Elsevier

Neuroscience

Volume 247, 5 September 2013, Pages 201-212
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2013.05.023Get rights and content

Highlights

  • ROS enhance excitatory synaptic transmission in rat spinal dorsal horn neurons.

  • These excitatory effects are induced by the activation of TRPA1 and TRPV1 channels.

  • ROS in the spinal cord can play an important role in central sensitization.

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-BOOH-induced 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 channels by ROS may induce central sensitization in the spinal cord and result in chronic pain such as that following SCI.

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 (O2radical dot) and hydroxyl radical (HOrad)] 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 (Dröge, 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 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 whole-cell patch-clamp recording technique, and we analyzed the role of TRPV1 and TRPA1 channels in these ROS-mediated effects.

Section snippets

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.

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

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

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.

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