GABA, but not opioids, mediates the anti-hyperalgesic effects of 5-HT₇ receptor activation in rats suffering from neuropathic pain

Abstract

Among receptors mediating serotonin actions in pain control, the 5-HT₇R is of special interest because it is expressed by primary afferent fibers and intrinsic GABAergic and opioidergic interneurons within the spinal dorsal horn. Herein, we investigated whether GABA and/or opioids contribute to 5-HT₇R-mediated control of neuropathic pain caused by nerve ligation. Acute administration of 5-HT₇R agonists (AS-19, MSD-5a, E-55888) was found to markedly reduce mechanical and thermal hyperalgesia in rats with unilateral constriction injury to the sciatic nerve (CCI-SN). In contrast, mechanical hypersensitivity caused by unilateral constriction injury to the infraorbital nerve was essentially unaffected by these ligands. Further characterization of the anti-hyperalgesic effect of 5-HT₇R activation by the selective agonist E-55888 showed that it was associated with a decrease in IL-1ß mRNA overexpression in ipsilateral L4–L6 dorsal root ganglia and lumbar dorsal horn in CCI-SN rats. In addition, E-55888 diminished CCI-SN-associated increase in c-Fos immunolabeling in superficial laminae of the lumbar dorsal horn and the locus coeruleus, but increased c-Fos immunolabeling in the nucleus tractus solitarius and the parabrachial area in both control and CCI-SN rats. When injected intrathecally (i.t.), bicuculline (3 μg i.t.), but neither phaclofen (5 μg i.t.) nor naloxone (10 μg i.t.), significantly reduced the anti-hyperalgesic effects of 5-HT₇R activation (E-55888, 10 mg/kg s.c.) in CCI-SN rats. These data support the idea that 5-HT₇R-mediated inhibitory control of neuropathic pain is underlain by excitation of GABAergic interneurons within the dorsal horn. In addition, 5-HT₇R activation-induced c-Fos increase in the nucleus tractus solitarius and the parabrachial area suggests that supraspinal mechanisms might also be involved.

Introduction

Neuropathic pain is a major problem for clinicians because of the limited efficacy of drugs currently used for its alleviation (Finnerup et al., 2010). Thus, the development of new therapeutic tools is of particular importance in order to relieve pain in medical practice. In this regard, monoaminergic neurotransmission is still a relevant target as shown by the recent development of innovative antinociceptive drugs (Finnerup et al., 2010; Riemsma et al., 2011). Among monoamines, serotonin (5-hydroxytryptamine, 5-HT) is of particular interest because it can mediate both antinociceptive and pronociceptive actions at the spinal level (Bardin, 2011; Kayser et al., 2010a; Millan, 2002; Suzuki et al., 2004). This bidirectional effect depends not only on the type, duration, intensity of the nociceptive stimulus, the location and extent of the injury, but also on the multiplicity of 5-HT receptors (Bardin, 2011; Kayser et al., 2010a, Kayser et al., 2010b; Suzuki et al., 2004). As a consequence, the understanding of the actual role of 5-HT in pain control requires the knowledge of the functional implication of the different 5-HT receptor types.

One of the most recently identified 5-HT receptors among those that may play a role in pain processing is the 5-HT₇R. Indeed, several lines of evidence support the idea that this receptor is most probably involved in pain control at peripheral, spinal and/or supraspinal levels (Amaya-Castellanos et al., 2011; Brenchat et al., 2009, 2010, 2012; Dogrul and Seyrek, 2006; Dogrul et al., 2009; Meuser et al., 2002; Rocha-Gonzalez et al., 2005; Yanarates et al., 2010). Its mRNA is expressed in dorsal root and trigeminal ganglia (Pierce et al., 1996; Terron et al., 2001). In the dorsal horn of the spinal cord, 5-HT₇R is present in laminae I–II and III on A∂ and C primary afferent fibers, on peptidergic interneurons and on glial cells (Doly et al., 2005; Neumaier et al., 2001). Its expression at the supraspinal level in regions related to pain control and/or perception, such as cortical areas, thalamic nuclei and several nuclei in midbrain, pons and medulla, is also consistent with its implication in pain control (Harte et al., 2005; Neumaier et al., 2001). As a whole, available data point to an antinociceptive effect of central 5-HT₇R activation, whereas peripheral 5-HT₇R would mediate pronociceptive effects of 5-HT (Brenchat et al., 2009, 2010, 2012; Dogrul and Seyrek, 2006; Dogrul et al., 2009; Meuser et al., 2002; Rocha-Gonzalez et al., 2005; Yanarates et al., 2010). In conditions involving central sensitization, the overall effect of systemically administered 5-HT₇R agonists seems to be analgesic (Brenchat et al., 2009, 2010, 2012).

In order to further investigate mechanisms underlying 5-HT₇R-mediated modulation of neuropathic pain, we used validated rat models which consist of chronic constriction injury to the sciatic nerve (CCI-SN; Bennett and Xie, 1988) or the infraorbital nerve (Vos et al., 1994). Mechanical and thermal nociceptive tests, immunohistochemical labeling of c-Fos expression (used as neuronal activity marker) and qRT-PCR determinations of mRNAs encoding pro-inflammatory cytokines were used to characterize the effects of 5-HT₇R stimulation (mainly by the selective agonist E-55888; Brenchat et al., 2010) in these models. On the other hand, because 5-HT₇R activation causes neuronal excitation (Leopoldo et al., 2011; Matthys et al., 2012) and interferes with opioidergic and GABAergic mechanisms (Dogrul and Seyrek, 2006; Tokarski et al., 2011), pharmacological experiments were carried out to assess whether 5-HT₇R-mediated excitation of inhibitory opioidergic and/or GABAergic interneurons could underlie the effects of 5-HT₇R agonists.