Spinal NMDA receptor phosphorylation correlates with the presence of neuropathic signs following peripheral nerve injury in the rat

Neuroscience Letters 399 (2006) 85–90

Spinal NMDA receptor phosphorylation correlates with the presence of neuropathic signs following peripheral nerve injury in the rat Camilla Ultenius ∗ , Bengt Linderoth, Bj¨orn A. Meyerson, Johan Wallin Department of Clinical Neuroscience, Section of Clinical CNS Research, Karolinska Institutet, 171 76 Stockholm, Sweden Received 20 October 2005; received in revised form 14 December 2005; accepted 13 January 2006

Abstract Substantial evidence has established that activation of the NMDA receptor in the spinal dorsal horn is essential for central sensitization—a phenomenon which comprises various pathophysiological mechanisms underlying neuropathic pain-like signs in animal models. In the present study, a partial sciatic nerve ligation in the rat was used to produce a model of nerve injury-induced pain represented by hypersensitivity to innocuous stimuli (“allodynia”). The aim was to assess whether alteration of NMDA receptor expression correlates with the presence of neuropathic signs. Our approach was to compare spinal NMDA receptor subunit expression and especially subunit 1 phosphorylation, assessed with immunohistochemistry and Western blot at late postoperative times, between nerve-injured rats with marked signs of neuropathy in terms of mechanical and cold hypersensitivity and nerve-injured rats that lacked robust behavioral signs of neuropathy. Quantification of receptor expression was based on comparisons between the dorsal horns ispi- and contralateral to the nerve lesion. The phosphorylated NR1 subunit of the NMDA receptor was found to be significantly increased in the ipsilateral dorsal horn in hypersensitive, but not in non-hypersensitive nerve-injured rats. We did not detect any differences in immunoreactivity in any of the non-phosphorylated NR1, NR2A, NR2B, NR2C or the NR2D subunits. These data suggest that phosphorylation of the NMDA receptor 1 subunit is correlated to the presence of signs of neuropathy (stimulus evoked pain-like behavior) and possibly also to persistent pain following nerve injury. This may represent a mechanism involved in spinal sensitization. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Immunohistochemistry; Nerve injury-induced pain; NMDA receptor; Phosphorylation; Rat; Western blot

Noxious stimulation or nerve injury may lead to a long-lasting increase in synaptic efficacy (i.e. central sensitization) in the spinal dorsal horn, which in turn causes an amplification of the response to sensory input. Such plasticity is presumably associated with both pre- and postsynaptic mechanisms and it has been shown that the activation of NMDA receptors is critical for the development of central sensitization in the dorsal horn [9,10,23,24]. Several studies suggest that phosphorylation of the NMDA receptor is an important component in receptor facilitation, probably contributing to central sensitization as a base for persistent pain [2,6,7,18,25,26]. Experimental nerve injury models present with a variety of behavioral responses that may differ regarding incidence, degree of hypersensitivity and between sensory modalities involved [1,24]. In the present study, rats were subjected to partial sciatic nerve injury according to Seltzer et al. [17] and subsequently

∗

Corresponding author. Tel.: +46 8 517 758 06; fax: +46 8 517 717 78. E-mail address: [email protected] (C. Ultenius).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.01.018

classified as hypersensitive or non-hypersensitive based on paw withdrawal thresholds to innocuous mechanical and cold stimuli. In order to establish potential correlations between expression of neuropathic signs and molecular changes following nerve injury, we believe it is important to adopt an experimental design, which focuses on the expression of such signs rather than on whether or not the animal has sustained a nerve injury. We hypothesized that NMDA receptor activation by phosphorylation, as well as expression of different receptor subunits, would be correlated with the presence of neuropathic signs. The experiments were performed in male Sprague–Dawley rats (n = 57, 200–350 g, B&K Universal AB, Sweden) in accordance with the ethical guidelines of the Committee For Research and Ethical Issues of the IASP [20]. The experimental protocols were examined and approved by the regional ethical committee for animal research. Surgery was performed under halothane anesthesia delivered through an open mask system. Anesthesia was induced by 4% Fluothane (AstraZeneca, Sweden) and maintained with 1–1.5% in a 1:1 mixture of air and oxygen at a flow-rate of 2 l/min.

86

C. Ultenius et al. / Neuroscience Letters 399 (2006) 85–90

Adequate level of anesthesia was verified by assuring areflexia to painful pinch stimuli of the fore paws. A heating pad connected to an automatic temperature controller (CMA/150, CMA Microdialysis AB, Sweden) was used to keep body temperature at 37 ± 0.5 ◦ C during surgery. A unilateral partial sciatic nerve lesion, originally described by Seltzer et al. [17], was produced to create a model of nerve injury-induced evoked pain. In brief, the left sciatic nerve was exposed at the proximal thigh level. An 8/0 silk suture was inserted into the dorsal part of the nerve and strictly tied, severing 1/2–1/3 of the nerve diameter. The soft tissue was then sutured in layers and the rat returned to its cage to recover. Approximately 1 week after nerve injury, the animals were reanesthetized as above and subjected to an intraneural injection into the intact right sciatic nerve of 1.5–2 ␮l 5% Fluoro-Gold® (FG; Fluorochrome Inc, USA), which is taken up by damaged fibers and retrogradely transported to the corresponding cell bodies (i.e. lamina IX motor neurons in the ventral horn). FG labeling was done in order to exactly localize the spinal cord segments where the sciatic nerve fibers terminate, as well as to distinguish between the ipsi- and contralateral sides. The behavioral tests were conducted under standardized conditions in a separate, quiet room. The animal was placed in a circular Plexi Glass cage with a metal mesh floor and was allowed to adapt to the environment for at least 15 min before the tests. The sensory tests were performed immediately prior to euthanasia at 2–3 weeks after the induction of nerve injury, when maximum hypersensitivity generally is observed [cf. 5,17]. Mechanical hypersensitivity in terms of reduced withdrawal thresholds to static stimuli (WT) was assessed with von Frey nylon filaments. Regularly calibrated filaments with stiffness corresponding to 1–30 g were alternately applied to the midplantar intact (contralateral) and nerve-injured (ipsilateral) hind paws. The test was started with a 4 g-filament and continued in ascending or descending order of stiffness after a negative or positive response, respectively. The softest filament provoking paw withdrawal at least five times out of 10 applications (50% probability of withdrawal) determined the WT, and based on these measurements, the rats were divided into a hypersensitive and a non-hypersensitive group. Rats with a WT of 7 g or less were considered hypersensitive (HS) and rats with a WT of 18 g or more were defined as non-hypersensitive (non-HS) [11,22]. Cold sensitivity was assessed with ethyl chloride spray (R¨onnings Europa AB, Medikema, Sweden). The responses were scored according to a cold sensitivity rank scale (CS): 0—no response; 1—paw withdrawal; 2—paw shaking; 3—paw licking; 4—vocalization and other generalized aversive reactions [cf. 8]. Prior to testing, a few bursts of ethyl chloride were sprayed next to the cage in order to habituate the animal to the noise of the discharge. Cold sensitivity was defined as the mean score response of three quick burst stimuli applied with 5min recovery periods. No untreated animals or “sham operated group” were used in this study since such animals, in several previous studies, have been found to retain normal paw sensibility [22], and unchanged NMDA receptor subunit expression patterns in their dorsal horns [6].

For immunohistochemistry the animals were anesthetized with a lethal dose of pentobarbitone (250 mg/kg, i.p., Apoteket APL, Sweden) and euthanized by transcardial perfusion with 200 ml, 37 ◦ C saline followed by 500 ml, 4 ◦ C paraformaldehyde (4%) in phosphate buffer solution (PBS). A laminectomy was performed at vertebrae T12–L1 and the exposed part of the spinal cord excised. Post-fixation was made in 4 ◦ C paraformaldehyde (4%) in PBS for 60 min followed by plain PBS overnight in the refrigerator. The tissue was immersed in 15% (w/v) sucrose in PBS for 24 h and finally cross-sectioned in 14 ␮m sections. Prior to immunolabeling, the spinal cord sections were examined for FG uptake by fluorescence microscopy and FG negative sections were excluded from further analyses. The FG positive sections were immunolabeled using routine protocols for immunohistochemistry. In brief, sections were incubated overnight at 4 ◦ C with primary antibodies (P-NR1 #06–640; NR1 #06–311; NR2A #06–313; NR2B #06-600 Upstate Biotech, NR2C #AB1592P Chemicon, NR2D #sc-1471 Santa Cruz Biotech and NeuN, Chemicon #MAB377) and the following day with the corresponding biotinylated secondary antibody. Subsequently, ABC (Vectastain Elite ABC kit, Vector Laboratories Inc, USA) and diaminobenzidine-Ni (DAB substrate kit for peroxidase with NiCl2 , Vector Laboratories Inc) protocols were used to reveal immunoreactivity (IR). Control experiments in which the primary or secondary antibodies were omitted showed no immunostaining. In order to confirm the specificity of the immunohistochemical labeling Western blot analysis was performed on frozen spinal cord tissue. The dorsal half of the L4–L5 spinal cord segments was removed, divided into ipsi- and contralateral sides, and immediately frozen. Tissue from the ipsilateral dorsal horn was homogenized in solubilization buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1%NP–40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM Na3 VO4 ) containing protease inhibitor (Protease Inhibitor Cocktail, Sigma, St Louis, MO, USA) and centrifuged twice at 10,000 × g for 10 min at 4 ◦ C. The supernatant was decanted and protein concentration was determined using an RC DC Protein Assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (50 ␮g) was separated by SDS–PAGE (Bio-Rad) and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences, UK). Nonspecific binding was blocked by incubating the membrane in 3% drymilk in TBS buffer prior to incubating the membrane for 1 h at RT with immunoaffinity-purified anti-phospho NR1 (1:1000; Upstate Biotechnology) and as a loading control, monoclonal mouse anti-ß-actin (1:2000, Sigma, St. Louis, Missouri). The membrane was washed with TBS-T buffer and incubated for 1 h at RT with horseradish peroxidase conjugated IgG (1:3000, donkey anti rabbit and sheep anti mouse, Amersham Biosciences). Immunoreactive bands were detected using Enhanced Chemiluminescence (ECL) and exposing the blot to Hyperfilm ECL (Amersham Biosciences). The IRs of NR1, phosphorylated NR1 (P-NR1), NR2A, NR2B, NR2C, NR2D receptor subunits and Neuronal Nuclei (NeuN) in the spinal dorsal horn were quantified with Scion Image for Windows (Scion Corporation, USA). The

C. Ultenius et al. / Neuroscience Letters 399 (2006) 85–90

phosphorylation site-specific NR1 antibody used in the current study recognizes either single phosphorylation at serine 896 or dual phosphorylation at serine 896 and 897. Areas comprising essentially laminae I–IV of the ipsi-and contralateral dorsal horns were photographed. These photomicrographs were then analyzed and the amount of immunostaining was estimated in terms of labeled pixel area using the “density slice tool” of the Scion Image software, which allows labeled structures to be segmented on the bases of gray level while background noise is ignored. Subsequently, a ratio (X) between the ipsiand contralateral dorsal horn immunolabeling was calculated for each animal and section. Thus, X > 1 would be interpreted as higher IR ipsi- than contralateral, X = 1 as equal IR ipsi- as contralateral and X < 1 would indicate higher IR contra- than ipsilateral. The ipsi/contra IR ratios of non-hypersensitive rats were then statistically compared with the corresponding ratios of the hypersensitive. By this approach, the threshold parameters, which define the labeled pixel area, could be adjusted for each spinal cord section to reduce the influence of background noise as much as possible. It should be noted, however, that identical threshold values were used for analyzing the respective ipsiand contralateral sides of a single section. Although the analysis excluded the possibility to compare pixel raw data between different sections, the assessed accuracy of immunostaining of each individual section was increased [cf. 19]. Graphics and calculations were done using GraphPad PRISM (GraphPad, USA). The films from Western blot analysis where scanned and the intensity of immunoreactive bands of interest was quantified using Kodak 1D Analysis Software (Eastman Kodak Company). Data from HS and non-HS rats is expressed as a ratio between the pNR1 and anti-ß-actin densities in the ipsilateral dorsal horn. The Mann–Whitney U-test was used for statistical comparisons for both behavioral tests, immunohistochemistry and Western blot; in all instances p < 0.05 was considered as significant. A total of 49 animals were subjected to nerve injury, and 21 (43%) of them were classified as HS (WT ≤ 7 g), 20 (41%) as non-HS (WT ≥ 18 g) and the remaining 8 (16%) (7 < WT < 18 g) were excluded from further experiments. In the group of HS animals, reduced WTs could be observed from 3 to 7 days and hypersensitivity generally peaked at 2–3 weeks post injury when the animals were euthanized. The cold scores (CSs) were also significantly different (p < 0.01; Mann–Whitney U-test) between the groups, although they overlapped to some extent. There was a clear co-variation between WTs and CSs (Fig. 1), implying that mechanically hypersensitive rats tended to display also an increased response to cold stimulation. Median WTs and CSs with 95% confidence intervals of the nerve-injured hind paws were 24 (22–27) g and 0.3 (0.2–1.0) in the group of non-hypersensitive rats and 3 (2.5–4.7) g and 1.7 (1.2–2.5) in hypersensitive rats, respectively. No mirror effects in terms of significant increases in mechanical or cold sensitivity of the contralateral, intact hind paws were observed. Scatterplots showing WTs and CSs of the nerve-injured hind paws of both groups are presented in Fig. 1A and B. Traces of Fluoro-Gold (FG) were observed in the lateral part of the lumbar ventral horn. No effects on WTs or CSs were observed as a result of the intraneural FG injection. The NeuN

87

Fig. 1. Mechanical (A) and cold (B) sensitivity in terms of withdrawal threshold and cold reactivity scores, respectively. Based on mechanical sensitivity, the rats were divided into a hypersensitive (HS) and a non-hypersensitive (non-HS) group. Although partially overlapping, CSs were significantly different between HS and non-HS rats (p < 0.01; Mann–Whitney U-test).

antibody, which specifically recognizes neuronal nuclei (Fig. 2A and B), was used as a control to examine whether the nerve lesion would have caused neuronal cell loss in the ipsilateral dorsal horn. As presented in Fig. 3C, the labeled pixel area of NeuN immunoreactive structures did not differ between ipsiand contralateral sides or groups, suggesting that the number of laminae I–IV dorsal horn neurons was unchanged following nerve injury. Strong bilateral immunoreactivity was observed in the lumbar spinal dorsal horn for both the NR1 and P-NR1. Whereas the dorsal horn NR1-IR and NR2B was notably dense and somewhat indistinct, suggesting staining of both cell bodies and neuropil (Fig. 2C and D), P-NR1-IR seemed to be confined mainly to cell bodies (Fig. 2E and H). In nerve injured rats displaying pronounced mechanical and/or cold hypersensitivity, P-NR1IR was significantly increased in the ipsilateral dorsal horn as compared to the contralateral one (Fig. 3A). This enhancement in phosphorylation differed significantly from the group of rats that lacked signs of neuropathy (p < 0.01; Mann–Whitney Utest). In contrast, NR1-IR, as well as NR2A, NR2B, NR2C and NR2D did not differ significantly between ipsi- and contralateral sides or between the two groups.

88

C. Ultenius et al. / Neuroscience Letters 399 (2006) 85–90

Fig. 2. Examples of NeuN (A and B), NR1 (C and D) and P-NR1 (E and F) immunolabeling in the lumbar spinal dorsal horns of hypersensitive (HS) rats photographed with ×5 magnification and P-NR1 (G and H) photographed with ×20 magnification. Images to the left show the ipsilateral (corresponding to the nerve-injured side) and images to the right show the contralateral (corresponding to the intact side) dorsal horns. P-NR1-IR was found to be significantly increased in the ipsilateral dorsal horn as compared to the contralateral one. Scale bars: 100 ␮m.

Western blot was performed on the ipsilateral side of segments L4-5 in nerve lesioned non-HS and HS rats. The analysis demonstrated a band at the expected molecular weight for phosphorylated NR1 (∼120 kDa). Approximately 14 days after sciatic nerve lesion the immunoblot showed a significant increase in density in the ipsilateral L4-5 dorsal horn of the HS group but not in the non-HS group (p < 0.05, Mann–Whitney U-test; Fig. 3D). To date, several studies have demonstrated that phosphorylation of the NMDA receptor by intracellular kinases is an important post-translational mechanism behind sustained facilitation

of receptor function [12,21], hence contributing to spinal central sensitization [10,16,26]. Enhanced dorsal horn NMDA receptor phosphorylation has been demonstrated in various experimental conditions involving central sensitization in the spinal cord [2,7,18,25]. Moreover, when protein kinase encoding genes are deleted, hypersensitivity following nerve injury or inflammation does not develop [3]. In a recent study by Gao et al. [6], it was shown that an increase in P-NR1 immunoreactive neurons in the spinal dorsal horn coincides with mechanical hypersensitivity in nerve injured rats when compared to healthy intact animals. It is well known from clinical practice that peripheral

C. Ultenius et al. / Neuroscience Letters 399 (2006) 85–90

89

Fig. 3. Quantification of P-NR1 (A), NR1 (B) and NeuN (C) immunoreactivity in the spinal dorsal horn of hypersensitive (HS) and non-hypersensitive (non-HS) rats (n = 7–11 per group). Immunostaining is presented as a ratio between the ipsi- and contralateral side in labeled pixel area. The ipsi/contra ratio of P-NR1-IR was significantly higher (** p < 0.01; Mann–Whitney U-test) in hypersensitive as compared to non-hypersensitive rats. (D) Western blot analysis of ipsilateral L4-5 dorsal horn tissue in hypersensitive (HS) and non-hypersensitive (non-HS) rats (n = 5 per group). Quantification of immunoblot data is expressed as the ratio of pNR1 density to anti-ß-actin density. The amount of pNR1 density was significantly increased in the ipsilateral dorsal horn of HS compared to non-HS rats approximately 14 days after sciatic nerve lesion (p < 0.05; Mann–Whitney U-test).

nerve injury is associated with sensibility abnormality and pain in only a fraction of the subjects [15]. Therefore, the current study was designed to explore whether the nerve injury per se relates to enhancement of NMDA receptor expression or if the presence of signs of neuropathy is a critical condition. Our results show a persistent increase in the expression of P-NR1 receptors in the ipsilateral spinal dorsal horn in nerve-injured rats only when they presented with definite neuropathic signs in the form of pronounced mechanical and/or cold hypersensitivity. On the contrary, in nerve injured rats lacking a clear hypersensitivity, no alterations in P-NR1 receptor expression were detected. This relationship does not seem to hold true for any of the nonphosphorylated NMDA receptor subunits examined at the time point selected in the present study. Although NMDA receptor phosphorylation constitutes a plausible mechanism involved in prolonged synaptic facilitation of importance for e.g. memory formation and pain [10,13], this process is generally regarded to be reversible and comparatively rapid [4,14]. The time course of phosphorylation in relation to the development of hypersensitivity signs cannot be determined on the basis of the present experiments, but it is noteworthy that upregulated P-NR1 levels are observed when the signs of neuropathy peaked. In our experience, hypersensitivity signs induced by the type of nerve lesion used in this study generally aggravate in a progressive manner from about 3 to 7 days until 2–3 weeks post injury and

last for approximately 8–10 weeks [cf. 5,17]. This time course is comparable to that reported in the study by Gao et al. [6] where enhanced P-NR1 expression was associated with mechanical hypersensitivity maintained 3, 7 and 28 days following L5 spinal nerve ligation. In conclusion, the present data confirm that signs of neuropathy following nerve injury is associated with enhanced NR1 phosphorylation, which probably represents one mechanism involved in central sensitization and which does not relate to the presence of a nerve injury per se. Acknowledgements The authors are indebted to Ernst Brodin and Gabriel von Euler for valuable discussions and to Britt Meijer for excellent technical assistance. This study was supported by unrestricted grants from The Magnus Bergvall Foundation, Medtronic Europe SA and The Swedish Society for Medical Research. References [1] G.J. Bennett, An animal model of neuropathic pain: a review, Muscle Nerve 16 (1993) 1040–1048. [2] R.M. Caudle, F.M. Perez, C. King, C.G. Yu, R.P. Yezierski, N-Methyld-aspartate receptor subunit expression and phosphorylation following excitotoxic spinal cord injury in rats, Neurosci. Lett. 349 (2003) 37–40.

90

C. Ultenius et al. / Neuroscience Letters 399 (2006) 85–90

[3] S.R. Chaplan, A.B. Malmberg, T.L. Yaksh, Efficacy of spinal NMDA receptor antagonism in formalin hyperalgesia and nerve injury evoked allodynia in the rat, J. Pharmacol. Exp. Ther. 280 (1997) 829–838. [4] P. Cohen, The origins of protein phosphorylation, Nat. Cell. Biol. 4 (2002) E127–E130. [5] J.C. Cui, B. Linderoth, B.A. Meyerson, Incidence of mononeuropathy in rats is influenced by pre-emptive alteration of spinal excitability, Eur. J. Pain. 1 (1997) 53–59. [6] X. Gao, H.K. Kim, J.M. Chung, K. Chung, Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats, Pain 116 (2005) 62–72. [7] W. Guo, S. Zou, Y. Guan, T. Ikeda, M. Tal, R. Dubner, K. Ren, Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia, J. Neurosci. 22 (2002) 6208–6217. [8] J.-X. Hao, I.S. Xu, Z. Wiesenfeld-Hallin, Effects of intrathecal morphine, clonidine and baclofen on allodynia after partial sciatic nerve injury in the rat, Acta Anaesthesiol. Scand. 43 (1999) 1027–1034. [9] G.E. Hardingham, H. Bading, The Yin and Yang of NMDA receptor signalling, Trends Neurosci. 26 (2003) 81–89. [10] R.-R. Ji, T. Kohno, K.A. Moore, C.J. Woolf, Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci. 26 (2003) 696–705. [11] V.K. Kontinen, L.C. Stanfa, A. Basu, A.H. Dickenson, Electrophysiologic evidence for increased endogenous gabaergic but not glycinergic inhibitory tone in the rat spinal nerve ligation model of neuropathy, Anesthesiology 94 (2001) 333–339. [12] W.Y. Lu, Z.G. Xiong, S. Lei, B.A. Orser, E. Dudek, M.D. Browning, J.F. MacDonald, G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors, Nat. Neurosci. 2 (1999) 331–338. [13] R.C. Malenka, R.A. Nicoll, Long-term potentiation–a decade of progress? Science 285 (1999) 1870–1874. [14] J.W. Mandell, Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology, Am. J. Pathol. 163 (2003) 1687–1698. [15] M. Otto, S. Bak, F.W. Bach, T.S. Jensen, S.H. Sindrup, Pain phenomena and possible mechanisms in patients with painful polyneuropathy, Pain 101 (2003) 187–192.

[16] A.B. Petrenko, T. Yamakura, H. Baba, K. Shimoji, The role of N-methyld-aspartate (NMDA) receptors in pain: a review, Anesth. Analg. 97 (2003) 1108–1116. [17] Z. Seltzer, R. Dubner, Y. Shir, A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury, Pain 43 (1990) 205–218. [18] S.E. Slack, S.W. Thompson, Brain-derived neurotrophic factor induces NMDA receptor 1 phosphorylation in rat spinal cord, Neuroreport 13 (2002) 1967–1970. [19] L.S. Stone, L. Vulchanova, M.S. Riedl, J. Wang, F.G. Williams, G.L. Wilcox, R. Elde, Effects of peripheral nerve injury on alpha-2A and alpha-2C adrenergic receptor immunoreactivity in the rat spinal cord, Neuroscience 93 (1999) 1399–1407. [20] The Committee for Research and Ethical Issues of the Internation Association for the Study of Pain, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (1983) 109– 110. [21] W.G. Tingley, M.D. Ehlers, K. Kameyama, C. Doherty, J.B. Ptak, C.T. Riley, R.L. Huganir, Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-d-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies, J. Biol. Chem. 272 (1997) 5157–5166. [22] J. Wallin, J.G. Cui, V. Yakhnitsa, G. Schechtmann, B.A. Meyerson, B. Linderoth, Gabapentin and pregabalin suppress tactile allodynia and potentiate spinal cord stimulation in a model of neuropathy, Eur. J. Pain 6 (2002) 261–272. [23] C.J. Woolf, M.W. Salter, Neuronal plasticity: increasing the gain in pain, Science 288 (2000) 1765–1769. [24] M. Zimmermann, Pathobiology of neuropathic pain, Eur. J. Pharmacol. 429 (2001) 23–37. [25] X. Zou, Q. Lin, W.D. Willis, Enhanced phosphorylation of NMDA receptor 1 subunits in spinal cord dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats, J. Neurosci. 20 (2000) 6989–6997. [26] X. Zou, Q. Lin, W.D. Willis, Role of protein kinase A in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats, Neuroscience 115 (2002) 775–786.