Role of α5-containing nicotinic receptors in neuropathic pain and response to nicotine
Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated channels formed from multiple α (α2–α10) and β subunits (β2–β4) in various combinations that are widely but not uniformly distributed in the peripheral and central nervous system. Heteropentameric nAChRs with an α3β4 backbone prevail in the PNS, whereas α4β2 receptors are more numerous in most parts of the CNS. Both the pharmacological and biochemical properties of nAChRs are critically determined by their subunit composition. Multiple neurobehavioral changes and effects have been attributed to nicotinic receptors in the CNS (Jacob et al., 2013, Dani and Bertrand, 2007, Hurst et al., 2013), including analgesia, allodynia, and pathological pain (Lawand et al., 1999, Bartolini et al., 2011, Umana et al., 2013, Hurst et al., 2013).
Various nicotinic agonists, e.g. epibatidine and related compounds, are potent analgesics acting at the spinal and supraspinal level (Khan et al., 1998, Khan et al., 2001, Bannon et al., 1998, Damaj et al., 1998). Substances such as epibatidine and ABT-594 have been known for quite some time to be equally or more potent analgesics than morphine, depending on the assay (Bannon and Jarboe, 1978). Nicotinic agonist antinociceptive effects have also been shown in animal models of postoperative (Rowley et al., 2008) and of neuropathic pain (Di Cesare et al., 2013, Abdin et al., 2006, Pacini et al., 2010). To date, several types of nAChRs have been implicated in mediating these effects, namely receptors containing the subunits α4 and β2 (Marubio et al., 1999, Khan et al., 2001), α3 (Young et al., 2008, Albers et al., 2014), α5 (Jackson et al., 2010) and α7 (Feuerbach et al., 2009). In vivo evidence for receptors containing the above subunits has been provided by the use of receptor-selective agonists and antagonists, as well as with mice carrying deletions of distinct nAChR subunit genes. Based on molecular modeling, desensitization of α4β2α5 receptors has recently been proposed as the mechanism which mediates the analgesic effect of nicotinic agonists (Zhang et al., 2012). Paradoxically, positive allosteric modulation using novel compounds acting on various nAChRs have also been shown to have potent effects in animal behavioral studies (Uteshev, 2014, Pandya and Yakel, 2013, Rode et al., 2012). For example, the positive α4β2 allosteric modulator NS-9283 can potentiate the analgesic efficacy of the epibatidine analogue ABT-594 (Zhu et al., 2011). Although analgesic effects have to date most often been reported to be due to action at α4β2 containing receptors, recent studies suggest that this subunit combination can be deemed as necessary but not necessarily sufficient to produce analgesia (Gao et al., 2010).
A number of studies have furthermore suggested that nAChRs are directly involved in the pain processing of noxious stimuli and in neuropathic pain. Hence, deletion of the β2 subunit lowers the mechanical and thermal nociceptive thresholds in β2-KO mice (Yalcin et al., 2011), knockdown of α5-containing receptors by intrathecal antisense oligonucleotides moderately reduces allodynia (Vincler and Eisenach, 2005), and hyperalgesia in a nicotine withdrawal model is lost in α7-KO mice (Jackson et al., 2008). After spinal nerve ligation in rats, spinal α5 receptor upregulation has also been reported (Vincler and Eisenach, 2004, Young et al., 2008).
Our work focuses on further studying the role of α5-containing receptors in neuropathic pain and in mediating the analgesic effects of nicotine. α5 is considered an accessory subunit as it can only form functional receptors when co-expressed with a principal subunit (such as α2, α3, or α4) and one complementary subunit (β2 or β4, e.g. as α4β2α5 or α3β4α5 receptors) (Wang et al., 1996, Gerzanich et al., 1998, Ramirez-Latorre et al., 1996). Recent studies using specific antibodies have localized the α5 subunit in various CNS regions, including the substantia nigra pars compacta, medial habenula, interpeduncular nucleus (IPN), striatum, thalamus, prefrontal cortex, hippocampus, and the spinal cord in both rats and mice (Mao et al., 2008, David et al., 2010, Grady et al., 2009, Scholze et al., 2012, Beiranvand et al., 2014). α5 assembles into α3β4 receptors in the superior cervical ganglion (SCG) (Mao et al., 2006, David et al., 2010), whereas in CNS regions such as the hippocampus, the striatum, the cerebral cortex, or the thalamus, α5 is found in combination with the subunits α4 and β2 (Mao et al., 2008). In the habenula, α5 co-assembles with both β2 and β4 to form the α3α5β4β2 complex (Grady et al., 2009, Scholze et al., 2012), while in the IPN α5 subunits co-assemble with β2, but not β4 (Grady et al., 2009, Beiranvand et al., 2014). The presence of α5 can profoundly impact the overall pharmacological and physiological properties of the receptor complex. Effects include altered calcium permeability, increased sensitivity to allosteric modulators, altered receptor desensitization, altered single-channel properties, or altered agonist-mediated responses such as effects on the potency and efficacy of agonists (Ciuraszkiewicz et al., 2013, Tapia et al., 2007, Kuryatov et al., 2008). Two tests for thermal sensitivity testing involving spinal and supraspinal mechanisms show that effects of nicotine are largely reduced in α5-KO mice (Jackson et al., 2010).
In the current study, we test whether α5-KO mice differ from their WT littermates in two well-established models of neuropathic pain and in their responses to analgesic doses of nicotine. We furthermore measure the overall number of hetero-pentameric nAChRs and the expression of distinct receptors containing the subunits β2-, β4-, and α5 by means of immunoprecipitation in the lumbar spinal cord, thalamus, hippocampus, habenula, striatum, and the IPN after peripheral nerve injury. We found no differences in the development of neuropathic pain between WT and α5-KO mice, and only minor changes in the expression of nicotinic receptors after peripheral nerve injury. The thermal analgesic effects of acute nicotine administration were also only marginally different. However, when tested in unoperated mice, WT animals developed tolerance to nicotine-induced analgesia to a larger extent than α5-KO mice.
Section snippets
Animals
For behavioral experiments (see exception below) and all biochemical assays, adult male littermate WT mice and mice with a deletion of the α5 nAChR subunit gene (α5-KO) (Wang et al., 2002) were used. Mice used in this study were backcrossed into C57Bl/6J background for at least 7 generations after germ line transmission. For most of the experiments, KO and WT mice were littermates from heterozygous breeding pairs and genotyped at weaning (18 days after birth). When probing for nicotine
Development of neuropathic pain behaviors is not altered in mice with deletions of the α5 nicotinic receptor subunit
Throughout all experiments, WT and α5-KO animals could not be distinguished by the blinded experimenter and did not show any differences in weight gain. Furthermore, prior to any procedure, control WT and α5-KO mice did not significantly differ in their reaction to heat, cold, or mechanical stimuli (Fig. 1). The development and maintenance of neuropathic pain was compared in α5-KO animals and their WT littermates in two different, widely used animal models: chronic constriction injury (CCI) and
Discussion
The use of nicotinic agonists as an alternative to conventional analgesics for the treatment of acute and pathologic pain is of major clinical interest. It is known that chronic pain patients have high smoking rates (Fishbain et al., 2013) and that smokers are more likely to show chronic pain disorders and have more intense pain (Palmer et al., 2003, Patterson et al., 2012). Studies conducted particularly in postoperative pain have shown mixed significant clinical analgesic effects of nicotine (
Acknowledgments
We would like to thank Gabriele Koth and Karin Schwarz for technical assistance with immunoprecipitation experiments and Jürgen Sandkühler for initial project discussions. Generation of the subunit-specific nAChR antibodies was supported by a grant from the Austrian Science Fund (P19325-B09 to P.S.).
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