Award details

Role of voltage-dependent calcium channels in nociceptive transmission under normal and neuropathic conditions

Reference	BB/D008832/1
Principal Investigator / Supervisor	Professor Gary Stephens
Co-Investigators / Co-Supervisors	Professor Anthony Dickenson
Institution	University of Reading
Department	Pharmacy
Funding type	Research
Value (£)	232,992
Status	Completed
Type	Research Grant
Start date	01/01/2007
End date	31/12/2009
Duration	36 months

Abstract

Our initial objective is to determine the relative contribution of voltage-dependent calcium channel (VDCC) subtypes to basal nociceptive transmission at the whole-animal level. We will make extracellular recordings from dorsal horn neurones using in vivo electrophysiology techniques, well-established in the laboratory. Such systems-based studies offer the best opportunity to assess the contribution of different receptors and ion channels to nociception. Based on our preliminary data with the selective antagonist SNX-482, we will focus on VDCC CaV2.3 subunits, which remains poorly understood in comparison to CaV2.1 and CaV2.2 subunits. A major aim is to correlate pharmacological antagonism of CaV2.3 with genetic ablation of the subunit, permitted by access to CaV2.3(-/-) mice. We will make the first in vivo recordings in CaV2.3(-/-) mice to determine any changes in basal neuronal responses compared to wild-type mice. A major objective is to examine the contribution of VDCCs to nociception using the spinal nerve ligation (SNL) model of neuropathy, also used routinely in the laboratory. In direct relation to this project, evidence suggests that neuropathy causes an increase in CaV2.2 subunit expression (Matthews & Dickenson, 2001a). Our preliminary data indicates a similar up-regulation of CaV2.3 in SNL rats (Stephens et al., 2003, Soc. Neurosci. Abstracts 589.10; Matthews et al., IASP 2005 meeting), which we will investigate further in this project. A further major aim is to perform in vivo electrophysiology in SNL mice. We will correlate electrophysiology with pain-related behavioural tests. SNX-482 will be injected at spinal nerves L3 and L4 in naive and SNL rats. The effects of SNX-482 on threshold for mechanical (von Frey), thermal (Hargreave's test) and cooling (acetone test) stimuli will be determined, together with effects on motor function (Rotorod test). We have recently performed a pilot study on the effects of SNX-482 on behavioural responses in rats and contributed to similar studies in mice (Nassar et al., 2004; Stirling et al., 2005). Therefore, we will extend our studies to CaV2.3(-/-) and wild-type mice and, also, examine the effects of neuropathy. In order to determine specific cellular locations in nociceptive pathways where changes in VDCC expression may underlie whole animal responses, we will extend our studies to in vitro electrophysiology. Rat spinal cord slices with intact dorsal roots will be cut. Evoked excitatory postsynaptic currents (EPSCs) will be measured in spinal laminae I neurones using intracellular patch clamp recording. Effects of selective VDCC blockers on EPSCs will be determined in naive and SNL rats using methods established in the laboratory (Stephens et al., 2001). These experiments aim to determine if pre- and/or postsynaptic VDCC mechanisms contribute to extracellular dorsal horn neuronal responses recorded at the whole animal level. A longer-term objective is to make spinal cord slices from CaV2.3(-/-) and wild type mice and, also, to examine the effects of neuropathy here. The patch clamp technique will also be used to record calcium currents in 'small' DRG neurones (that predominantly supply nociceptive information) from naive and SNL rats. These studies will be supported by RT-PCR in DRG neurones, to allow us to compare expression levels of VDCC subtypes. This work will be usefully extended to DRG cells from CaV2.3(-/-) mice to determine the contribution of CaV2.3 to R-type calcium current and, subsequently, to the effects of neuropathy on this contribution. Together, these experiments will determine the contribution of VDCCs to nociception at a sensory neurone level. Overall, this project will contribute basic research on the role of VDCCs in nociceptive pathways and investigate the effects of neuropathy. The current interest in the therapeutic potential of VDCCs in this area suggests this project can contribute to significant advances in human healthcare.

Summary

This project seeks to increase our understanding of how living organisms function and behave and, also, to provide basic knowledge that can be used to develop new products for medicine. The basic knowledge here relates to how the function of specific proteins involved in nerve cell signalling may be affected in an animal model of disease. The proteins involved are called calcium channels and the disease that we model is pain caused by nerve damage. Everybody suffers from pain at some point in their lives, it is estimated that two-thirds of the elderly experience some form of pain on a daily basis. In fact, the costs of symptoms such as lower back pain are greater than heart disease, stroke and depression. Pain can arise from a variety of causes including injury, childbirth, headache, surgery or disease. Of course, pain is the body's warning system, vital to prevent us from prolonged exposure to harmful agents. Pain may be short-lived, disappearing when the cause is withdrawn, this type is usually called 'nociceptive' pain. However, longer-term or 'chronic' pain can arise from damage to nerves themselves, called 'neuropathic' pain. The real problem with neuropathic pain is that medicines that effectively treat nociceptive pain, such as common drugs like aspirin and even stronger painkillers like morphine, are largely ineffective. In fact, the major medicine for neuropathic pain, gabapentin, only works in about one-third of patients, and then only reduces the pain by around half. Therefore, it is clear that the quality of life in the UK would benefit from basic research into changes occurring during neuropathy, as ultimately this knowledge may lead to the introduction of much-need novel medicines. To address these issues, we will use a common animal model of neuropathy, which introduces injury by tying off spinal nerves. Once damage is established, we can monitor responses from the nerve cells that transmit messages to the brain alerting us of pain. A drug thatsuccessfully blocks these signals has the potential to treat neuropathic pain. Nerve cells communicate with their neighbours by releasing neurotransmitter substances. This release is controlled by calcium ions, which enter the cell via proteins called calcium channels. Using our animal model, we have applied a chemical isolated from tarantula spider venom which blocks a single class of calcium channels selectively. This chemical was found to reduce communication only in rats suffering from nerve damage, and not in non-injured rats. Therefore, if we were to give the same chemical to patients suffering neuropathic pain, their symptoms may be reduced. Of course, it may not be wise to give people a chemical found in the venom of a poisonous spider. However, chemists can make subtle changes to the structure of the chemical to make it safer (or more selective) if necessary. For instance, a similar chemical, which blocks a different type of calcium channel, has recently been developed and given approval to treat neuropathic pain. In addition to blocking calcium channels with drugs, we can also remove them genetically by 'knocking-out' the specific gene responsible for generating the channel. We can now see if responses are changed in animals that uniquely lack this particular calcium channel. We also want to find out the points in the pain signalling pathway where different calcium channels are important and, also, how these may be affected in our animal model. These studies will allow us to target particular channels selectively and avoid any side-effects of medication. By combining these techniques we will provide not only basic research, but may also contribute to knowledge about human medicine and healthcare that will improve quality of life in the UK.

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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