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Partial involvement of NMDA receptors and glial cells in the nociceptive behaviors induced by intrathecally administered histamine
Neuroscience Letters 495 (2011) 83–87
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Partial involvement of NMDA receptors and glial cells in the nociceptive behaviors induced by intrathecally administered histamine Hirokazu Mizoguchi a , Takaaki Komatsu b , Yoko Iwata a , Chizuko Watanabe a , Hiroyuki Watanabe a , Tohru Orito a , Soh Katsuyama c , Akihiko Yonezawa a , Kenji Onodera d , Tsukasa Sakurada b,1 , Shinobu Sakurada a,∗,1 a
Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan c Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan d Department of Clinical Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan b
a r t i c l e
i n f o
Article history: Received 10 January 2011 Received in revised form 15 February 2011 Accepted 16 February 2011 Keywords: Glial cell Histamine Nociception NMDA receptor NK1 receptor Spinal cord
a b s t r a c t The involvement of spinal glial cells in the nociceptive behaviors induced by 800 pmol of histamine was determined in mice. Histamine at 800 pmol injected intrathecally (i.t.) produced nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with tachykinin NK1 receptor antagonist CP99,994 or competitive antagonist for N-methyl-d-aspartate (NMDA) receptor d-(−)-2-amino-5-phosphonovaleric acid (d-APV). The i.t. pretreatment with the glial cell inhibitor dl-fluorocitric acid or minocycline failed to affect the nociceptive behaviors induced by histamine. However, in mice pretreated i.t. with dlfluorocitric acid or minocycline, the nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with CP99,994 but not d-APV. In Western blot analysis using lumbar spinal cords, i.t. treatment with 800 pmol of histamine increased the phosphorylation of the NR1 subunit of NMDA receptors. The increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was abolished by i.t. pretreatment with dl-fluorocitric acid or minocycline. The present results suggest that histamine at 800 pmol elicits nociceptive behaviors through activation of the neuronal NK1 receptor and the NR1 subunit-containing NMDA receptors on glial cells in the spinal cord. © 2011 Elsevier Ireland Ltd. All rights reserved.
Histamine is a neurotransmitter that modulates pain transmission in the spinal dorsal horn [9,10]. We have previously reported that intrathecal (i.t.) administration of histamine produced nociceptive behaviors in mice, consisting of scratching, biting and licking [8,15]. Interestingly, the mechanism of the nociceptive behaviors induced by histamine depends on the doses used. The nociceptive behaviors induced by 800 pmol of histamine were suppressed by co-administration of histamine H1 receptor antagonists and tachykinin NK1 receptor antagonists and by pretreatment with an antiserum against substance P, suggesting that 800 pmol of histamine produces nociceptive behaviors by increasing the release of substance P from the primary afferent nerve fibers via activation of the histamine H1 receptor [8]. On the contrary, the nociceptive behaviors induced by 1600 pmol of histamine were suppressed by co-administration of N-methyl-d-aspartate (NMDA) receptor antagonists and antagonists for the polyamine recognition site
∗ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 1 These authors contributed equally. 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.02.038
on the NR1 subunit of NMDA receptors, but not histamine H1 receptor antagonists and tachykinin NK1 receptor antagonists, suggesting that 1600 pmol of histamine elicits nociceptive behaviors by activating the NR1 subunit-containing NMDA receptors via the polyamine recognition site that histamine can bind [1,15]. The NMDA receptor, an ionotropic receptor for glutamate, is ubiquitously distributed in all laminae of the spinal cord [7] and plays an important role in pain transmission in the spinal cord [2]. In fact, glutamate that is contained in and released from the primary afferent nerve fibers [13] binds to NMDA receptors on the cell body of the second-order neurons for pain transmission. Notably, NMDA receptors have been identified in glial cells, as well as in neural cells [4,14]. We recently found that the nociceptive behaviors induced by 1600 pmol of histamine injected i.t., which are elicited by the activation of NMDA receptors via binding of histamine to the polyamine recognition site on the NR1 subunit of NMDA receptors [15], were mediated through the activation of spinal glial cells [6]. The activation of NR1 subunit-containing NMDA receptors induces phosphorylation of the NR1 subunit of NMDA receptors and results in the increased probability of ion channel opening in NMDA receptors [3]. Indeed, 1600 pmol of histamine administered
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i.t. induced increased phosphorylation of the NR1 subunit of NMDA receptors [6], as well as the NMDA receptor-mediated nociceptive behaviors [15]. Moreover, similar to histamine-induced nociceptive behaviors, increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was eliminated by the suppression of glial cells [6]. The evidence suggests that the NMDA receptor containing a polyamine recognition site on its NR1 subunit are located on glial cells, which surround or float around the synapses that connect the primary afferent nerve fibers to the second-order neurons in the dorsal horn of the spinal cord. Histamine at 1600 pmol may elicit nociceptive behaviors via activation of the NR1 subunit-containing NMDA receptor on glial cells. As mentioned above, the involvement of spinal NMDA receptors and glial cells in the nociceptive behaviors induced by 1600 pmol of histamine administered i.t. was recently investigated. Therefore, in the present study, the involvement of spinal NMDA receptors and glial cells in the nociceptive behaviors induced by 800 pmol of histamine administered i.t. was determined in mice. Moreover, the involvement of spinal glial cells in the increased phosphorylation of NR1 subunits induced by histamine was also investigated using Western blot analysis. All experiments were performed with the approval of the Ethics Committee for Animal Experiments at Tohoku Pharmaceutical University and according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize the number of animals sacrificed and to limit animal suffering. Male ddY mice (Japan SLC, Hamamatsu, Japan) weighing 22–25 g were used. The animals were housed in a room maintained at 22–23 ◦ C and at 50–60% relative humidity with an alternating 12-h light/dark cycle. Food and water were available ad libitum. Mice were used only once. The following drugs were used for the experiments: histamine dihydrochloride (Sigma–Aldrich Chemical Co., St. Louis, MO), CP99,994 (obtained through courtesy of Pfizer Pharmaceuticals), d-(−)-2-amino-5-phosphonovaleric acid (d-APV: Sigma–Aldrich Chemical Co.), dl-fluorocitric acid barium salt (Sigma–Aldrich Chemical Co.), and minocycline hydrochloride (Sigma–Aldrich Chemical Co.). These drugs were dissolved in sterile artificial cerebrospinal fluid (aCSF) containing 126.6 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2 , and 1.3 mM CaCl2 and were injected into the mice intrathecally (i.t.) according to the methods described by Hylden and Wilcox [5], using a 29-gauge stainless-steel needle attached to a 50-l Hamilton microsyringe. The volume for the i.t. injection was 5 l. Approximately 1 h before the i.t. injection, the mice were adapted to an individual plastic cage (22.0 cm × 15.0 cm × 12.5 cm), which also served as the observation chamber. Immediately after the i.t. injection of histamine, each mouse was placed into the transparent cage and nociceptive behaviors induced by histamine were observed for 20 min. The nociceptive behaviors observed were caudally directed biting and licking, as well as reciprocal hindlimb scratching. The total response times of each of these nociceptive behaviors were pooled and recorded as a single value for each animal. The time spent in nociceptive behaviors was presented as the mean ± S.E.M. for 10 mice. The statistical significance of the differences between groups was assessed with a one-way analysis of variance (ANOVA) followed by Bonferroni’s test. The phosphorylated NR1 subunit of NMDA receptors was measured by Western blot analysis. At 13 min after the injection of histamine, the mice were sacrificed by decapitation, and the dorsal part of the lumbar spinal cord was rapidly excised at 4 ◦ C. Tissue samples were homogenized in 0.1 ml of lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris–HCl, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mM sodium vanadate, and 1 mM EDTA; pH 8.0) and centrifuged at 16,000 × g for 30 min at 4 ◦ C. Supernatants were collected and total protein was measured using the Protein Assay Reagent (Bio-Rad, Hercules, CA). Thirty g of sample in sample
Fig. 1. Effects of NK1 receptor antagonist and NMDA receptor antagonist on the nociceptive behaviors induced by i.t.-administered histamine in mice. Groups of mice were treated i.t. with aCSF, histamine (800 pmol), or histamine in combination with either the NK1 receptor antagonist CP99,994 (2 nmol) or the competitive NMDA receptor antagonist d-APV (250 pmol), and the nociceptive behaviors induced by histamine were observed for 20 min. Each column represents the mean ± S.E.M. for 10 mice. The statistical significance of the differences between groups was assessed with a one-way ANOVA followed by Bonferroni’s test. The F-values of the oneway ANOVA are F[3,36] = 15.26 (P < 0.001). ***P < 0.001 vs. the group treated with histamine alone.
buffer (100 mM Tris–HCl, 2.5% sodium dodecyl sulfate (SDS), 20% glycerol, 0.006% bromophenol blue, and 10% -mercaptoethanol; pH 6.8) were boiled, electrophoresed in a 10% SDS polyacrylamide gel (Bio-Rad) and then transferred to a Hybond-P membrane (GE Healthcare UK Ltd., Buckinghamshire, UK). The blotted membrane was then incubated overnight with 5% skim milk (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in phosphate-buffered saline (80 mM Na2 HPO4 , 20 mM NaH2 PO4 and 100 mM NaCl; pH 7.5) with 0.1% (v/v) Tween 20 (T-PBS). After washing, the membranes were incubated for 2 h at room temperature with a rabbit antiserum for phospho-NR-1 (1:1000 in T-PBS; Millipore Corp., Billerica, MA) or a mouse antiserum for -actin (1:5000 in T-PBS; Sigma–Aldrich Chemical Co.). The membranes were washed extensively with TPBS and incubated for 2 h with a peroxidase-conjugated anti-rabbit IgG (1:5000 in T-PBS; GE Healthcare UK Ltd.) or a peroxidaseconjugated anti-mouse IgG (1:5000 in T-PBS; GE Healthcare UK Ltd.). After washing, the proteins were detected using the ECLPlus Western blotting detection system (GE Healthcare UK Ltd.) and visualized with the Dolphine-Chemi Image System (Wealtec Corp., Sparks, NV). MagicMark Western protein standard (Invitrogen Corp., Carlsbad, CA) was simultaneously resolved on the gel, and the molecular weight of the protein was estimated. The fold changes in the levels of the phosphorylated NR1 subunits for each drug treatment were expressed as the ratio to those in the group treated with aCSF in each independent set of experiments. Each value represents the mean ± S.E.M. for at least three independent sets of experiments. The statistical significance of the differences between groups was assessed with a Student’s t-test. The i.t. administration of 800 pmol of histamine evoked characteristic nociceptive behaviors that consisted mainly of vigorous biting and/or licking with some scratching. These behavioral responses peaked at 10–15 min and had disappeared at 20–25 min after the injection. In agreement with previous data [8], the nociceptive behaviors induced by 800 pmol of histamine were significantly suppressed by co-administration of NK1 receptor antagonist CP99,994 (2 nmol) (Fig. 1). Moreover, the nociceptive behaviors induced by 800 pmol of histamine were also significantly
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Fig. 2. Effects of glial cell inhibitors on the inhibiting effects of NK1 receptor antagonist and NMDA receptor antagonist with respect to the nociceptive behaviors induced by i.t.-administered histamine in mice. Groups of mice were treated i.t. with histamine (800 pmol), histamine in combination with either the NK1 receptor antagonist CP99,994 (2 nmol) or the competitive NMDA receptor antagonist d-APV (250 pmol), and the nociceptive behaviors induced by histamine were observed for 20 min. The glial cell inhibitors dl-fluorocitric acid (A: 40 pmol) or minocycline (B: 2 nmol) were injected i.t. 1 h before the histamine treatment. Each column represents the mean ± S.E.M. for 10 mice. The statistical significance of the differences between groups was assessed with a one-way ANOVA followed by Bonferroni’s test. The F-values of the one-way ANOVA are (A) F[3,36] = 27.00 (P < 0.001) and (B) F[3,36] = 23.95 (P < 0.001). ***P < 0.001 vs. the group treated with histamine in combination with glial cell inhibitors.
suppressed by co-administration of d-APV (250 pmol), a competitive antagonist for NMDA receptor (Fig. 1). To elucidate the role of spinal glial cells in the nociceptive behaviors induced by 800 pmol of histamine, the effects of 1-h pretreatment with the glial cell inhibitor dl-fluorocitric acid or minocycline on the nociceptive behaviors induced by 800 pmol of histamine were determined. The nociceptive behaviors induced by 800 pmol of histamine were not significantly affected by i.t. pretreatment with either dl-fluorocitric acid (40 pmol) or minocycline (2 nmol) (Fig. 2), which completely eliminated the nociceptive behaviors induced by i.t.-administered 1600 pmol of histamine [6]. In the condition that spinal glial cells were suppressed by pretreatment with dl-fluorocitric acid and minocycline, the
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nociceptive behaviors induced by 800 pmol of histamine were significantly suppressed by co-administration of CP99,994 (2 nmol), but co-administration of d-APV (250 pmol) failed to suppress the nociceptive behaviors induced by 800 pmol of histamine (Fig. 2). The activation of NR1 subunit-containing NMDA receptors results in phosphorylation of the NR1 subunit of NMDA receptors [3]. To assess histamine-induced activation of the NR1 subunit of NMDA receptors, the level of NR1 subunit phosphorylation after i.t. treatment with 800 pmol of histamine was measured by Western blot analysis. In the dorsal part of the lumbar spinal cord of mice that were treated i.t. with 800 pmol of histamine, the phosphorylation of NR1 subunits increased approximately 50% compared to levels in naïve mice (Fig. 3). To investigate the involvement of spinal glial cells in the increase in NR1 subunit phosphorylation after histamine treatment (800 pmol, i.t.), the effects of i.t. pretreatment with dl-fluorocitric acid (40 pmol) or minocycline (2 nmol) on the histamine-induced increase in NR1 subunit phosphorylation were determined. The increase in NR1 subunit phosphorylation by 800 pmol of histamine was significantly suppressed by 1-h pretreatment with dl-fluorocitric acid or minocycline (Fig. 3). We have previously reported that nociceptive behaviors induced by 800 pmol of histamine injected i.t. were mediated by activation of tachykinin NK1 receptors, which was mediated by substance P released from the primary afferent nerve fibers via activation of the histamine H1 receptor [8]. In the present study, the nociceptive behaviors induced by 800 pmol of histamine were suppressed by i.t. co-administration of tachykinin NK1 receptor antagonist CP99,994. Interestingly, the nociceptive behaviors induced by 800 pmol of histamine were also suppressed by i.t. co-administration of competitive NMDA receptor antagonist d-APV. The involvement of spinal NMDA receptors in the nociceptive behaviors induced by 800 pmol of histamine have not been described previously. In the present study, we found that the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of spinal NMDA receptors. As we reported recently, the nociceptive behaviors induced by 1600 pmol of histamine were mediated through the activation of NR1 subunit-containing NMDA receptors via binding of histamine to the polyamine recognition site on the NR1 subunit of NMDA receptors in the spinal cord [15]. The activation of NR1 subunitcontaining NMDA receptors results in phosphorylation of the NR1 subunit of NMDA receptors [3]. In fact, i.t. treatment with histamine at 1600 pmol induced a 50% increase in NR1 subunit phosphorylation in the dorsal part of lumbar spinal cord [6]. The increased phosphorylation of NR1 subunits of NMDA receptors by 1600 pmol of histamine was completely suppressed by pretreatment with the glial cell inhibitor minocycline or dl-fluorocitric acid, suggesting that the increase in NR1 subunit phosphorylation induced by 1600 pmol of histamine was mediated through the activation of glial cells in the spinal cord. Because NMDA receptors have been identified in glial cells as well as in neural cells [4,14], NMDA receptors stimulated by the treatment with 1600 pmol of histamine (likely, NMDA receptors containing the polyamine recognition site) are probably located on the glial cells surrounding or floating around the synapses that connect the primary afferent nerve fibers to the second-order neurons in the dorsal horn of the spinal cord. In the present study, i.t. treatment with histamine at 800 pmol induced a 50% increase in NR1 subunit phosphorylation in the dorsal part of lumbar spinal cord. The result is more direct evidence that supports our hypothesis that the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NR1 subunit-containing NMDA receptors in the spinal cord. The increase in NR1 subunit phosphorylation induced by 800 pmol of histamine was completely suppressed by pretreatment with minocycline or dl-fluorocitric acid, suggesting that the increased phosphorylation of NR1 subunit induced by 800 pmol or
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Fig. 3. Effects of glial cell inhibitors on the histamine-induced increase of NR1 subunit phosphorylation in the dorsal part of the mouse lumbar spinal cord. Groups of mice were treated i.t. with aCSF or histamine (800 pmol) 13 min before decapitation. The glial cell inhibitors dl-fluorocitric acid (A: 40 pmol) or minocycline (B: 2 nmol) were injected i.t. 1 h before the treatment with histamine. The dorsal part of the mouse lumbar spinal cord was rapidly excised and prepared for Western blot analysis. The levels of NR1 subunit phosphorylation were measured by Western blot analysis. Fold changes in the levels of the phosphorylated NR1 subunits for each drug treatment were expressed as the ratio to those in the group treated with aCSF in each independent set of experiments. Each column represents the mean ± S.E.M. for at least three independent sets of experiments. The statistical significance of the differences between the groups was assessed with Student’s t-test. (A) The t-value is 5.024 (P < 0.001). (B) The t-value is 3.846 (P < 0.01). **P < 0.01, ***P < 0.001 vs. the group treated with histamine alone.
1600 pmol of histamine is mediated through the activation of glial cells in the spinal cord. The NMDA receptor subtype stimulated by 800 pmol of histamine may be the same subtype (probably NMDA receptors containing the polyamine recognition site) stimulated by 1600 pmol of histamine. This type of NMDA receptor may be located on glial cells in the dorsal horn of the spinal cord. In contrast to the increased phosphorylation of NR1 subunits of NMDA receptors, in the present study, the nociceptive behaviors induced by 800 pmol of histamine were not affected by pretreatment with the glial cell inhibitor minocycline or dl-fluorocitric acid. We have previously reported that the nociceptive behaviors induced by 800 pmol of histamine injected i.t. were mediated through the release of substance P from primary afferent nerves via activation of histamine H1 receptors located on the terminals of the primary afferent nerve fibers [8,10]. In fact, the nociceptive behaviors induced by 800 pmol of histamine were significantly suppressed by pretreatment with a high dose of capsaicin [6], which induces the depletion of substance P and glutamate in the primary afferent nerves and decreases the release of substance P and glutamate from primary afferent nerve fibers [11,12]. Although the nociceptive behaviors induced by 800 pmol of histamine are partially mediated through the activation of NR1 subunit-containing NMDA receptors in spinal glial cells, the nociceptive behaviors induced by 800 pmol of histamine may be mainly mediated by substance P released from the primary afferent nerve, which subsequently stimulates the NK1 receptors. At variance with the nociceptive behaviors induced by 800 pmol of histamine, the nociceptive behaviors induced by 1600 pmol of histamine were completely suppressed by the pretreatment with minocycline or dl-fluorocitric acid [6]. Because the depletion of substance P and glutamate in the primary afferent nerves by the pretreatment with a high dose of capsaicin did not alter the nociceptive behaviors induced by 1600 pmol of histamine [6], the nociceptive behaviors induced by 1600 pmol of histamine may be mediated strictly by activation of NR1 subunit-containing NMDA receptors in the spinal glial cells but not by the release of substance P and glutamate from primary afferent nerve fibers. As described above, the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NK1 receptors and NMDA receptors in the spinal cord. However, in the present study, the suppression of the 800 pmol histamine-induced nociceptive behaviors by NMDA receptor antagonist, but not NK1 receptor antagonist, was eliminated in mice pretreated i.t. with glial cell inhibitors. This result clearly suggests that, when spinal glial cells are inhibited, the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NK1 receptors but not NMDA receptors. This supports our hypothesis that the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NK1 receptors on the neurons and the NR1 subunit-containing NMDA receptors on glial cells in the spinal cord. In conclusion, the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of neuronal NK1 receptor and the glial NMDA receptors in the spinal cord. With respect to the nociceptive behaviors induced by 800 pmol of histamine, the contribution of NK1 receptors on neurons may be more important than that of NR1 subunit-containing NMDA receptors on glial cells. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) [KAKENHI 18613015, 21600013 and 22600009] from the Japan Society for the Promotion of Science and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology Japan (2010–2014).
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Partial involvement of NMDA receptors and glial cells in the nociceptive behaviors induced by intrathecally administered histamine Hirokazu Mizoguchi a , Takaaki Komatsu b , Yoko Iwata a , Chizuko Watanabe a , Hiroyuki Watanabe a , Tohru Orito a , Soh Katsuyama c , Akihiko Yonezawa a , Kenji Onodera d , Tsukasa Sakurada b,1 , Shinobu Sakurada a,∗,1 a
Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan c Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan d Department of Clinical Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan b
a r t i c l e
i n f o
Article history: Received 10 January 2011 Received in revised form 15 February 2011 Accepted 16 February 2011 Keywords: Glial cell Histamine Nociception NMDA receptor NK1 receptor Spinal cord
a b s t r a c t The involvement of spinal glial cells in the nociceptive behaviors induced by 800 pmol of histamine was determined in mice. Histamine at 800 pmol injected intrathecally (i.t.) produced nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with tachykinin NK1 receptor antagonist CP99,994 or competitive antagonist for N-methyl-d-aspartate (NMDA) receptor d-(−)-2-amino-5-phosphonovaleric acid (d-APV). The i.t. pretreatment with the glial cell inhibitor dl-fluorocitric acid or minocycline failed to affect the nociceptive behaviors induced by histamine. However, in mice pretreated i.t. with dlfluorocitric acid or minocycline, the nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with CP99,994 but not d-APV. In Western blot analysis using lumbar spinal cords, i.t. treatment with 800 pmol of histamine increased the phosphorylation of the NR1 subunit of NMDA receptors. The increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was abolished by i.t. pretreatment with dl-fluorocitric acid or minocycline. The present results suggest that histamine at 800 pmol elicits nociceptive behaviors through activation of the neuronal NK1 receptor and the NR1 subunit-containing NMDA receptors on glial cells in the spinal cord. © 2011 Elsevier Ireland Ltd. All rights reserved.
Histamine is a neurotransmitter that modulates pain transmission in the spinal dorsal horn [9,10]. We have previously reported that intrathecal (i.t.) administration of histamine produced nociceptive behaviors in mice, consisting of scratching, biting and licking [8,15]. Interestingly, the mechanism of the nociceptive behaviors induced by histamine depends on the doses used. The nociceptive behaviors induced by 800 pmol of histamine were suppressed by co-administration of histamine H1 receptor antagonists and tachykinin NK1 receptor antagonists and by pretreatment with an antiserum against substance P, suggesting that 800 pmol of histamine produces nociceptive behaviors by increasing the release of substance P from the primary afferent nerve fibers via activation of the histamine H1 receptor [8]. On the contrary, the nociceptive behaviors induced by 1600 pmol of histamine were suppressed by co-administration of N-methyl-d-aspartate (NMDA) receptor antagonists and antagonists for the polyamine recognition site
∗ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 1 These authors contributed equally. 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.02.038
on the NR1 subunit of NMDA receptors, but not histamine H1 receptor antagonists and tachykinin NK1 receptor antagonists, suggesting that 1600 pmol of histamine elicits nociceptive behaviors by activating the NR1 subunit-containing NMDA receptors via the polyamine recognition site that histamine can bind [1,15]. The NMDA receptor, an ionotropic receptor for glutamate, is ubiquitously distributed in all laminae of the spinal cord [7] and plays an important role in pain transmission in the spinal cord [2]. In fact, glutamate that is contained in and released from the primary afferent nerve fibers [13] binds to NMDA receptors on the cell body of the second-order neurons for pain transmission. Notably, NMDA receptors have been identified in glial cells, as well as in neural cells [4,14]. We recently found that the nociceptive behaviors induced by 1600 pmol of histamine injected i.t., which are elicited by the activation of NMDA receptors via binding of histamine to the polyamine recognition site on the NR1 subunit of NMDA receptors [15], were mediated through the activation of spinal glial cells [6]. The activation of NR1 subunit-containing NMDA receptors induces phosphorylation of the NR1 subunit of NMDA receptors and results in the increased probability of ion channel opening in NMDA receptors [3]. Indeed, 1600 pmol of histamine administered
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i.t. induced increased phosphorylation of the NR1 subunit of NMDA receptors [6], as well as the NMDA receptor-mediated nociceptive behaviors [15]. Moreover, similar to histamine-induced nociceptive behaviors, increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was eliminated by the suppression of glial cells [6]. The evidence suggests that the NMDA receptor containing a polyamine recognition site on its NR1 subunit are located on glial cells, which surround or float around the synapses that connect the primary afferent nerve fibers to the second-order neurons in the dorsal horn of the spinal cord. Histamine at 1600 pmol may elicit nociceptive behaviors via activation of the NR1 subunit-containing NMDA receptor on glial cells. As mentioned above, the involvement of spinal NMDA receptors and glial cells in the nociceptive behaviors induced by 1600 pmol of histamine administered i.t. was recently investigated. Therefore, in the present study, the involvement of spinal NMDA receptors and glial cells in the nociceptive behaviors induced by 800 pmol of histamine administered i.t. was determined in mice. Moreover, the involvement of spinal glial cells in the increased phosphorylation of NR1 subunits induced by histamine was also investigated using Western blot analysis. All experiments were performed with the approval of the Ethics Committee for Animal Experiments at Tohoku Pharmaceutical University and according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize the number of animals sacrificed and to limit animal suffering. Male ddY mice (Japan SLC, Hamamatsu, Japan) weighing 22–25 g were used. The animals were housed in a room maintained at 22–23 ◦ C and at 50–60% relative humidity with an alternating 12-h light/dark cycle. Food and water were available ad libitum. Mice were used only once. The following drugs were used for the experiments: histamine dihydrochloride (Sigma–Aldrich Chemical Co., St. Louis, MO), CP99,994 (obtained through courtesy of Pfizer Pharmaceuticals), d-(−)-2-amino-5-phosphonovaleric acid (d-APV: Sigma–Aldrich Chemical Co.), dl-fluorocitric acid barium salt (Sigma–Aldrich Chemical Co.), and minocycline hydrochloride (Sigma–Aldrich Chemical Co.). These drugs were dissolved in sterile artificial cerebrospinal fluid (aCSF) containing 126.6 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2 , and 1.3 mM CaCl2 and were injected into the mice intrathecally (i.t.) according to the methods described by Hylden and Wilcox [5], using a 29-gauge stainless-steel needle attached to a 50-l Hamilton microsyringe. The volume for the i.t. injection was 5 l. Approximately 1 h before the i.t. injection, the mice were adapted to an individual plastic cage (22.0 cm × 15.0 cm × 12.5 cm), which also served as the observation chamber. Immediately after the i.t. injection of histamine, each mouse was placed into the transparent cage and nociceptive behaviors induced by histamine were observed for 20 min. The nociceptive behaviors observed were caudally directed biting and licking, as well as reciprocal hindlimb scratching. The total response times of each of these nociceptive behaviors were pooled and recorded as a single value for each animal. The time spent in nociceptive behaviors was presented as the mean ± S.E.M. for 10 mice. The statistical significance of the differences between groups was assessed with a one-way analysis of variance (ANOVA) followed by Bonferroni’s test. The phosphorylated NR1 subunit of NMDA receptors was measured by Western blot analysis. At 13 min after the injection of histamine, the mice were sacrificed by decapitation, and the dorsal part of the lumbar spinal cord was rapidly excised at 4 ◦ C. Tissue samples were homogenized in 0.1 ml of lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris–HCl, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mM sodium vanadate, and 1 mM EDTA; pH 8.0) and centrifuged at 16,000 × g for 30 min at 4 ◦ C. Supernatants were collected and total protein was measured using the Protein Assay Reagent (Bio-Rad, Hercules, CA). Thirty g of sample in sample
Fig. 1. Effects of NK1 receptor antagonist and NMDA receptor antagonist on the nociceptive behaviors induced by i.t.-administered histamine in mice. Groups of mice were treated i.t. with aCSF, histamine (800 pmol), or histamine in combination with either the NK1 receptor antagonist CP99,994 (2 nmol) or the competitive NMDA receptor antagonist d-APV (250 pmol), and the nociceptive behaviors induced by histamine were observed for 20 min. Each column represents the mean ± S.E.M. for 10 mice. The statistical significance of the differences between groups was assessed with a one-way ANOVA followed by Bonferroni’s test. The F-values of the oneway ANOVA are F[3,36] = 15.26 (P < 0.001). ***P < 0.001 vs. the group treated with histamine alone.
buffer (100 mM Tris–HCl, 2.5% sodium dodecyl sulfate (SDS), 20% glycerol, 0.006% bromophenol blue, and 10% -mercaptoethanol; pH 6.8) were boiled, electrophoresed in a 10% SDS polyacrylamide gel (Bio-Rad) and then transferred to a Hybond-P membrane (GE Healthcare UK Ltd., Buckinghamshire, UK). The blotted membrane was then incubated overnight with 5% skim milk (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in phosphate-buffered saline (80 mM Na2 HPO4 , 20 mM NaH2 PO4 and 100 mM NaCl; pH 7.5) with 0.1% (v/v) Tween 20 (T-PBS). After washing, the membranes were incubated for 2 h at room temperature with a rabbit antiserum for phospho-NR-1 (1:1000 in T-PBS; Millipore Corp., Billerica, MA) or a mouse antiserum for -actin (1:5000 in T-PBS; Sigma–Aldrich Chemical Co.). The membranes were washed extensively with TPBS and incubated for 2 h with a peroxidase-conjugated anti-rabbit IgG (1:5000 in T-PBS; GE Healthcare UK Ltd.) or a peroxidaseconjugated anti-mouse IgG (1:5000 in T-PBS; GE Healthcare UK Ltd.). After washing, the proteins were detected using the ECLPlus Western blotting detection system (GE Healthcare UK Ltd.) and visualized with the Dolphine-Chemi Image System (Wealtec Corp., Sparks, NV). MagicMark Western protein standard (Invitrogen Corp., Carlsbad, CA) was simultaneously resolved on the gel, and the molecular weight of the protein was estimated. The fold changes in the levels of the phosphorylated NR1 subunits for each drug treatment were expressed as the ratio to those in the group treated with aCSF in each independent set of experiments. Each value represents the mean ± S.E.M. for at least three independent sets of experiments. The statistical significance of the differences between groups was assessed with a Student’s t-test. The i.t. administration of 800 pmol of histamine evoked characteristic nociceptive behaviors that consisted mainly of vigorous biting and/or licking with some scratching. These behavioral responses peaked at 10–15 min and had disappeared at 20–25 min after the injection. In agreement with previous data [8], the nociceptive behaviors induced by 800 pmol of histamine were significantly suppressed by co-administration of NK1 receptor antagonist CP99,994 (2 nmol) (Fig. 1). Moreover, the nociceptive behaviors induced by 800 pmol of histamine were also significantly
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Fig. 2. Effects of glial cell inhibitors on the inhibiting effects of NK1 receptor antagonist and NMDA receptor antagonist with respect to the nociceptive behaviors induced by i.t.-administered histamine in mice. Groups of mice were treated i.t. with histamine (800 pmol), histamine in combination with either the NK1 receptor antagonist CP99,994 (2 nmol) or the competitive NMDA receptor antagonist d-APV (250 pmol), and the nociceptive behaviors induced by histamine were observed for 20 min. The glial cell inhibitors dl-fluorocitric acid (A: 40 pmol) or minocycline (B: 2 nmol) were injected i.t. 1 h before the histamine treatment. Each column represents the mean ± S.E.M. for 10 mice. The statistical significance of the differences between groups was assessed with a one-way ANOVA followed by Bonferroni’s test. The F-values of the one-way ANOVA are (A) F[3,36] = 27.00 (P < 0.001) and (B) F[3,36] = 23.95 (P < 0.001). ***P < 0.001 vs. the group treated with histamine in combination with glial cell inhibitors.
suppressed by co-administration of d-APV (250 pmol), a competitive antagonist for NMDA receptor (Fig. 1). To elucidate the role of spinal glial cells in the nociceptive behaviors induced by 800 pmol of histamine, the effects of 1-h pretreatment with the glial cell inhibitor dl-fluorocitric acid or minocycline on the nociceptive behaviors induced by 800 pmol of histamine were determined. The nociceptive behaviors induced by 800 pmol of histamine were not significantly affected by i.t. pretreatment with either dl-fluorocitric acid (40 pmol) or minocycline (2 nmol) (Fig. 2), which completely eliminated the nociceptive behaviors induced by i.t.-administered 1600 pmol of histamine [6]. In the condition that spinal glial cells were suppressed by pretreatment with dl-fluorocitric acid and minocycline, the
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nociceptive behaviors induced by 800 pmol of histamine were significantly suppressed by co-administration of CP99,994 (2 nmol), but co-administration of d-APV (250 pmol) failed to suppress the nociceptive behaviors induced by 800 pmol of histamine (Fig. 2). The activation of NR1 subunit-containing NMDA receptors results in phosphorylation of the NR1 subunit of NMDA receptors [3]. To assess histamine-induced activation of the NR1 subunit of NMDA receptors, the level of NR1 subunit phosphorylation after i.t. treatment with 800 pmol of histamine was measured by Western blot analysis. In the dorsal part of the lumbar spinal cord of mice that were treated i.t. with 800 pmol of histamine, the phosphorylation of NR1 subunits increased approximately 50% compared to levels in naïve mice (Fig. 3). To investigate the involvement of spinal glial cells in the increase in NR1 subunit phosphorylation after histamine treatment (800 pmol, i.t.), the effects of i.t. pretreatment with dl-fluorocitric acid (40 pmol) or minocycline (2 nmol) on the histamine-induced increase in NR1 subunit phosphorylation were determined. The increase in NR1 subunit phosphorylation by 800 pmol of histamine was significantly suppressed by 1-h pretreatment with dl-fluorocitric acid or minocycline (Fig. 3). We have previously reported that nociceptive behaviors induced by 800 pmol of histamine injected i.t. were mediated by activation of tachykinin NK1 receptors, which was mediated by substance P released from the primary afferent nerve fibers via activation of the histamine H1 receptor [8]. In the present study, the nociceptive behaviors induced by 800 pmol of histamine were suppressed by i.t. co-administration of tachykinin NK1 receptor antagonist CP99,994. Interestingly, the nociceptive behaviors induced by 800 pmol of histamine were also suppressed by i.t. co-administration of competitive NMDA receptor antagonist d-APV. The involvement of spinal NMDA receptors in the nociceptive behaviors induced by 800 pmol of histamine have not been described previously. In the present study, we found that the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of spinal NMDA receptors. As we reported recently, the nociceptive behaviors induced by 1600 pmol of histamine were mediated through the activation of NR1 subunit-containing NMDA receptors via binding of histamine to the polyamine recognition site on the NR1 subunit of NMDA receptors in the spinal cord [15]. The activation of NR1 subunitcontaining NMDA receptors results in phosphorylation of the NR1 subunit of NMDA receptors [3]. In fact, i.t. treatment with histamine at 1600 pmol induced a 50% increase in NR1 subunit phosphorylation in the dorsal part of lumbar spinal cord [6]. The increased phosphorylation of NR1 subunits of NMDA receptors by 1600 pmol of histamine was completely suppressed by pretreatment with the glial cell inhibitor minocycline or dl-fluorocitric acid, suggesting that the increase in NR1 subunit phosphorylation induced by 1600 pmol of histamine was mediated through the activation of glial cells in the spinal cord. Because NMDA receptors have been identified in glial cells as well as in neural cells [4,14], NMDA receptors stimulated by the treatment with 1600 pmol of histamine (likely, NMDA receptors containing the polyamine recognition site) are probably located on the glial cells surrounding or floating around the synapses that connect the primary afferent nerve fibers to the second-order neurons in the dorsal horn of the spinal cord. In the present study, i.t. treatment with histamine at 800 pmol induced a 50% increase in NR1 subunit phosphorylation in the dorsal part of lumbar spinal cord. The result is more direct evidence that supports our hypothesis that the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NR1 subunit-containing NMDA receptors in the spinal cord. The increase in NR1 subunit phosphorylation induced by 800 pmol of histamine was completely suppressed by pretreatment with minocycline or dl-fluorocitric acid, suggesting that the increased phosphorylation of NR1 subunit induced by 800 pmol or
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Fig. 3. Effects of glial cell inhibitors on the histamine-induced increase of NR1 subunit phosphorylation in the dorsal part of the mouse lumbar spinal cord. Groups of mice were treated i.t. with aCSF or histamine (800 pmol) 13 min before decapitation. The glial cell inhibitors dl-fluorocitric acid (A: 40 pmol) or minocycline (B: 2 nmol) were injected i.t. 1 h before the treatment with histamine. The dorsal part of the mouse lumbar spinal cord was rapidly excised and prepared for Western blot analysis. The levels of NR1 subunit phosphorylation were measured by Western blot analysis. Fold changes in the levels of the phosphorylated NR1 subunits for each drug treatment were expressed as the ratio to those in the group treated with aCSF in each independent set of experiments. Each column represents the mean ± S.E.M. for at least three independent sets of experiments. The statistical significance of the differences between the groups was assessed with Student’s t-test. (A) The t-value is 5.024 (P < 0.001). (B) The t-value is 3.846 (P < 0.01). **P < 0.01, ***P < 0.001 vs. the group treated with histamine alone.
1600 pmol of histamine is mediated through the activation of glial cells in the spinal cord. The NMDA receptor subtype stimulated by 800 pmol of histamine may be the same subtype (probably NMDA receptors containing the polyamine recognition site) stimulated by 1600 pmol of histamine. This type of NMDA receptor may be located on glial cells in the dorsal horn of the spinal cord. In contrast to the increased phosphorylation of NR1 subunits of NMDA receptors, in the present study, the nociceptive behaviors induced by 800 pmol of histamine were not affected by pretreatment with the glial cell inhibitor minocycline or dl-fluorocitric acid. We have previously reported that the nociceptive behaviors induced by 800 pmol of histamine injected i.t. were mediated through the release of substance P from primary afferent nerves via activation of histamine H1 receptors located on the terminals of the primary afferent nerve fibers [8,10]. In fact, the nociceptive behaviors induced by 800 pmol of histamine were significantly suppressed by pretreatment with a high dose of capsaicin [6], which induces the depletion of substance P and glutamate in the primary afferent nerves and decreases the release of substance P and glutamate from primary afferent nerve fibers [11,12]. Although the nociceptive behaviors induced by 800 pmol of histamine are partially mediated through the activation of NR1 subunit-containing NMDA receptors in spinal glial cells, the nociceptive behaviors induced by 800 pmol of histamine may be mainly mediated by substance P released from the primary afferent nerve, which subsequently stimulates the NK1 receptors. At variance with the nociceptive behaviors induced by 800 pmol of histamine, the nociceptive behaviors induced by 1600 pmol of histamine were completely suppressed by the pretreatment with minocycline or dl-fluorocitric acid [6]. Because the depletion of substance P and glutamate in the primary afferent nerves by the pretreatment with a high dose of capsaicin did not alter the nociceptive behaviors induced by 1600 pmol of histamine [6], the nociceptive behaviors induced by 1600 pmol of histamine may be mediated strictly by activation of NR1 subunit-containing NMDA receptors in the spinal glial cells but not by the release of substance P and glutamate from primary afferent nerve fibers. As described above, the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NK1 receptors and NMDA receptors in the spinal cord. However, in the present study, the suppression of the 800 pmol histamine-induced nociceptive behaviors by NMDA receptor antagonist, but not NK1 receptor antagonist, was eliminated in mice pretreated i.t. with glial cell inhibitors. This result clearly suggests that, when spinal glial cells are inhibited, the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NK1 receptors but not NMDA receptors. This supports our hypothesis that the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of NK1 receptors on the neurons and the NR1 subunit-containing NMDA receptors on glial cells in the spinal cord. In conclusion, the nociceptive behaviors induced by 800 pmol of histamine are mediated through the activation of neuronal NK1 receptor and the glial NMDA receptors in the spinal cord. With respect to the nociceptive behaviors induced by 800 pmol of histamine, the contribution of NK1 receptors on neurons may be more important than that of NR1 subunit-containing NMDA receptors on glial cells. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) [KAKENHI 18613015, 21600013 and 22600009] from the Japan Society for the Promotion of Science and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology Japan (2010–2014).
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