Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain
Highlights
► Intrathecal α-conotoxins reduce mechanical allodynia in a neuropathic pain model. ► Primary afferent eEPSCs are not strongly modulated by α-conotoxins MII, AuIB or Vc1.1. ► Actions of intrathecal α-conotoxins not likely related to activity at GABAB receptors. ► α3-containing nAChR are potential targets for neuropathic pain.
Introduction
A limitation to effective clinical pain management is the lack of highly specific analgesics that exhibit tolerable side effects. The enormous diversity of peptides from Conus snails that target ion channels, receptors, and transporters known to be involved in neuropathic pain has led to the search for better analgesics based on venom-derived peptides (Lewis and Garcia, 2003). The α-conotoxins represent one such family that specifically target nicotinic acetylcholine receptor (nAChR) subtypes. Owing to the combination of a large variety of nAChR subunit assemblies and subunit-selective α-conotoxins, many potential novel analgesics have recently been identified (Dutton and Craik, 2001; Alonso et al., 2003; Sandall et al., 2003; Lang et al., 2005; Satkunanathan et al., 2005; Olivera et al., 2008; McIntosh et al., 2009) α-Conotoxins, including Vc1.1, RgIA, MII and AuIB, have all been reported to potently reverse signs of neuropathic pain, particularly tactile allodynia, in animal models when administered systemically (Satkunanathan et al., 2005; Klimis et al., 2011).
Some controversy exists as to the mechanisms of anti-allodynia among α-conotoxins. Early studies suggested that interaction with α3 subunit-containing nAChRs may mediate these actions (Livett et al., 2006), but the affinity of Vc1.1 and AuIB for these subtypes is rather weak (Clark et al., 2006; Vincler et al., 2006). Vc1.1 and RgIA are both potent antagonists of α9α10 nAChRs, suggesting this may be the anti-allodynia target (Vincler et al., 2006). However, MII and AuIB are both devoid of activity at α9α10 nAChRs (McIntosh et al., 1999; Callaghan et al., 2008; Azam and McIntosh, 2009; Callaghan and Adams, 2010; Klimis et al., 2011) and other α-conotoxin analogues that act on these nAChRs fail to inhibit allodynia (Nevin et al., 2007). Moreover, α9α10 nAChRs show very limited tissue distribution, being expressed predominantly in the olivochochlear system (Vetter et al., 2007) and their role in sensory nerve function is unclear. We have recently shown that Vc1.1, AuIB and RgIA inhibit N-type calcium channels in dorsal root ganglion (DRG) neurons through a novel GABAB receptor-dependent mechanism distinct from the well-known modulation of these channels by G-protein βγ subunits (Callaghan et al., 2008; Callaghan and Adams, 2010; Klimis et al., 2011). MII is inactive at this target, although it produces partial reversal of allodynia in nerve injured rats (Klimis et al., 2011), suggesting this is not the only mechanism. Taken together, these findings suggest N-type calcium channels and possibly α3 subunit-containing nAChR may both be important, but it is unlikely that α9α10 nAChRs are responsible for pain relief after systemic administration. However, α-conotoxins exhibit varying and incompletely characterized selectivity for nAChR comprising combinations of α3-, α5- and α6 – and α7-subunits together with different β-subunits (Clark et al., 2006; Vincler and McIntosh, 2007). Many of these subunits are expressed by sensory neurons (eg. Khan et al., 2003).
The present study was designed to determine if α-conotoxins with distinct activity profiles at α3- (but possibly other α-subunits) or α9α10-containing nAChRs differentially relieve allodynia following intrathecal administration in a neuropathic pain model and, in parallel, if inhibition of N-type calcium channels in primary afferent nerve terminals through a novel GABAB-receptor-dependent mechanism is responsible. The α-conotoxins MII, AuIB and Vc1.1 all displayed long-lasting (up to 6 h) anti-allodynic activity. In vitro electrophysiological recordings of primary afferent-stimulated evoked excitatory post-synaptic currents (eEPSCs) onto superficial dorsal horn neurons revealed that none of these peptides substantially inhibited primary afferent activity, although a conventional GABAB receptor agonist produced profound presynaptic inhibition. The findings suggest that neither α9α10 nAChRs nor GABAB-receptor-dependent inhibition of N-type calcium channels in primary afferent synapses is the mechanism of action, but intrathecal delivery of α-conotoxins appears to be a promising therapeutic avenue.
Section snippets
Rodents and surgical procedures for establishing neuropathic pain
All experiments involving animals were approved by the University of Sydney or Royal North Shore Hospital/University of Technology Animal Ethics Committees. Experiments were performed under the guidelines of the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council, Australia, 7th Edition). Great care was taken to minimise animal suffering during these experiments whenever possible. In vivo experiments were performed on
Effects of Vc1.1, AuIB and MII on PNL-induced mechanical allodynia
Partial nerve ligation-induced, long-lasting mechanical allodynia that was maximal by 10–12 days after surgery (data not shown) as determined by changes to the paw withdrawal threshold (PWT). At day 12–14 post-PNL, α-conotoxins were administered to conscious rats via a chronically implanted intrathecal catheter. The small apparent differences between animals randomly assigned to the different treatment groups for pre-PNL PWT and post-PNL PWT before injecting conotoxins were not significant.
Discussion
The present study has shown that intrathecal delivery of α-conotoxins differentially targeting α3 subunit-containing, α9α10 nAChR channels and GABAB receptors/N-type calcium channels are anti-allodynic in rodent models of neuropathic pain. These anti-allodynic effects were not confounded by motor deficits because rotarod performance was not impaired.
Previous studies have shown that nAChR agonists such as nicotine (α4β2) and epibatadine (non- α4β2) display anti-allodynic activity in neuropathic
Conclusions
Here we have shown that α-conotoxins which are applied intrathecally and are known to interact with α3∗ nAChRs, but not α9α10 nAChRs or GABAB receptors/N-type calcium channels, display anti-allodynic activity in vivo in a neuropathic pain model. This finding implies that drugs (including α-conotoxins) targeting α3-containing nAChRs, or perhaps other nAChR subunit combinations that these conotoxins interact with, may prove to be clinically relevant in the treatment of neuropathic pain.
Acknowledgements
This work was supported by the NHMRC Program Grant 351446. MJC is supported by an NHMRC Fellowship (SPRF, 511914). DJA is supported by an ARC Australian Professorial Fellowship.
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Present address: Health Innovations Research Institute, RMIT University, Melbourne, 3083 Victoria, Australia.