ω-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn

Abstract

A number of ω-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. ω-Conotoxins CVID and CVIB, venom peptides from Conus catus, inhibit N-type and N/P/Q-type VGCCs, respectively, in rat dorsal root ganglion sensory neurons. In the present study, we tested the effects of five different ω-conotoxins, CVID, CVIB, MVIIA, MVIIC and GVIA, on excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurons of rat spinal cord. The N-type VGCC antagonists CVID (200 nM) and MVIIA (500 nM) completely and irreversibly inhibited excitatory postsynaptic currents (EPSCs) in the dorsal horn superficial lamina. The N- and P/Q-type VGCC antagonist CVIB (200 nM) reversibly reduced evoked EPSC amplitude an average of 34 ± 8%, whereas MVIIC (200 nM) had no effect on excitatory synaptic transmission. In neurons receiving polysynaptic input, CVIB reduced both the EPSC amplitude and the “success rate” calculated as the relative number of primary afferent stimulations that resulted in postsynaptic responses. These results indicate that (i) the analgesic action of ω-conotoxins that antagonise N-type VGCCs may be attributed to inhibition of neurotransmission between primary afferents and superficial dorsal horn neurons, (ii) nociceptive synaptic transmission between primary afferents and superficial lamina neurons is mediated predominantly by N-type VGCCs, and (iii) in contrast to the irreversible inhibition by CVID, MVIIA and GVIA, the inhibition of excitatory monosynaptic transmission by CVIB is reversible.

Introduction

Voltage-gated calcium channels (VGCCs) are critical in the transduction of nociceptive information from the periphery to the central nervous system. Action potential propagation along the primary afferents conducts nociceptive information from the periphery, depolarising the presynaptic terminal. This results in the opening of VGCCs, inducing the release of a neurotransmitter into the synaptic cleft. It has been demonstrated that the N-type VGCC is the major class of VGCC associated with nociceptive synaptic transmission (Heinke et al., 2004), and N-type VGCCs are situated on both pre- and post-synaptic membranes of the synapse between primary nociceptive afferents and superficial lamina neurons of the substantia gelatinosa (SG) (Bao et al., 1998, Soeda et al., 2002, Westenbroek et al., 1998).

ω-Conotoxins are a large family of structurally related peptides containing a characteristic cystine knot motif with a wide range of specificities for inhibition of different types of VGCCs (Uchitel, 1997, Schroeder et al., 2004). An important application of VGCCs antagonists is related to their use as anti-nociceptive agents for the management of chronic pain (Scott et al., 2002). In spite of difficulties with delivery, the use of conotoxins in chronic pain management may have some advantages compared to opiates in terms of habituation and tolerance (Miljanich, 2004). Previous studies of conotoxins as anti-nociceptive agents have largely focused on the VGCC antagonists GVIA or MVIIA (Ziconotide) which potently and irreversibly inhibit N-type VGCCs. However, a number of peptides that antagonise VGCCs have been isolated from the predatory cone snail Conus catus (Lewis at al., 2000), including ω-conotoxin CVID which exhibits the highest selectivity for N- over P/Q-type VGCCs in radioligand binding assays. CVID (AM336) inhibits the release of substance P in rat spinal cord slices and has potent anti-nociceptive activity (Smith et al., 2002). ω-Conotoxin CVIB, a non-selective N- and P/Q-type antagonist, has recently been shown to block N-type VGCCs reversibly and P/Q-type VGCCs irreversibly in dorsal root ganglion (DRG) neurons (Motin et al., 2007a). However, the effectiveness of these ω-conotoxins for inhibition of nociceptive synaptic transmission mediated by primary afferent nerve stimulation in the spinal cord has not been examined.

The aim of the present study was to compare the effects of known anti-nociceptive ω-conotoxins CVID, MVIIA and GVIA and the non-selective N- and P/Q-type VGCC antagonists, ω-conotoxins CVIB and MVIIC, on excitatory synaptic transmission between Aδ and C fibres of the dorsal root and lamina I–II neurons of the superficial dorsal horn. A preliminary report of some of these results has been presented in abstract form (Motin et al., 2007b).