ω-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn
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).
Section snippets
Spinal cord slice preparation
Briefly, 8–15 day old Wistar rats were killed by cervical dislocation in accordance with guidelines of the University of Queensland Animal Ethics Committee. The spinal cord was removed in ice-cold oxygenated artificial cerebrospinal fluid (ACSF). Transverse 500–600 μm slices from L4–5 segments with attached 2–4 mm dorsal root were cut using a NVSLM1 motorised vibroslice (World Precision Instruments, Sarasota, FL, USA).
Patch-clamp recordings
A spinal cord slice was placed on the bottom of the recording chamber and
Results
In the present study, we used an experimental model mimicking nociceptive synaptic transmission in the rat spinal cord to elucidate the effects of different ω-conotoxins. Primary afferent fibres that are responsible for the conduction of nociceptive signals were electrically stimulated and recordings made primarily from lamina I and II neurons of the dorsal horn. Synaptic events were therefore monitored at the site where the primary nociceptive afferents terminate and the first synaptic relay
Discussion
The present findings provide new information on the inhibition of excitatory synaptic transmission between primary afferents and superficial lamina neurons in the dorsal horn of the rat spinal cord by five different ω-conotoxins: CVID, MVIIA and GVIA, highly selective antagonists for N-type VGCCs, and CVIB and MVIIC which inhibit both N- and P/Q-type VGCCs (Uchitel, 1997, Schroeder et al., 2004). The selective N-type VGCC antagonist CVID has been shown to effectively inhibit VGCCs in rat DRG
Acknowledgements
We thank the laboratory of Prof. Paul Alewood and Dr Richard Lewis (Institute for Molecular Bioscience, University of Queensland) for preparing ω-conotoxin samples. Supported by a National Health and Medical Research Council (NHMRC) Program Grant.
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