Reactive oxygen species enhance excitatory synaptic transmission in rat spinal dorsal horn neurons by activating TRPA1 and TRPV1 channels

Neuroscience 247 (2013) 201–212

REACTIVE OXYGEN SPECIES ENHANCE EXCITATORY SYNAPTIC TRANSMISSION IN RAT SPINAL DORSAL HORN NEURONS BY ACTIVATING TRPA1 AND TRPV1 CHANNELS N. NISHIO, a,b W. TANIGUCHI, a* Y. K. SUGIMURA, a,b N. TAKIGUCHI, b M. YAMANAKA, b Y. KIYOYUKI, a H. YAMADA, b N. MIYAZAKI, b M. YOSHIDA b AND T. NAKATSUKA a

nels by ROS may induce central sensitization in the spinal cord and result in chronic pain such as that following SCI. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

a Pain Research Center, Kansai University of Health Sciences, Kumatori, Osaka 590-0482, Japan

Key words: ROS, spinal cord, pain, TRPV1, TRPA1, central neuropathic pain.

b

Department of Orthopaedic Surgery, Wakayama Medical University, Wakayama 641-8510, Japan

Abstract—Central neuropathic pain (CNP) in the spinal cord, such as chronic pain after spinal cord injury (SCI), is an incurable ailment. However, little is known about the spinal cord mechanisms underlying CNP. Recently, reactive oxygen species (ROS) have been recognized to play an important role in CNP of the spinal cord. However, it is unclear how ROS affect synaptic transmission in the dorsal horn of the spinal cord. To clarify how ROS impact on synaptic transmission, we investigated the effects of ROS on synaptic transmission in rat spinal cord substantia gelatinosa (SG) neurons using whole-cell patch-clamp recordings. Administration of tert-butyl hydroperoxide (t-BOOH), an ROS donor, into the spinal cord markedly increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in SG neurons. This t-BOOHinduced enhancement was not suppressed by the Na+ channel blocker tetrodotoxin. However, in the presence of a non-N-methyl-D-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, t-BOOH did not generate any sEPSCs. Furthermore, in the presence of a transient receptor potential ankyrin 1 (TRPA1) channel antagonist (HC-030031) or a transient receptor potential vanilloid 1 (TRPV1) channel antagonist (capsazepine or AMG9810), the t-BOOH-induced increase in the frequency of sEPSCs was inhibited. These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons through TRPA1 and TRPV1 channel activation. Excessive activation of these ion chan-

INTRODUCTION Central neuropathic pain (CNP) in the spinal cord, such as chronic pain after spinal cord injury (SCI), is an incurable disease for which the underlying molecular mechanisms have not been elucidated. As there is no effective treatment for CNP, a large number of individuals suffer from this form of severe chronic pain. A major feature of CNP in the spinal cord is central sensitization induced by neuronal plasticity in substantia gelatinosa (SG) neurons. However, neuronal plasticity in the spinal cord is a complex process impacted by numerous factors. Several studies suggest that reactive oxygen species (ROS) can cause central sensitization in the spinal cord, and are involved in persistent pain (Wang et al., 2004; Salvemini et al., 2011). ROS are highly reactive molecules derived from O2, and include free radicals [e.g. superoxide (O2) and hydroxyl radical (HO)] and other reactive species [e.g. hydrogen peroxide (H2O2) and peroxynitrite (ONOO )]. A major function of ROS is immunological host defense. However, while ROS are essential for health, high levels of ROS can cause various disorders, such as cancer, arteriosclerosis, hypertension and neurodegenerative diseases (Dro¨ge, 2002; Valko et al., 2007). At excess levels, the normal physiological roles of ROS in cellular metabolism and signal transduction are supplanted by toxicity (Lander, 1997). Recently, ROS have been implicated in the etiology of chronic pain, including neuropathic and inflammatory pain (Salvemini et al., 2011). For example, it has been reported that ROS are involved in long-term potentiation (LTP) in the dorsal horn (Lee et al., 2010) and in capsaicin-induced secondary hyperalgesia (Schwartz et al., 2008). Therefore, ROS may mediate nociceptive signaling in the dorsal horn of the spinal cord, in addition to functioning as neuromodulators. It is well known that in spinal cord trauma, the release of ROS often induces second injury; therefore, it is thought that ROS are involved in the pathogenesis of post-SCI pain (Hulsebosch et al., 2009). However, the cellular

*Corresponding author. Tel: +81-(72)-498-1194; fax: +81-(72)-4530276. E-mail address: [email protected] (W. Taniguchi). Abbreviations: AITC, allyl isothiocyanate; AMPA, a-amino-3-hydroxy5-methyl-4-isozazole propionate; CNP, central neuropathic pain; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DRG, dorsal root ganglion; EPSC, excitatory postsynaptic currents; IPSC, inhibitory postsynaptic currents; LTP, long-term potentiation; mEPSCs, miniature EPSCs; NAC, N-acetylcysteine; NMDA, N-methyl-D-aspartate; PBN, phenyl-N-tert-butylnitrone; RNS, reactive nitrogen species; ROS, reactive oxygen species; SCI, spinal cord injury; sEPSC, spontaneous excitatory postsynaptic currents; SG, substantia gelatinosa; t-BOOH, tert-butyl hydroperoxide; TEMPOL, 4-hydroxy2,2,6,6-tetramethylpiperidine 1-oxyl; TRP, transient receptor potential; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1; TTX, tetrodotoxin.

0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.05.023 201

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mechanisms underlying the effects of ROS in the spinal cord are still unclear. Moreover, recent reports have suggested that transient receptor potential (TRP) channels are involved in CNP in the spinal cord (Kanai et al., 2005; Patapoutian et al., 2009; Patwardhan et al., 2009; Kim et al., 2012). TRP channels belong to a family of ion channels that are activated by temperature and which are expressed in primary sensory nerve terminals, where they provide information about thermal changes in the environment (Tominaga, 2007; Vay et al., 2012). There are six thermosensitive ion channels in mammals (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1), all of which belong to the TRP superfamily. These channels are involved in chemical, mechanical and thermal nociception. TRP channels are drug targets for the relief of pain, including neuropathic pain (Levine and Alessandri-Haber, 2007; Patapoutian et al., 2009; Stucky et al., 2009; Holzer, 2011; Wei et al., 2011b). In particular, TRPV1 (TRP vanilloid 1) and TRPA1 (TRP ankyrin 1) have been a major focus of research into the mechanisms of inflammatory and neuropathic pain. Therefore, to clarify the mechanisms underlying neuropathic pain, we investigated the effects of ROS on glutamatergic excitatory synaptic transmission in SG neurons in adult rat spinal cord slices using the wholecell patch-clamp recording technique, and we analyzed the role of TRPV1 and TRPA1 channels in these ROSmediated effects.

EXPERIMENTAL PROCEDURES All of the experimental procedures involving the use of animals were approved by the Ethics Committee on Animal Experiments, Kansai University of Health Sciences, and were in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and associated guidelines. Spinal cord slice preparation The methods used for obtaining adult rat spinal cord slice preparations have been described previously (Nakatsuka et al., 1999). In brief, male adult Sprague–Dawley rats (5– 6 weeks of age, 170–200 g) were deeply anesthetized with urethane (1.2 g/kg, IP), and then lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1–S3) was removed and placed in preoxygenated Krebs solution at 1–3 °C. Immediately after the removal of the spinal cord, the rats were given an overdose of urethane and were then killed by exsanguination. The pia-arachnoid membrane was removed after cutting all of the ventral and dorsal roots near the root entry zone. The spinal cord was mounted on a microslicer and a 600-lm-thick transverse slice was cut from the lumbar region containing the L4 or L5 dorsal root entry zone. The slice was placed on a nylon mesh in the recording chamber, which had a volume of 0.5 ml, and then perfused at a rate of 10–15 ml/min with Krebs solution saturated with 95% O2 and 5% CO2, and maintained at 36 ± 1 °C. The Krebs solution contained the following (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2

MgCl2, 1.2 NaH2PO4, 25 NaHCO3 and 11 glucose, pH 7.4. Patch-clamp recordings from SG neurons Blind whole-cell patch-clamp recordings were made from SG neurons with patch-pipette electrodes having a resistance of 5–10 MX. The patch-pipette solution used to record excitatory postsynaptic currents (EPSCs) was composed of the following (in mM): 135 potassium gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES and 5 ATP-Mg, pH 7.2. Membrane potentials were held at 70 mV in voltage-clamp mode. After making a gigaohm seal, the membrane patch was ruptured by a brief period of more negative pressure, thus resulting in a whole cell configuration. Signals were acquired with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Sunnyvale, CA, USA). Data were digitized with an analog-to-digital converter (Digidata 1440A; Molecular Devices) and stored on a personal computer using the pCLAMP 10 data acquisition program (Molecular Devices). They were analyzed using Mini Analysis 6.0 software (Synaptosoft, Fort Lee, NJ, USA) and the pCLAMP 10 data acquisition program. SG neurons were viable for up to 24 h in slices perfused with pre-oxygenated Krebs solution. However, all of the recordings described here were obtained within 12 h. Whole-cell patch-clamp recordings were stable for up to 4 h. The membrane potentials were not corrected for the liquid junction potential between the Krebs and patchpipette solutions. Application of drug Drugs were dissolved in Krebs solution and applied by perfusion via a three-way stopcock without any change in the perfusion rate or the temperature. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord was 30 s. The drugs used in this study were tert-butyl hydroperoxide (tBOOH), phenyl-N-tert-butylnitrone (PBN), 4-hydroxy2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), Nacetylcysteine (NAC), HC-030031, capsazepine, AMG9810 (Sigma, St. Louis, MO, USA), tetrodotoxin (TTX) (Latoxan, Valence, France) and 6-cyano-7nitroquinoxaline-2,3-dione (CNQX) (TOCRIS, Bristol, UK). CNQX, TEMPOL, HC-030031, capsazepine and AMG9810 were dissolved in dimethyl sulfoxide as 1000 stock solutions. TTX, PBN and NAC were dissolved in distilled water as 1000 stock solutions. These drugs were diluted to the final concentration in Krebs solution immediately before use. The osmotic pressure of nominally Ca2+-free, high-Mg2+ (5 mm) Krebs solution was adjusted by lowering the Na+ concentration. Statistical analysis All numerical data were expressed as the mean ± S.E.M. Paired Student’s t test or Welch’s t test was used to determine the statistical significance between means, and the Kolmogorov–Smirnov test was used to compare

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cumulative distributions of EPSC parameters in the absence and presence of the test drugs. P < 0.05 was considered significant for these tests. In electrophysiological data, n refers to the number of neurons studied.

RESULTS ROS enhance excitatory synaptic transmission in SG neurons All SG neurons tested exhibited spontaneous EPSCs (sEPSCs) at a holding potential (VH) of 70 mV, at which no inhibitory postsynaptic currents (IPSCs) were observed because the reversal potential for IPSCs was near 70 mV (Yoshimura and Nishi, 1995). To examine the effect of ROS on excitatory synaptic transmission, we used t-BOOH as an ROS donor in the present study. Superfusing t-BOOH (10 mM) for 5 min resulted in a significant increase in the frequency and amplitude of sEPSCs in all 20 neurons recorded (Fig. 1A). Moreover, this effect was sometimes accompanied by a slow inward current, as shown in Fig. 1A. t-BOOH produced an inward current (>5 pA) at 70 mV in 11 (55%) of 20 neurons examined (Fig. 1A). The average peak amplitude of the t-BOOH-induced inward current was 14.0 ± 2.0 pA (n = 11). The average increases in sEPSC frequency and amplitude mediated by t-BOOH were 331.4 ± 49.9 and 176.7 ± 11.9% (n = 20), respectively (Fig. 1C). Fig. 1B demonstrates the effects of t-BOOH (10 mM) on the cumulative distributions of the inter-event interval and amplitude of sEPSCs. tBOOH increased the proportion of sEPSCs having a significantly shorter inter-event interval (P < 0.05) and a significantly larger amplitude (P < 0.05) compared with the control. When t-BOOH (10 mM) was applied repeatedly at 25-min intervals, it induced a similar increase in sEPSC frequency and amplitude (Fig. 2A). The average increases in sEPSC frequency and amplitude by the secondary application of t-BOOH were 416.9 ± 142.1 and 184.7 ± 26.0% (n = 4), respectively; these values were not substantially different from those elicited by the first application of tBOOH (374.7 ± 135.1 and 181.2 ± 36.9%, respectively) (Fig. 2B). Analysis of presynaptic ROS effects in SG neurons We investigated the effect of ROS on the central terminals of fibers, which were synaptically connected with the recording SG neuron. This was done by determining the effects of ROS on miniature EPSCs (mEPSCs) in the presence of a Na+ channel blocker, TTX. In the presence of TTX (1 lM), t-BOOH (10 mM) markedly increased both mEPSC frequency and amplitude (Fig. 3A). The average increases in mEPSC frequency and amplitude induced by the application of t-BOOH were 878.38 ± 333.8 and 191.6 ± 39.1% (n = 7), respectively; these values were significantly different from those in the absence of TTX (P < 0.05) (Fig. 3B). Fig. 3C demonstrates the effects of t-BOOH on the cumulative distributions of the inter-event intervals and

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amplitudes of mEPSCs. t-BOOH increased the proportion of mEPSCs having a significantly shorter inter-event interval (P < 0.05) and a significantly larger amplitude (P < 0.05) compared with the control. Next, we constructed amplitude histograms of mEPSCs before and during t-BOOH perfusion. Fig. 3D shows a typical result of a neuron exhibiting a large increase in mEPSC frequency and amplitude following the application of t-BOOH. This histogram reveals that tBOOH shifted the mEPSC amplitude from low to high. Furthermore, a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, CNQX (20 lM), suppressed sEPSCs, not only in the control, but also in the presence of t-BOOH (10 mM) in all four neurons examined (Fig. 4A), indicating that t-BOOH caused robust glutamate release onto SG neurons. Subsequently, we examined whether the t-BOOHmediated increases in sEPSC frequency and amplitude were dependent on extracellular Ca2+. In a Ca2+-free bath solution, the superfusing t-BOOH (10 mM) did not significantly increase sEPSC frequency or amplitude (Fig. 4B). The application of ROS in a Ca2+-free bath solution increased the frequency and amplitude of sEPSCs by 129.9 ± 9.8% and 134.5 ± 11.9% (n = 10) (Fig. 4C) compared with control, respectively; these values were significantly different from those obtained using normal Krebs solution (P < 0.05). These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons. Moreover, the t-BOOH induced inward current (>5 pA) was suppressed in a Ca2+-free bath solution in all recording neurons (n = 10) (Fig. 4B). An ROS scavenger reduces t-BOOH-induced sEPSC enhancement We investigated the effect of ROS scavengers on tBOOH-induced excitation of SG neurons. PBN, TEMPOL, and NAC were used as ROS scavengers. PBN is a nonspecific ROS scavenger, while TEMPOL has generally been considered a superoxide dismutase mimetic. However, recently it has been discovered that TEMPOL can also reduce the levels of other radicals (i.e., hydroxyl radicals and peroxynitrite), allowing it to be categorized as a nonspecific scavenger (Muscoli et al., 2003; Salvemini et al., 2011). NAC is an antioxidant that functions as a direct universal ROS scavenger. The t-BOOH-induced increases in sEPSC frequency and amplitude were blocked by PBN (10 mM) (Fig. 5A). The t-BOOH-induced increases in sEPSC frequency and amplitude in the presence of PBN averaged 142.0 ± 12.3% and 132.5 ± 10.8% (n = 7), respectively; these values were significantly smaller than in normal Krebs solution (P < 0.05) (Fig. 5D). In comparison, in the presence of TEMPOL (10 mM), the tBOOH-induced increases in sEPSC frequency and amplitude averaged 223.9 ± 38.3% and 189.9 ± 42.0% (n = 5), respectively. In the presence of TEMPOL, the frequency of sEPSCs was lower than in the absence of TEMPOL; however, this value was not significantly different from that obtained with normal Krebs solution (P > 0.05) (Fig. 5B, D). Moreover, the t-BOOH-induced

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Fig. 1. Effects of an ROS donor, t-BOOH, on excitatory synaptic transmission in SG neurons. (A) A continuous chart recording of glutamatergic sEPSCs before and during the application of t-BOOH (10 mM: upper). Three consecutive traces of sEPSCs are shown in an expanded scale in time, before (lower left) and during the application of t-BOOH (lower right). In the voltage-clamp mode (VH = 70 mV), the application of t-BOOH markedly increased the frequency and amplitude of sEPSCs. Note a slow inward current that is accompanied by increases in sEPSC frequency and amplitude (upper). (B) Cumulative distributions of the inter-event interval (left) and amplitude (right) of sEPSCs, before (dotted line) and during (continuous line) the action of t-BOOH. t-BOOH shifted the inter-event interval and amplitude to a shorter and a larger one, respectively (P < 0.05; Kolmogorov–Smirnov test). Data in A and B were obtained from the same neuron. C, Summary of sEPSC frequency (left) and amplitude (right) under the action of t-BOOH (n = 20) relative to those in the control. In this and subsequent figures, vertical lines accompanied by bars show SEM. Statistical significance between data shown by bars is indicated by an asterisk; ⁄P < 0.05; n.s., not significant.

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Fig. 2. The effects of t-BOOH on SG neurons are reversible following short-term exposure. (A) When t-BOOH (10 mM) was applied repeatedly at 25-min intervals, it produced similar increases in sEPSC frequency and amplitude. (B) Summary of sEPSC frequency (left) and amplitude (right) under the first [t-BOOH (1st)] and second application of t-BOOH [t-BOOH (2nd)] (n = 4).

increase in sEPSC frequency was reduced by NAC (10 mM) (Fig. 5C). The t-BOOH-induced increases in sEPSC frequency and amplitude in the presence of NAC averaged 127.0 ± 10.0% and 158.6 ± 29.2% (n = 4), respectively. The t-BOOH-induced increase in frequency was significantly smaller than in normal Krebs solution (P < 0.05) (Fig. 5D).

TRPA1 and TRPV1 channel antagonists reduce tBOOH induced sEPSC enhancement Next, we examined whether TRP channel antagonists could inhibit the t-BOOH-induced increases in sEPSC frequency and amplitude. In the presence of a selective TRPV1 channel antagonist, capsazepine (100 lM), the t-BOOH (10 mM)-induced increase in sEPSC frequency was partially blocked (Fig. 6A). The t-BOOH-induced increases in sEPSC frequency and amplitude in the presence of capsazepine averaged 178.7 ± 26.7% and 223.2 ± 58.7% (n = 7), respectively; this increase in frequency was significantly small compared with that obtained in normal Krebs solution (P < 0.05) (Fig. 6D). Moreover, we examined whether another TRPV1 antagonist, AMG9810, could inhibit the t-BOOH-induced sEPSC enhancement. The t-BOOH-induced increases in sEPSC frequency and amplitude in the presence of AMG9810 (100 lM) averaged 160.0 ± 25.4% and 214.0 ± 43.1% (n = 4), respectively. As with capsazepine, this increase in frequency was significantly smaller than that obtained in normal Krebs solution

(Fig. 6B). Next, in the presence of a selective TRPA1 channel antagonist, HC-030031 (100 lM), the t-BOOHinduced enhancement of sEPSC was robustly blocked (Fig. 6C). The t-BOOH-induced increases in sEPSC frequency and amplitude in the presence of HC-030031 averaged 129.5 ± 9.5% and 137.9 ± 20.0% (n = 8), respectively; this increase in frequency was significantly smaller than that obtained in normal Krebs solution (P < 0.05) (Fig. 6D). Furthermore, we investigated whether the inhibitory effects of capsazepine and HC030031 are dose-dependent. The t-BOOH-induced increase in sEPSC frequency was 144.1 ± 14.9% in the presence of 10 lM capsazepine (n = 5), while it was 155.5 ± 11.9% in the presence of 300 lM capsazepine (n = 4). With both 10 and 300 lM capsazepine, the increase in frequency was significantly smaller than that obtained in normal Krebs solution (P < 0.05) (Fig. 6E). The t-BOOH-induced increase in frequency was 223.4 ± 41.4% in the presence of 10 lM HC-030031 (n = 5), while it was 131.0 ± 18.1% in the presence of 300 lM HC-030031 (n = 3). With the 300 lM concentration, the increase in frequency was significantly smaller than that obtained in normal Krebs solution (P < 0.05) (Fig. 6E). These results show that the inhibitory effect of capsazepine plateaus at 10 lM, while that of HC-030031 plateaus at 100 lM (Fig. 6E). tBOOH-induced inward currents in all 8 recorded neurons were blocked (<5 pA) in the presence of HC-030031, but three of seven recorded neurons (42.8%) had inward currents (10.3 ± 2.3 pA) in the presence of capsazepine.

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Fig. 3. The t-BOOH-induced increases in mEPSC frequency and amplitude were resistant to the voltage-gated Na+ channel blocker TTX. (A) Effect of t-BOOH (10 mM) on mEPSCs in the presence of TTX (1 lM). The application of t-BOOH markedly increased the frequency and amplitude of mEPSCs in the presence of TTX. (B) Summary of mEPSC frequency (left) and amplitude (right) under the action of t-BOOH (n = 7) relative to those in the control. (C) Cumulative distributions of the inter-event interval (left) and amplitude (right) of mEPSCs, before (dotted line) and during (continuous line) the action of t-BOOH. t-BOOH shifted the inter-event interval and amplitude to a shorter and a larger one, respectively (P < 0.05; Kolmogorov–Smirnov test). (D) Distributions of mEPSC amplitude before and during (white bar and gray bar, respectively) the action of t-BOOH. The bin width is 1 pA. Data in A, B and D were obtained from the same neuron. VH = 70 mV.

DISCUSSION ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons SCI increases the levels of highly toxic ROS, which can damage neural, glial and microvascular elements, resulting in sensory and motor neuron apoptosis (Xu et al., 2005; Hall and Bosken, 2009). Damaged cells release ROS into the extracellular space, and may induce excitatory enhancement of SG neurons. However, there is little evidence that ROS can directly affect SG neurons and lead to severe pain in animal models of SCI. In this study, we investigated the effect of ROS on glutamatergic excitatory synaptic

transmission in SG neurons in adult rat spinal cord slices, using the whole-cell patch-clamp technique, to elucidate the cellular mechanisms underlying the action of ROS on SG neurons. The ROS donor t-BOOH markedly increased the frequency and amplitude of spontaneous sEPSCs. In the presence of a Na+ channel blocker, TTX, t-BOOH also significantly increased the frequency and amplitude of mEPSCs. However, in the presence of a non-NMDA glutamate receptor antagonist, CNQX, t-BOOH did not produce any sEPSCs. In addition, the t-BOOH-induced increase in sEPSC frequency and amplitude was dependent on extracellular Ca2+. Furthermore, the t-BOOH-induced increase in the frequency and amplitude of sEPSCs was

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Fig. 4. ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons. (A) Effect of t-BOOH (10 mM) on sEPSCs in the presence of CNQX (20 lM). CNQX blocked sEPSCs not only in the absence of t-BOOH, but also in the presence of t-BOOH. (B) Effect of t-BOOH on sEPSCs in Ca2+-free solution. t-BOOH did not substantially change sEPSC frequency or amplitude in a Ca2+-free solution. (C) Summary of t-BOOH-mediated increases in sEPSC frequency (left) and amplitude (right) in Ca2+-free solution (n = 4), relative to those in the control (n = 20).

inhibited by a nonspecific ROS scavenger, PBN or NAC. These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons, and excessive ROS may produce central sensitization in the spinal cord and result in chronic pain. In fact, intrathecal administration of ROS produces hyperalgesia (Schwartz et al., 2008; Wei et al., 2011a). Numerous reports have shown that ROS also play substantial roles in central sensitization of the spinal cord in neuropathic pain caused by peripheral nerve injury (Tal 1996; Kim et al., 2004, 2006; Park et al., 2006; Gao et al., 2007; Mao et al., 2009; Kallenborn-Gerhardt et al., 2012), inflammation (Wang et al., 2004; Gao et al., 2007; Lee et al., 2007; Schwartz et al., 2008; Lu et al., 2012), ischemia/ reperfusion injury (Kwak et al., 2009; Ryu et al., 2010), opioid-induced hyperalgesia and anti-nociceptive tolerance (Muscoli et al., 2007), and chemotherapy (Kim et al., 2010a,b; Fidanboylu et al., 2011; Doyle et al., 2012). Intrathecal injection or systemic administration of ROS scavengers or antioxidants reduces central sensitization and pain in these experimental models (Tal 1996; Kim et al., 2004, 2006; Gao et al., 2007; Lee et al., 2007; Schwartz et al., 2008; Mao et al., 2009; Lu

et al., 2012). Little is known about the primary source of ROS in these pain models. However, the mitochondrion is a major source of ROS at the intracellular level (Stowe and Camara, 2009). Indeed, levels of mitochondrially-derived ROS in dorsal horn neurons are increased in various models of pain, including inflammatory pain (Wang et al., 2004; Schwartz et al., 2009), neuropathic pain (Park et al., 2006) and spinal mitochondrial superoxide-induced mechanical hyperalgesia (Kim et al., 2008). Interestingly, a number of reports have shown that the major sources of ROS are dorsal root ganglion (DRG) neurons (KallenbornGerhardt et al., 2012) and microglia in the spinal cord (Kim et al., 2010a,b). Salvemini’s group reported that reactive nitrogen species (RNS), especially peroxynitrite, contribute to CNP in the spinal cord (see review by Salvemini et al., 2011). Peroxynitrite is a non-radical formed from the reaction of superoxide with nitric oxide. Thus, in addition to ROS, RNS (peroxynitrite) may also have enhanced sEPSCs in the present study. Excessive amounts of ROS produce central sensitization in the spinal cord, resulting in CNP. It is worth noting that, in the present study, owing to the short duration of ROS exposure, the effects of ROS on SG neurons were

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Fig. 5. Effects of ROS scavengers on sEPSC frequency and amplitude. (A) Effect of t-BOOH (10 mM) on sEPSCs in the presence of PBN (10 mM). PBN suppressed the t-BOOH-induced increases in sEPSC frequency and amplitude. (B) Effect of t-BOOH (10 mM) on sEPSCs in the presence of TEMPOL (10 mM). TEMPOL did not substantially suppress the t-BOOH-induced increases in sEPSC frequency and amplitude. (C) In the presence of NAC (10 mM), the t-BOOH-induced increase in sEPSC frequency was suppressed. (D) Summary of sEPSC frequency (upper) and amplitude (lower) under the action of t-BOOH only (n = 20), t-BOOH + TEMPOL (n = 5), t-BOOH + PBN (n = 7) and t-BOOH + NAC (n = 4) relative to those in the control.

reversible. In contrast, long-term exposure to ROS has the potential to elicit plastic changes in SG neurons and result in chronic severe pain. This hypothesis is supported by reports that ROS participate in LTP in the dorsal horn of the spinal cord and is a contributing factor in chronic pain (Lee et al., 2010; Kim et al., 2011).

ROS enhance excitatory synaptic transmission in SG neurons via TRPA1 and TRPV1 channel activation In this study, ROS-induced sEPSC enhancement in SG neurons was inhibited by a selective TRPA1 channel antagonist, HC-030031, as well as by a selective TRPV1 channel antagonist, capsazepine or AMG9810.

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Fig. 6. Effects of TRP channel antagonists on sEPSC frequency and amplitude. (A) Effect of t-BOOH (10 mM) on sEPSCs in the presence of capsazepine (100 lM). Capsazepine suppressed the t-BOOH-induced increases in sEPSC frequency. (B) In the presence of AMG9810 (100 lM), the t-BOOH-induced increase in sEPSC frequency was significantly suppressed. (C) Effect of t-BOOH (10 mM) on sEPSCs in the presence of the HC-030031 (100 lM). HC-030031 significantly suppressed the t-BOOH-induced increases in sEPSC frequency. (D) Summary of sEPSC frequency (upper) and amplitude (lower) under the action of t-BOOH only (n = 20), t-BOOH + HC-030031 (n = 8), t-BOOH + capsazepine (n = 7), and tBOOH + AMG9810 (100 lM) relative to those in the control. (E) Histogram showing the dose-dependency of the inhibitory effects of capsazepine (upper) and HC-030031 (lower) at 10, 100 and 300 lM.

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This indicates that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons through the activation of TRPA1 and TRPV1 channels. In addition to their expression at peripheral nerve endings, it was revealed by whole-cell patch-clamp recording that TRPV1 and TRPA1 are expressed at the central terminals of primary afferent fibers, and that their activation facilitates excitatory synaptic transmission in SG neurons by increasing glutamate release (Yang et al., 1998; Kosugi et al., 2007; Uta et al., 2010). In this study, we show that ROS activate TRPV1 and TRPA1 channels at the central terminals of primary afferent fibers. Therefore, excessive activation of these channels by ROS may induce central sensitization in the spinal cord. It has recently been reported that TRPA1 is activated by endogenous and exogenous oxidative stressors, including NO, H2O2 and a,b-unsaturated aldehydes (Trevisani et al., 2007; Andersson et al., 2008; Andre` et al., 2008; Bessac et al., 2008; Sawada et al., 2008; Takahashi et al., 2008). In particular, H2O2, a ROS, activates TRPA1 channels directly. H2O2 is able to induce Ca2+ influx and inward currents in cultured cells and DRG neurons expressing TRPA1 (Andersson et al., 2008; Sawada et al., 2008). These findings support our result that SG neurons are stimulated by ROS through TRPA1 channel activation. There are a number of insightful observations on the relationship between TRPV1 and ROS. H2O2 sensitizes TRPV1 by covalent cysteine modification (Chuang and Lin, 2009). Furthermore, oxidative stress increases responsiveness to thermal stimuli in cultured cells via TRPV1 (Susankova et al., 2006). TRPV1 and ROS play critical roles in the spinal cord in an arthritic inflammation model (Westlund et al., 2010). TRPV1 activation increases the expression of TNF receptor type1 in DRG neurons through a ROS-dependent signaling pathway (Ma et al., 2009). NADPH oxidase accelerates the translocation of PKC in DRG neurons, thereby enhancing TRPV1 (Ibi et al., 2008). Although, in our study, the inhibitory effect of the TRPV1 antagonist was smaller than that of the TRPA1 antagonist, ROS also activated TRPV1 channels in central afferent fiber terminals. We also found that non-specific ROS scavengers, PBN and NAC, significantly suppressed the t-BOOH-induced enhancement of sEPSCs. However, another non-specific scavenger, TEMPOL, only slightly suppressed the t-BOOH-induced enhancement of sEPSCs. It is unclear why the suppressive effect of TEMPOL was minor compared with PBN or NAC. Although not statistically significant, the sEPSC frequency in the presence of TEMPOL was lower than in its absence. TEMPOL is a nonspecific ROS scavenger; however it acts mainly as an SOD mimetic. We hypothesize that H2O2 concentrations transiently rise owing to TEMPOL-mediated conversion of superoxide into H2O2. The high H2O2 levels then activate TRPA1. This may be one of the reasons why TEMPOL’s suppressive effect was minimal compared with PBN or NAC. In fact, TEMPOL infusion into the renal medulla fails to prevent ROS-induced hypertension, unless catalase is co-administered

(Makino et al., 2003). This shows that TEMPOL produces H2O2 from superoxide, increasing levels of ROS. In line with our findings, there are reports showing that t-BOOH co-administration reverses TEMPOLinduced analgesia in formalin-induced pain (Hacimuftuoglu et al., 2006), and that TEMPOL’s analgesic effect is smaller than that of PBN in chemotherapy-induced pain (Fidanbpylu et al., 2011). ROS induce inward currents in SG neurons via TRPA1 channels In our study, t-BOOH occasionally produced an inward current, but this inward current was suppressed in the presence of not only a TRPA1 antagonist HC-030031, but also by Ca2+-free bath solution. This is likely the same inward current that was induced in SG neurons by the selective TRPA1 agonist allyl isothiocyanate (AITC) in our previous study (Kosugi et al., 2007). We suggest that AITC-induced inward currents are involved in NMDA receptor activation. The TRPV1 agonist capsaicin has also been reported to induce an inward current in SG neurons (Yang et al., 1998; Yang et al., 2000), although the mechanism of this inward current is unclear. In our study, t-BOOH-induced inward currents were incompletely blocked by a TRPV1 antagonist, capsazepine. Therefore t-BOOH-induced inward currents may have been produced mainly via TRPA1 activation. Although we did not investigate the relationship between ROS, NMDA receptors and TRPA1, there are several reports showing that ROS activate NMDA receptors (Muscoli et al., 2004; Ryu et al., 2010). In the present study, we did not examine the effect of tBOOH on IPSCs. However, t-BOOH was previously shown to decrease the frequency of miniature IPSCs (mIPSCs) in SG neurons in the mouse spinal cord (Yowtak et al., 2011). In contrast, another group reported that H2O2 increased the frequency of GABAergic mIPSCs in SG neurons in the mouse spinal cord (Takahashi et al., 2007). Thus, the effect of ROS on inhibitory transmission in SG neurons in the spinal cord is controversial and unclear. Finally, we focused on synaptic analysis of SG neurons in this study, rather than on intracellular mechanisms, although ROS can activate excitatory signaling through intracellular mechanisms as well. Previous literature has shown that mitochondrial ROS are activated by metabotropic glutamate receptors (mGluRs) in amygdaloid neurons to increase pain behavior (Li et al., 2011). Several studies reported that ROS convert polyunsaturated fatty acids into a,bunsaturated aldehydes, which activate TRPA1 channels (Trevisani et al., 2007; Andre` et al., 2008). ROS modulate a-amino-3-hydroxy-5-methyl-4-isozazole propionate (AMPA) receptor phosphorylation and regulate the cell surface localization of AMPA receptors (Lee et al., 2012). Further research is required to clarify the molecular mechanisms underlying the ability of ROS to modulate the activity of SG neurons. Conclusion This is the first report to show that ROS enhance excitatory synaptic transmission in rat spinal dorsal horn

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neurons via TRPA1 and TRPV1 channels. Our results suggest that ROS play an important role in the activation of SG neurons and provide insight into our understanding of the mechanisms of CNP in the spinal cord, such as that following SCI.

CONFLICT OF INTEREST STATEMENT The authors report no conflicts of interest regarding this study. Acknowledgments—This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) KAKENHI 2279139 to W.T., MEXT KAKENHI 22591647 to T.N., MEXT KAKENHI 23592173 to M.Y., and a grant from the Japan Orthopaedics and Traumatology Foundation, Inc. No. 274 to W.T.

REFERENCES Andersson DA, Gentry C, Moss S, Bevan S (2008) Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J Neurosci 28:2485–2494. Andre` E, Campi B, Materazzi S, Trevisani M, Amadesi S, Massi D, Creminon C, Vaksman N, Nassini R, Civelli M, Baraldi PG, Poole DP, Bunnett NW, Geppetti P, Patacchini R (2008) Cigarette smoke-induced neurogenic inflammation is mediated by alpha, beta-unsaturated aldehydes and the TRPA1 receptor in rodents. J Clin Invest 118:2574–2582. Bessac BF, Sivula M, von Hehn CA, Escalera J, Cohn L, Jordt SE (2008) TRPA1 is a major oxidant sensor in murine airway sensory neurons. J Clin Invest 118:1899–1910. Chuang HH, Lin S (2009) Oxidative challenges sensitize the capsaicin receptor by covalent cysteine modification. Proc Natl Acad Sci U S A 106:20097–20102. Doyle T, Chen Z, Muscoli C, Bryant L, Esposito E, Cuzzocrea S, Dagostino C, Ryerse J, Rausaria S, Kamadulski A, Neumann WL, Salvemini D (2012) Targeting the overproduction of peroxynitrite for the prevention and reversal of paclitaxel-induced neuropathic pain. J Neurosci 32:6149–6160. Dro¨ge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82:47–95. Fidanboylu M, Griffiths LA, Flatters SJ (2011) Global inhibition of reactive oxygen species (ROS) inhibits paclitaxel-induced painful peripheral neuropathy. PLoS One 6:e25212. Gao X, Kim HK, Chung JM, Chung K (2007) Reactive oxygen species (ROS) are involved in enhancement of NMDA-receptor phosphorylation in animal models of pain. Pain 131:262–271. Hacimuftuoglu A, Handy CR, Goettl VM, Lin CG, Dane S, Stephens Jr RL (2006) Antioxidants attenuate multiple phases of formalininduced nociceptive response in mice. Behav Brain Res 173:211–216. Hall ED, Bosken JM (2009) Measurement of oxygen radicals and lipid peroxidation in neural tissues. Curr Protoc Neurosci Chapter 7:Unit 7.17.1-51. Holzer P (2011) Transient receptor potential (TRP) channels as drug targets for diseases of the digestive system. Pharmacol Ther 131:142–170. Hulsebosch CE, Hains BC, Crown ED, Carlton SM (2009) Mechanisms of chronic central neuropathic pain after spinal cord injury. Brain Res Rev 60:202–213. Ibi M, Matsuno K, Shiba D, Katsuyama M, Iwata K, Kakehi T, Nakagawa T, Sango K, Shirai Y, Yokoyama T, Kaneko S, Saito N, Yabe-Nishimura C (2008) Reactive oxygen species derived from NOX1/NADPH oxidase enhance inflammatory pain. J Neurosci 28:9486–9494. Kallenborn-Gerhardt W, Schro¨der K, Del Turco D, Lu R, Kynast K, Kosowski J, Niederberger E, Shah AM, Brandes RP, Geisslinger

211

G, Schmidtko A (2012) NADPH oxidase-4 maintains neuropathic pain after peripheral nerve injury. J Neurosci 32:10136–10145. Kanai Y, Nakazato E, Fujiuchi A, Hara T, Imai A (2005) Involvement of an increased spinal TRPV1 sensitization through its up-regulation in mechanical allodynia of CCI rats. Neuropharmacology 49:977–984. Kim HK, Park SK, Zhou JL, Taglialatela G, Chung K, Coggeshall RE, Chung JM (2004) Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain 111:116–124. Kim HK, Kim JH, Gao X, Zhou JL, Lee I, Chung K, Chung JM (2006) Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain. Pain 122:53–62. Kim HY, Chung JM, Chung K (2008) Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: the effect of mitochondrial electron transport complex inhibitors. Neurosci Lett 447:87–91. Kim D, You B, Jo E-K, Han S-K, Simon MI, Lee SJ (2010a) NADPH oxidase 2-derived reactive oxygen species in spinal cord microglia contribute to peripheral nerve injury-induced neuropathic pain. Proc Natl Acad Sci U S A 107:14851–14856. Kim HK, Zhang YP, Gwak YS, Abdi S (2010b) Phenyl N-tertbutylnitrone, a free radical scavenger, reduces mechanical allodynia in chemotherapy-induced neuropathic pain in rats. Anesthesiology 112:432–439. Kim HY, Lee KY, Lu Y, Wang J, Cui L, Kim SJ, Chung JM, Chung K (2011) Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain. J Neurosci 31:12982–12991. Kim YH, Back SK, Davies AJ, Jeong H, Jo HJ, Chung G, Na HS, Bae YC, Kim SJ, Kim JS, Jung SJ, Oh SB (2012) TRPV1 in GABAergic interneurons mediates neuropathic mechanical allodynia and disinhibition of the nociceptive circuitry in the spinal cord. Neuron 74:640–647. Kosugi M, Nakatsuka T, Fujita T, Kuroda Y, Kumamoto E (2007) Activation of TRPA1 channel facilitates excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord. J Neurosci 27:4443–4451. Kwak KH, Han CG, Lee SH, Jeon Y, Park SS, Kim SO, Baek WY, Hong JG, Lim DG (2009) Reactive oxygen species in rats with chronic post-ischemia pain. Acta Anaesthesiol Scand 53:648–656. Lander HM (1997) An essential role for free radicals and derived species in signal transduction. FASEB J 11:118–124. Lee I, Kim HK, Kim JH, Chung K, Chung JM (2007) The role of reactive oxygen species in capsaicin-induced mechanical hyperalgesia and in the activities of dorsal horn neurons. Pain 133:9–17. Lee KY, Chung K, Chung JM (2010) Involvement of reactive oxygen species in long-term potentiation in the spinal cord dorsal horn. J Neurophysiol 103:382–391. Lee DZ, Chung JM, Chung K, Kang MG (2012) Reactive oxygen species (ROS) modulate AMPA receptor phosphorylation and cell-surface localization in concert with pain-related behavior. Pain 153:1905–1915. Levine JD, Alessandri-Haber N (2007) TRP channels: targets for the relief of pain. Biochim Biophys Acta 1772:989–1003. Li Z, Ji G, Neugebauer V (2011) Mitochondrial reactive oxygen species are activated by mGluR5 through IP3 and activate ERK and PKA to increase excitability of amygdala neurons and pain behavior. J Neurosci 31:1114–1127. Lu JM, Gong N, Wang YC, Wang YX (2012) D-Amino acid oxidasemediated increase in spinal hydrogen peroxide is mainly responsible for formalin-induced tonic pain. Br J Pharmacol 165:1941–1955. Ma F, Zhang L, Westlund KN (2009) Reactive oxygen species mediate TNFR1 increase after TRPV1 activation in mouse DRG neurons. Mol Pain 5:31. Makino A, Skelton MM, Zou AP, Cowley Jr AW (2003) Increased renal medullary H2O2 leads to hypertension. Hypertension 42:25–30. Mao Y-F, Yan N, Xu H, Sun JH, Xiong YC, Deng XM (2009) Edaravone, a free radical scavenger, is effective on neuropathic pain in rats. Brain Res 1248:68–75.

212

N. Nishio et al. / Neuroscience 247 (2013) 201–212

Muscoli C, Cuzzocrea S, Riley DP, Zweier JL, Thiemermann C, Wang ZQ, Salvemini D (2003) On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies. Br J Pharmacol 40:445–460. Muscoli C, Mollace V, Wheatley J, Masini E, Ndengele M, Wang ZQ, Salvemini D (2004) Superoxide-mediated nitration of spinal manganese superoxide dismutase: a novel pathway in Nmethyl-D-aspartate-mediated hyperalgesia. Pain 111:96–103. Muscoli C, Cuzzocrea S, Ndengele MM, Mollace V, Porreca F, Fabrizi F, Esposito E, Masini E, Matuschak GM, Salvemini D (2007) Therapeutic manipulation of peroxynitrite attenuates the development of opiate-induced antinociceptive tolerance in mice. J Clin Invest 117:3530–3539. Nakatsuka T, Park JS, Kumamoto E, Tamaki T, Yoshimura M (1999) Plastic changes in sensory inputs to rat substantia gelatinosa neurons following peripheral inflammation. Pain 82:39–47. Park ES, Gao X, Chung JM, Chung K (2006) Levels of mitochondrial reactive oxygen species increase in rat neuropathic spinal dorsal horn neurons. Neurosci Lett 391:108–111. Patapoutian A, Tate S, Woolf C (2009) Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov 8:55–68. Patwardhan AM, Scotland PE, Akopian AN, Hargreaves KM (2009) Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia. Proc Natl Acad Sci U S A 106:18820–18824. Ryu TH, Jung KY, Ha MJ, Kwak KH, Lim DG, Hong JG (2010) Superoxide and nitric oxide involvement in enhancing of Nmethyl-D-aspartate receptor-mediated central sensitization in the chronic post-ischemia pain model. Korean J Pain 23:1–10. Salvemini D, Little JW, Dovle T, Neumann WL (2011) Roles of reactive oxygen and nitrogen species in pain. Free Radic Biol Med 51:951–966. Sawada Y, Hosokawa H, Matsumura K, Kobayashi S (2008) Activation of transient receptor potential ankyrin 1 by hydrogen peroxide. Eur J Neurosci 27:1131–1142. Schwartz ES, Lee I, Chung K, Chung JM (2008) Oxidative stress in the spinal cord is an important contributor in capsaicin-induced mechanical secondary hyperalgesia in mice. Pain 138:514–524. Schwartz ES, Kim HY, Wang J, Lee I, Klann E, Chung JM, Chung K (2009) Persistent pain is dependent on spinal mitochondrial antioxidant levels. J Neurosci 29:159–168. Stowe DF, Camara AK (2009) Mitochondrial reactive oxygen species production in excitable cells: modulators of mitochondrial and cell function. Antioxid Redox Signal 11:1373–1414. Stucky CL, Dubin AE, Jeske NA, Malin SA, McKemy DD, Story GM (2009) Roles of transient receptor potential channels in pain. Brain Res Rev 60:2–23. Susankova K, Tousova K, Vyklicky L, Teisinger J, Vlachova V (2006) Reducing and oxidizing agents sensitize heat-activated vanilloid receptor (TRPV1) current. Mol Pharmacol 70:383–394. Takahashi A, Mikami M, Yang J (2007) Hydrogen peroxide increases GABAergic mIPSC through presynaptic release of calcium from IP3 receptor-sensitive stores in spinal cord substantia gelatinosa neurons. Eur J Neurosci 25:705–716. Takahashi N, Mizuno Y, Kozai D, Yamamoto S, Kiyonaka S, Shibata T, Uchida K, Mori Y (2008) Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators. Channels (Austin) 2:287–298.

Tal M (1996) A novel antioxidant alleviates heat hyperalgesia in rats with an experimental painful peripheral neuropathy. Neuroreport 7:1382–1384. Tominaga M (2007) Nociception and TRP channels. Handb Exp Pharmacol:489–505. Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, Imamachi N, Andre` E, Patacchini R, Cottrell GS, Gatti R, Basbaum AI, Bunnett NW, Julius D, Geppetti P (2007) 4Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci U S A 104:13519–13524. Uta D, Furue H, Pickering AE, Rashid MH, Mizuguchi-Takase H, Katafuchi T, Imoto K, Yoshimura M (2010) TRPA1-expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord. Eur J Neurosci 31:1960–1973. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84. Vay L, Gu C, McNaughton PA (2012) The thermo-TRP ion channel family: properties and therapeutic implications. Br J Pharmacol 165:787–801. Wang ZQ, Porreca F, Cuzzocrea S, Galen K, Lightfoot R, Masini E, Muscoli C, Mollace V, Ndengele M, Ischiropoulos H, Salvemini D (2004) A newly identified role for superoxide in inflammatory pain. J Pharmacol Exp Ther 309:869–878. Wei H, Huang J-L, Hao B, Wang Y-C, Nian G, Ma A-N, Li X-Y, Wang Y-X, Pertovaara A (2011a) Intrathecal administration of antioxidants attenuates mechanical pain hypersensitivity induced by REM sleep deprivation in the rat. Scand J Pain 2:64–69. Wei H, Koivisto A, Saarnilehto M, Chapman H, Kuokkanen K, Hao B, Huang JL, Wang YX, Pertovaara A (2011b) Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat. Pain 152:582–591. Westlund KN, Kochukov MY, Lu Y, McNearney TA (2010) Impact of central and peripheral TRPV1 and ROS levels on proinflammatory mediators and nociceptive behavior. Mol Pain 6:46. Xu W, Chi L, Xu R, Ke Y, Luo C, Cai J, Qiu M, Gozal D, Liu R (2005) Increased production of reactive oxygen species contributes to motor neuron death in a compression mouse model of spinal cord injury. Spinal cord 43:204–213. Yang K, Kumamoto E, Furue H, Yoshimura M (1998) Capsaicin facilitates excitatory but not inhibitory synaptic transmission in substantia gelatinosa of the rat spinal cord. Neurosci Lett 255:135–138. Yang K, Kumamoto E, Furue H, Li YQ, Yoshimura M (2000) Capsaicin induces a slow inward current which is not mediated by substance P in substantia gelatinosa neurons of the rat spinal cord. Neuropharmacology 39:2185–2194. Yoshimura M, Nishi S (1995) Primary afferent-evoked glycine- and GABA-mediated IPSPs in substantia gelatinosa neurones in the rat spinal cord in vitro. J Physiol 482(Pt 1):29–38. Yowtak J, Lee KY, Kim HY, Wang J, Kim HK, Chung K, Chung JM (2011) Reactive oxygen species contribute to neuropathic pain by reducing spinal GABA release. Pain 152:844–852.

(Accepted 14 May 2013) (Available online 22 May 2013)