Elizabeth A. Matthews

Effects of spinally delivered N- and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model of neuropathy.

&NA; Neuropathic pain, due to peripheral nerve damage, can include allodynia (perception of innocuous stimuli as being painful), hyperalgesia (increased sensitivity to noxious stimuli) and spontaneous pain, often accompanied by sensory deficits. Plasticity in transmission and modulatory systems are implicated in the underlying mechanisms. The Kim and Chung rodent model of neuropathy (Kim and Chung, Pain 50 (1992) 355) employed here involves unilateral tight ligation of two (L5 and L6) of the three (L4, L5, and L6) spinal nerves of the sciatic nerve and reproducibly induced mechanical and cold allodynia in the ipsilateral hindpaw over the 14 day post‐operative period. In vivo electrophysiological techniques have then been used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation (SNL). Activation of voltage‐dependent calcium channels (VDCCs) is critical for neurotransmitter release and neuronal excitability, and antagonists can be antinociceptive. Here, for the first time, the effect of N‐ and P‐type VDCC antagonists (&ohgr;‐conotoxin‐GVIA and &ohgr;‐agatoxin‐IVA, respectively) on the evoked dorsal horn neuronal responses after neuropathy have been investigated. Spinal &ohgr;‐conotoxin‐GVIA (0.1–3.2 &mgr;g) produced prolonged inhibitions of both the electrically‐ and low‐ and high‐intensity naturally‐evoked neuronal responses in SNL and control rats. Spinal &ohgr;‐agatoxin‐IVA (0.1–3.2 &mgr;g) also had an inhibitory effect but to a lesser extent. After neuropathy the potency of &ohgr;‐conotoxin‐GVIA was increased at lower doses in comparison to control. This indicates an altered role for N‐type but not P‐type VDCCs in sensory transmission after neuropathy and selective plasticity in these channels after nerve injury. Both pre‐ and post‐synaptic VDCCs appear to be important.

Calcium Channel α2δ1 Subunit Mediates Spinal Hyperexcitability in Pain Modulation

&NA; Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage‐gated calcium channel (VGCC) &agr;2&dgr;1 subunit (Cav&agr;2&dgr;1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Cav&agr;2&dgr;1 in neuronal tissues. Cav&agr;2&dgr;1 overexpression resulted in enhanced currents, altered kinetics and voltage‐dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue‐injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Cav&agr;2&dgr;1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration‐dependently in transgenic, but not wild‐type, sensory neurons. Thus, elevated neuronal Cav&agr;2&dgr;1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.

Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice.

&NA; NaV1.8 is a voltage‐gated sodium channel expressed only in a subset of sensory neurons of which more than 85% are nociceptors. In order to delete genes in nociceptive neurons, we generated heterozygous transgenic mice expressing Cre recombinase under the control of the NaV1.8 promoter. Functional Cre recombinase expression replicated precisely the expression pattern of NaV1.8. Cre expression began at embryonic day 14 in small diameter neurons in dorsal root, trigeminal and nodose ganglia, but was absent in non‐neuronal or CNS tissues into adulthood. Sodium channel subtypes were normal in isolated DRG neurons. Pain behaviour in response to mechanical or thermal stimuli, and in acute, inflammatory and neuropathic pain was also normal. These data demonstrate that the heterozygous NaV1.8‐Cre mouse line is a useful tool to analyse the effects of deleting floxed genes on pain behaviour.

Neurobiology of neuropathic pain: mode of action of anticonvulsants

Anticonvulsants are widely used for the treatment of neuropathic pain. Here we review the evidence for a number of peripheral and central changes after nerve injury that may provide a basis for the mechanisms of action of anticonvulsant therapies. The roles of sodium channels, calcium channels, and central glutamate mechanisms are emphasized as the main targets for anticonvulsant drugs in neuropathic pain states. The focus of this article is on anticonvulsants; however, opioids and antidepressants can also be effective in increasing inhibitions to control of pain in a manner similar to that of the enhancement of γ‐aminobutyric acid (GABA) function by antiepileptic drugs. A brief account of these approaches to neuropathic pain is also given.

A combination of gabapentin and morphine mediates enhanced inhibitory effects on dorsal horn neuronal responses in a rat model of neuropathy.

Background Peripheral nerve damage can result in severe, long-lasting pain accompanied by sensory deficits. This neuropathic pain remains a clinical problem, and effective morphine analgesia is often limited by intolerable side effects. The antiepileptic gabapentin has recently emerged as an alternative chronic pain treatment. Improved management of the diverse symptoms and mechanisms of neuropathic pain may arise from combination therapy, based on multiple pharmacologic targets and low drug doses. Methods The authors used the Kim and Chung rodent model of neuropathy to induce mechanical and cold allodynia in the ipsilateral hind paw. In vivo electrophysiologic techniques were subsequently used to record evoked dorsal horn neuronal responses in which the effects of systemic morphine and gabapentin were investigated, both individually and in combination. Results Morphine (1 and 4 mg/kg) inhibited neuronal responses of control rats but not after neuropathy. Gabapentin (10 and 20 mg/kg) inhibited neuronal responses in nerve injured rats and to a lesser extent in sham rats but not in naive rats. In the presence of gabapentin (ineffective low dose of 10 mg/kg), morphine (1 and 3 mg/kg) mediated significant inhibitory effects in all experimental groups, with the greatest inhibitions observed in spinal nerve–ligated and sham-operated rats. After neuropathy, inhibitions mediated by morphine were significantly increased in the presence of gabapentin compared with morphine alone. Conclusions After spinal nerve ligation, the inhibitory effects of systemic morphine on evoked dorsal horn neuronal responses are reduced compared with control, whereas the effectiveness of systemic gabapentin is enhanced. In combination with low-dose gabapentin, significant improvement in the effectiveness of morphine is observed, which demonstrates a clinical potential for the use of morphine and gabapentin combinational treatment for neuropathic pain.

Differential Role of N-Type Calcium Channel Splice Isoforms in Pain

N-type calcium channels are essential mediators of spinal nociceptive transmission. The core subunit of the N-type channel is encoded by a single gene, and multiple N-type channel isoforms can be generated by alternate splicing. In particular, cell-specific inclusion of an alternatively spliced exon 37a generates a novel form of the N-type channel that is highly enriched in nociceptive neurons and, as we show here, downregulated in a neuropathic pain model. Splice isoform-specific small interfering RNA silencing in vivo reveals that channels containing exon 37a are specifically required for mediating basal thermal nociception and for developing thermal and mechanical hyperalgesia during inflammatory and neuropathic pain. In contrast, both N-type channel isoforms (e37a- and e37b-containing) contribute to tactile neuropathic allodynia. Hence, exon 37a acts as a molecular switch that tailors the channels toward specific roles in pain.

Comparison of the effects of MK-801, ketamine and memantine on responses of spinal dorsal horn neurones in a rat model of mononeuropathy.

&NA; Selective ligation of the L5/L6 spinal nerves produces a partial denervation of the hindpaw and has proved to be a useful model for studying the mechanisms underlying neuropathic pain. Two weeks after surgery, in vivo electrophysiological studies were performed in sham operated and nerve injured rats and the responses of spinal dorsal horn neurones to controlled electrical and natural (mechanical and heat) stimuli were recorded. The systemic effects of three N‐methyl‐D‐aspartate receptor (NMDA) antagonists, ketamine (1–10 mg/kg), memantine (1–20 mg/kg) and MK‐801 (0.1–5 mg/kg) were compared. Ketamine a clinically available NMDA receptor antagonist, produced greater reductions of the postdischarge, thermal (10 mg/kg, P=0.02), and mechanical evoked responses in spinal nerve ligated (SNL) rats (von Frey 9 g, 1 mg/kg, P=0.04; 5 mg/kg, P=0.01; 10 mg/kg, P=0.05; von Frey 50 g, 5 mg/kg, P=0.02; 10 mg/kg, P=0.003). The inhibition of wind‐up was comparable in both animal groups. Memantine produced powerful inhibitions of wind‐up after nerve injury with little effect in sham controls (5 mg/kg, P=0.02). The postdischarge, mechanical and thermal evoked responses were reduced to similar extents by memantine in both experimental groups. The effects of MK‐801 were comparable between SNL and sham operated rats for all neuronal measures (wind‐up, postdischarge, thermal and noxious mechanical evoked responses). The differential blocking abilities of these antagonists on the various neuronal responses may relate to the characteristics of their voltage‐dependent blockage of the channel associated with the receptor. The favourable side effect profile of memantine supports its potential use for the treatment of neuropathic pain.

Effects of ethosuximide, a T-type Ca2+ channel blocker, on dorsal horn neuronal responses in rats

Plasticity in transmission and modulatory systems are implicated in mechanisms of neuropathic pain. Studies demonstrate the importance of high voltage-activated Ca(2+) channels in pain transmission, but the role of low voltage-activated, T-type Ca(2+) channels in nociception has not been investigated. The Kim and Chung rodent model of neuropathy [Pain 50 (1992) 355] was used to induce mechanical and cold allodynia in the ipsilateral hindpaw. In vivo electrophysiological techniques were used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation. Spinal ethosuximide (5-1055 microg) exerted dose-related inhibitions of both the electrically and low- and high-intensity mechanical and thermal evoked neuronal responses and its profile remained unaltered after neuropathy. Measures of spinal cord hyperexcitability were most susceptible to ethosuximide. This study, for the first time, indicates a possible role for low voltage-activated Ca(2+) channels in sensory transmission.

Enlargement of the receptive field size to low intensity mechanical stimulation in the rat spinal nerve ligation model of neuropathy

One characteristic of plasticity after peripheral tissue or nerve damage is receptive field reorganization, and enlargement of receptive field size has been suggested to occur in certain models of neuropathic pain. The aim of the present study was to explore whether enlargement of neuronal receptive fields could contribute to the mechanical allodynia found on the ipsilateral paw in the spinal nerve ligation model of neuropathy. After ligation of L(5)-L(6) spinal nerves, all rats developed behavioral signs of mechanical allodynia, while the sham-operated control group displayed no such changes. The characteristics of the evoked responses of the neurones recorded in the dorsal horn of the rats were similar between the spinal nerve ligation, the sham operated control group, and the nonoperated control group, except for spontaneous activity, which was significantly increased in the spinal nerve ligation group. The mean size of the receptive field on the ipsilateral hindpaw, mapped using low-intensity stimulation with 9-g von Frey hair, was significantly increased in the spinal nerve ligation group, as compared to the sham-operated group. No significant difference was seen with 15- or 75-g von Frey hairs. The distribution of the receptive fields over the plantar surface of the paw was similar between the study groups. The enlargement of receptive field for non-noxious touch could be an indication of central sensitization in this model.

The N-methyl-d-aspartate receptor modulator GLYX-13 enhances learning and memory, in young adult and learning impaired aging rats

NMDA receptor (NMDAR) activity has been strongly implicated in both in vitro and in vivo learning models and the decline in cognitive function associated with aging and is linked to a decrease in NMDAR functional expression. GLYX-13 is a tetrapeptide (Thr-Pro-Pro-Thr) which acts as a NMDAR receptor partial agonist at the glycine site. GLYX-13 was administered to young adult (3 months old) and aged (27-32 months old) Fischer 344 X Brown Norway F1 rats (FBNF1), and behavioral learning tested in trace eye blink conditioning (tEBC), a movable platform version of the Morris water maze (MWM), and alternating t-maze tasks. GLYX-13 (1mg/kg, i.v.) enhanced learning in both young adult and aging animals for MWM and alternating t-maze, and increased tEBC in aging rats. We previously showed optimal enhancement of tEBC in young adult rats given GLYX-13 at the same dose. Of these learning tasks, the MWM showed the most robust age related deficit in learning. In the MWM, GLYX-13 enhancement of learning was greater in the old compared to the young adult animals. Examination of the induction of long-term potentiation (LTP) and depression (LTD) at Schaffer collateral-CA1 synapses in hippocampal slices showed that aged rats showed marked, selective impairment in the magnitude of LTP evoked by a sub-maximal tetanus, and that GLYX-13 significantly enhanced the magnitude of LTP in slices from both young adult and aged rats without affecting LTD. These data, combined with the observation that the GLYX-13 enhancement of learning was greater in old than in young adult animals, suggest that GLYX-13 may be a promising treatment for deficits in cognitive function associated with aging.