (lamina III) of the MDH contralateral to the FG injection into the posteromedial ventral thalamic nucleus (VPM), posterior thalamic nuclei (Po), or midline-intramedullary thalamic nuclei (ML-IL) Rat
Lamina I
Lamina II
Lamina III
R2, R5, R7 ( injected into VPM) FG-cells (mean6S.E.M.) PKC-cells (mean6S.E.M.) FG / PKC-cells (mean6S.E.M.) % Of FG / PKC-cells in FG-cells % Of FG / PKC-cells in PKC-cells
96.367.8 241.765.7 88.767.2 92.1 36.7
4.36 0.8 3027.76110.7 0 0 0
18.062.9 505.367.6 0.360.4 1.7 0.06
R9, R12, R16 ( injected into Po) FG-cells (mean6S.E.M.) PKC-cells (mean6S.E.M.) FG / PKC-cells (mean6S.E.M.) % Of FG / PKC-cells in FG-cells % Of FG / PKC-cells in PKC-cells
124.065.3 258.7616.1 115.36 4.6 93.0 44.6
3.360.5 3025.7696.2 0 0 0
28.763.9 514.367.9 0.760.7 2.4 0.01
R17, R20, R24 ( injected into ML-IL) FG-cells (mean6S.E.M.) PKC-cells (mean6S.E.M.) FG / PKC-cells (mean6S.E.M.) % Of FG / PKC-cells in FG-cells % Of FG / PKC-cells in PKC-cells
79.764.1 248.364.9 73.364.1 92.0 29.5
1.761.3 2863.06115.0 0 0 0
13.361.3 495.766.1 0.360.4 2.3 0.06
Counts in each rat were made by using ten sections from a series of every fourth serial section of 20-mm thickness.
Acknowledgements The authors thank Akira Uesugi and Keiko Okamoto of Kyoto University, Yue-Pin Yuan of The Fourth Military Medical University, and Nobuyuki Kobayashi and Hideki Itabashi of the Tokyo Metropolitan Institute for Neuroscience for their photographic help. This work was supported in part by Grants-in-Aid from the National Natural Science Foundation of China (39625011, 39800044, 39870262, 39970239), from the Foundation for University Key Teachers of the Ministry of Education of China, and from the Ministry of Education, Science, Sports and Culture of Japan (12680743).
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Short communication
Protein kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat a a a b c, Yun-Qing Li , Jin-Lian Li , Hui Li , Takeshi Kaneko , Noboru Mizuno * a
Department of Anatomy and K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’ an 710032, PR China b Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606 -8501, Japan c Office of the Director, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183 -8526, Japan Accepted 13 June 2001
Abstract We examined protein kinase C gamma-like immunoreactivity (PKCg-LI) of trigeminothalamic neurons in the rat medullary dorsal horn (MDH) after injecting a retrograde tracer, Fluoro-Gold (FG), into the thalamus. Over 90% of FG-labeled neurons in the marginal layer (lamina I) and a few FG-labeled neurons in the superficial part of the magnocellular layer (lamina III) showed PKCg-LI. No PKCg-neurons in the substantia gelatinosa (lamina II) were labeled with FG. PKCg-mediated regulation of trigeminothalamic neurons may contribute to the changes in MDH activity during persistent pain. 2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation, anatomy and physiology Keywords: Protein kinase C gamma; Medullary dorsal horn; Trigeminothalamic tract neuron; Immunohistochemistry; Rat
The protein kinase C (PKC) family, which is activated by 1,2-diacylglycerol in the presence of Ca 21 and phospholipids, is involved as a second messenger in signal transduction in various cellular processes [13–15] (for review, see Ref. [19]). A growing body of evidence indicates that the activation of protein kinase C gamma (PKCg) in the spinal cord is involved in spinal hyperexcitability and / or persistent pain state [7–10,16]. Immunohistochemical studies have shown that PKCg is expressed at a high concentration in neurons of the superficial laminae (laminae I–III) of the spinal dorsal horn (SDH) [7,9–11,16]. On the other hand, it has been reported that in adult normal rats, PKCg-like immunoreactivity (-LI) was observed in 20–30% of neurokinin 1 (NK1) receptor-containing neurons in laminae I and III of the SDH [16]. Since NK1 receptor-LI is expressed in some spinothalamic neurons in the superficial laminae of the rat SDH [5], and since substance P (SP), the main endogenous ligand for *Corresponding author. Tel.: 181-42-325-3881, ext. 4417; fax: 18142-321-8678. E-mail address: [email protected] (N. Mizuno).
NK1 receptor, is implicated in transmission of noxious information from the periphery to the central nervous system [1,2,6] (for reviews, see Refs. [4,21]), it appears to be likely that some PKCg-positive neurons with NK1 receptor may send projection fibers to the thalamic regions and are implicated in nociception. However, there is at present no direct evidence indicating expression of PKCg in pain-tract neurons. The medullary dorsal horn (MDH; spinal trigeminal nucleus caudalis), which is functionally analogous to the SDH [3], is enriched in SP-containing primary afferent fibers [18,20], and some trigeminothalamic neurons in the rat MDH receive synapses from SP-immunoreactive axon terminals [17]. It has also been shown that the rat MDH contains many NK1 receptor-immunoreactive neurons [12], and that some trigeminal thalamic neurons in the rat MDH express NK1 receptor-LI [5]. Thus, in the present study, we examined whether or not the trigeminothalamic neurons in the rat MDH (brainstem homologue of spinothalamic neurons in the rat SDH) might express PKCg-LI; a retrograde tract-tracing method with fluorescent dye, Fluoro-Gold (FG), was combined with immunofluorescence histochemistry for PKCg.
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02777-9
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Y.-Q. Li et al. / Brain Research 913 (2001) 159 – 164
A total of nine Sprague–Dawley male rats weighing 300–350 g were used for the present study. All procedures for the experiments were approved by the Animal Care and Use Committees at the Fourth Military Medical University (Xi’an, People’s Republic of China) and at the Graduate School of Medicine, Kyoto University (Kyoto, Japan). In the rats anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg body weight), stereotaxic injection of FG (Fluorochrome, Denver, CO) was made unilaterally into the posteromedial ventral thalamic nucleus (VPM), midline-intralaminar thalamic nuclei (ML-IL), or posterior thalamic nuclei (Po); each of the three kinds of injection was performed in three rats. A volume of 0.05– 0.2 ml of 4% FG which was dissolved in distilled water was injected by pressure over a period of 15–20 min through a glass micropipette (tip diameter: 25–30 mm) which was attached to a 1-ml Hamilton microsyringe mounted on a microdriver. After injection, the rats were allowed to survive for 72 h, then were anesthetized deeply with sodium pentobarbital (100 mg / kg body weight, i.p.) and perfused transcardially with 100 ml of 0.01 M phosphate-buffered saline (PBS; pH 7.3), followed with 500 ml of 0.1 M phosphate buffer (PB; pH 7.3) containing 4% (w / v) paraformaldehyde and 75% (v / v)-saturated picric acid. After perfusion, the brains were removed immediately, placed into the same fixative for 4 h at 48C, then immersed in 0.1 M PB containing 30% (w / v) sucrose overnight at 48C. Subsequently, the brainstems were cut transversely 20 mm thick on a freezing microtome. The sections were collected serially into four dishes containing PBS; each dish contained a series of every fourth section. The first series of sections was mounted onto gelatincoated glass slides and stained lightly with 0.5% Neutral Red. The second series of sections was mounted onto clean glass slides, air-dried, and then used for determination of the location and extent of the FG injection sites and for observation of FG-labeled neurons in the MDH. The third series of sections was processed for PKCg immunofluorescence histochemistry. Briefly, the sections were incubated at room temperature sequentially with: (i) rabbit antisera against PKCg (1:3000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight; (ii) biotinylated donkey antirabbit IgG antibody (1:200 dilution; Chemicon, Temecula, CA) for 4–6 h; and (iii) Texas Red-conjugated avidin D (1:1000 dilution; Vector, Burlingame, CA) for 2–3 h. Then the sections were mounted onto clean glass slides, airdried, and cover-slipped with PBS containing 50% (v / v) glycerin and 2.5% (w / v) triethylenediamine (anti-fading agent). Subsequently, the sections were observed with an epifluorescence microscope (Axiophoto; Zeiss, Oberkochen, Germany) under appropriate filters for FG-labeled neurons (excitation 360–370 nm; emission .397 nm) and Texas Red (excitation 530–585 nm; emission >615 nm). In each rat, the numbers of FG-labeled neurons, PKCg-like immunoreactive neurons, and FG-labeled neurons with PKCg-LI in the MDH contralateral to the FG injection
were counted in ten sections by using the third series of the every fourth serial section. The fourth series of sections was used for PKCg immunohistochemistry and for control purposes: some sections were incubated at room temperature sequentially with: (i) rabbit anti-PKCg IgG (1:3000 dilution; Santa Cruz Biotechnology) overnight; (ii) 10 mg / ml of donkey biotinylated anti-rabbit IgG antibody (Jackson, West Grove, PA) for 3 h; and (iii) avidin– biotinylated peroxidase complex (1:50; 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) NaN 3, and 5% (v / v) normal donkey serum in steps (i) and (ii), and by using 0.05 M PBS containing 0.3% (v / v) Triton X-100 in step (iii). Subsequently, the sections were reacted with 0.02% (w / v) 3,39-diaminobenzidine tetrahyrochloride and 0.002% (v / v) H 2 O 2 in 0.05 M Tris–HCl buffer (pH 7.6). In the control experiments, when the
Fig. 1. Photomicrographs of a section through a middle level of the medullary dorsal horn, showing PKCg-like immunoreactivity in low (a) and a higher magnification (b). I, lamina I (marginal layer); II, lamina II (substantia gelatinosa); III, lamina III (magnocellular layer). Scale bar represents 100 mm for (a) and 75 mm for (b).
Y.-Q. Li et al. / Brain Research 913 (2001) 159 – 164
antibody for PKCg was omitted or replaced with normal IgG, no immunoreactivity for PKCg was found. Immunoreactivity for PKCg in the MDH was most marked in lamina II, especially in its outer part (Fig. 1); it was localized within cell bodies and dendrites of MDH neurons. The number of MDH neurons with PKCg-LI was
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much larger in lamina II than in laminae I and III, and was much larger in lamina III than in lamina I (Table 1). In all nine rats injected with FG into the VPM, ML-IL, or Po (Fig. 2), many FG-labeled neurons were seen in the MDH bilaterally with a clear contralateral predominance. FG-labeled neurons in the MDH (Fig. 3a) were encountered more frequently in the marginal layer (lamina I) than in the substantia gelatinosa (lamina II) or the magnocellular layer (lamina III); only a small number of FG-labeled neurons were seen in the substantia gelatinosa (Table 1). Over 90% (92.0–93.0%) of FG-labeled neurons in lamina I showed PKCg-LI (Fig. 3; Table 1); these FGlabeled neurons with PKCg-LI constituted 29.5–44.6% of the total population of PKCg-like immunoreactive neurons in lamina I (Table 1). FG-labeled neurons with PKCg-LI were rarely encountered in lamina III and were not found in lamina II. Thus, in the present study, trigeminothalamic MDH neurons with PKCg-LI were substantially localized within the marginal layer, although MDH neurons with PKCg-L were distributed not only in the marginal layer (lamina I), but also in the substantia gelatinosa (lamina II), and superficial part of the magnocellular layer (MDH part homologous to lamina III of the SDH). The localization of PKCg-LI within cell bodies and dendrites implied a postsynaptic role of PKCg in these neurons. Previous reports of immunocytochemical studies concerning the distribution of PKCg-LI in the spinal cord of the rat have been somewhat variable: Mori et al. [11] and Malmberg et al. [7] observed PKCg-LI only in neurons in lamina II, particularly in lamina IIi, and Martin et al. [9] reported that spinal neurons with PKCg-LI were distributed mainly in laminae II and only occasionally in lamina ´ et al. [16] observed PKCgIII. On the other hand, Polgar like immunoreactive neurons in all superficial laminae (laminae I–III) of the rat SDH, and Miletic et al. [10] also reported that PKCg-LI was concentrated within laminae I and II. The present results in the MDH were consistent ´ et al. [16]. with those in the SDH reported by Polgar Malmberg et al. [7] have suggested that the activity of PKCg in interneurons in the inner part of laminae II was critical for the development of neuropathic pain after nerve injury. On the other hand, it has been assumed that PKCg
Fig. 2. Projection drawings of transverse sections through the thalamus, showing the Fluoro-Gold (FG) injection sites (blackened areas) in nine rats. The FG injections were centered on the posteromedial ventral nucleus (VPM) in R2, R5 and R7, on the posterior nuclei (Po) in R9, R12 and R16, or on the midline-intralaminar nuclear regions of the thalamus in R17, R20 and R24. The drawings of sections are arranged rostrocaudally (from top to bottom) in each rat. 3V, third ventricle; CL, centrolateral nucleus; CM, centromedial nucleus; f, fornix; fr, retroflexus fascicle; Hb, habenula; ic, internal capsule; IMD, intermediodorsal nucleus; MD, mediodorsal nucleus; ml, medial lemniscus; mt, mammillothalamic tract; opt, optic tract; PF, parafascicular nucleus; PV, paraventricular nucleus; Re, reuniens nucleus; Rh, rhomboid nucleus; VPL, posterolateral ventral nucleus.
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Fig. 3. Fluorescent photomicrographs showing FG-labeled (a) and PKCg-immunoreactive (b) neurons in the marginal layer (I), substantia gelatinosa (II) and magnocellular layer (III) of the medullary dorsal horn of a rat (R5), which was injected with FG contralaterally into the VPM (Fig. 1). The field in (a) is the same as that in (b). Double-arrowheads indicate FG-labeled neurons with PKCg-like immunoreactivity. Arrows indicate neurons which were PKCg-like immunoreactive but not labeled with FG Scale bar represents 35 mm.
predominated in excitatory interneurons in the superficial laminae of the rat SDH, although some GABAergic neurons in the superficial laminae also showed PKCg-LI [9,16]. In the present study, we observed PKCg-LI in some projection neurons (trigeminothalamic neurons) in lamina I of the MDH. Some of these neurons may express NK1 receptor [5,16] and be activated by substance P, which is released from primary nociceptive afferent fibers (for review, see Refs. [4,21]). In lamina II (substantia gelatinosa) which has been well known to be deeply involved in
nociception, the number of neurons with PKCg-LI was ten times more than that in lamina I, although no PKCg-like immunoreactive neurons in lamina II were labeled with FG injected into the thalamic regions (Table 1). These possible interneurons in lamina II with PKCg-LI may also be involved in nociception. Thus, it may be assumed that PKCg-mediated regulation of trigeminothalamic neurons and many possible interneurons in lamina II may contribute to the changes in MDH activity during persistent pain in the orofacial regions.
Y.-Q. Li et al. / Brain Research 913 (2001) 159 – 164
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Table 1 Numbers of FG-labeled neurons (FG-cells), neurons with PKCg-like immunoreactivity (PKC-cells) and FG-cells with PKCg-like immunoreactivity (FG / PKC-cells) in the marginal layer (lamina I), substantia gelatinosa (lamina II) and magnocellular layer (lamina III) of the MDH contralateral to the FG injection into the posteromedial ventral thalamic nucleus (VPM), posterior thalamic nuclei (Po), or midline-intramedullary thalamic nuclei (ML-IL) Rat
Lamina I
Lamina II
Lamina III
R2, R5, R7 ( injected into VPM) FG-cells (mean6S.E.M.) PKC-cells (mean6S.E.M.) FG / PKC-cells (mean6S.E.M.) % Of FG / PKC-cells in FG-cells % Of FG / PKC-cells in PKC-cells
96.367.8 241.765.7 88.767.2 92.1 36.7
4.36 0.8 3027.76110.7 0 0 0
18.062.9 505.367.6 0.360.4 1.7 0.06
R9, R12, R16 ( injected into Po) FG-cells (mean6S.E.M.) PKC-cells (mean6S.E.M.) FG / PKC-cells (mean6S.E.M.) % Of FG / PKC-cells in FG-cells % Of FG / PKC-cells in PKC-cells
124.065.3 258.7616.1 115.36 4.6 93.0 44.6
3.360.5 3025.7696.2 0 0 0
28.763.9 514.367.9 0.760.7 2.4 0.01
R17, R20, R24 ( injected into ML-IL) FG-cells (mean6S.E.M.) PKC-cells (mean6S.E.M.) FG / PKC-cells (mean6S.E.M.) % Of FG / PKC-cells in FG-cells % Of FG / PKC-cells in PKC-cells
79.764.1 248.364.9 73.364.1 92.0 29.5
1.761.3 2863.06115.0 0 0 0
13.361.3 495.766.1 0.360.4 2.3 0.06
Counts in each rat were made by using ten sections from a series of every fourth serial section of 20-mm thickness.
Acknowledgements The authors thank Akira Uesugi and Keiko Okamoto of Kyoto University, Yue-Pin Yuan of The Fourth Military Medical University, and Nobuyuki Kobayashi and Hideki Itabashi of the Tokyo Metropolitan Institute for Neuroscience for their photographic help. This work was supported in part by Grants-in-Aid from the National Natural Science Foundation of China (39625011, 39800044, 39870262, 39970239), from the Foundation for University Key Teachers of the Ministry of Education of China, and from the Ministry of Education, Science, Sports and Culture of Japan (12680743).
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