GABAA α5 subunit-containing receptors do not contribute to reversal of inflammatory-induced spinal sensitization as indicated by the unique selectivity profile of the GABAA receptor allosteric modulator NS16085.

GABAA receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain. Restoration of diminished spinal GABAA-α2 and -α3 subunit-containing receptor function is a principal contributor to this analgesia, albeit involvement of GABAA-α5-receptors has not been excluded. Thus, we compared NS11394 and TPA023 (PAMs with selectivity/efficacy at GABAA-α2/α3/α5 receptors) with TP003 (a reportedly GABAA-α3 selective PAM) against spinal sensitization. However, in-house electrophysiology studies designed to confirm the selectivity of TPA023 and TP003 for human GABAA receptors did not corroborate published data, with TP003 displaying considerable GABAA-α5 receptor efficacy. Therefore, we identified a novel PAM, NS16085, which possesses negligible efficacy at GABAA-α5 receptors, but with GABAA-α2/α3 efficacy equivalent to NS11394. At the GABAA-α1 receptor the compound gives low level of negative modulation further separating it from the other compounds. Rat pups with carrageenan-induced hindpaw inflammatory hyperalgesia were used to make ex vivo spinal dorsal root-evoked ventral root recordings. Some spontaneous activity and large numbers of spikes to repetitive stimulation of dorsal roots at C-fibre intensity, indicative of wind-up and sensitization were observed. Equimolar concentrations of NS11394, TP003 and NS16085 all attenuated wind-up to a similar degree; TPA023 was clearly less effective. In adult rats, NS16085 (3-30 mg/kg, p.o.) dose-dependently reduced formalin-induced hindpaw flinching with efficacy comparable to NS11394. Thus, potentiation of GABAA-α2 and-α3 receptors is sufficient to depress spinal sensitization and mediate analgesia after inflammatory injury. Positive modulation at GABAA-α5-receptors is apparently dispensable for this process, an important consideration given the role of this receptor subtype in cognitive function.

Axotomy-induced changes in activity-dependent slowing in peripheral nerve fibres: role of hyperpolarization-activated/HCN channel current.

BACKGROUND: Slowing refers to the gradual decrease in conduction velocity evoked by repetitive electrical stimuli. The underlying mechanisms are still poorly understood, and its physiological/pathological relevance scarcely discussed; however, changes in axonal conduction properties might unmask abnormal nociceptor function and alter the encoding time window at the spinal cord. METHODS: Here, we characterized and compared the slowing in isolated units recorded from intact and axotomized saphenous nerves from mice, in vitro. We evaluated the role of hyperpolarization-activated/HCN channel current, Ih , in the generation of slowing, by examining the effect of the specific Ih blocker ZD7288. RESULTS: Based on their degree of slowing, intact C-fibres were classified as presumed nociceptors or non-nociceptors (>13% or <7% latency increase, respectively). Upon ZD7288 treatment, slowing was significantly augmented in 19/25 of the presumed C-nociceptors. In nerve-end neuromas, axotomized C-fibres could not be classified by their degree of slowing, which, in addition, was unrelated to the presence of ectopic mechanosensitivity. Axotomized fibres showed a ∼2.5-fold reduction in their slowing as compared with intact units and the effects of ZD7288 were more prominent, both in magnitude and percentage of sensitive fibres. Interestingly, in control conditions, all fibres sensitive to ZD7288 were more resistant to slowing. CONCLUSIONS: Under our experimental conditions, slowing seems largely dependent on functional Ih . The marked decrease in slowing after axotomy in C-fibres fits with the increased expression of functional hyperpolarization-activated/HCN channel current and may underlie the analgesic effects of the specific Ih blocker ZD7288 previously described in neuropathic pain models.

Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization.

BACKGROUND AND PURPOSE: The sigma-1 (σ(1) ) receptor is a ligand-regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ(1) receptor ligands used as pharmacological tools are unclear and the demonstration that σ(1) receptor antagonists have efficacy in reversing central sensitization-related pain sensitivity is still missing. EXPERIMENTAL APPROACH: The pharmacological properties of a novel σ(1) receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ(1) receptors on in vivo nociception in sensitizing conditions and on in vitro spinal cord sensitization in mice. Drug levels and autoradiographic, ex vivo binding for σ(1) receptor occupancy were measured to substantiate behavioural data. KEY RESULTS: Formalin-induced nociception (both phases), capsaicin-induced mechanical hypersensitivity and sciatic nerve injury-induced mechanical and thermal hypersensitivity were dose-dependently inhibited by systemic administration of S1RA. Occupancy of σ(1) receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve-injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ(1) receptors attenuated the wind-up responses in spinal cords sensitized by repetitive nociceptive stimulation. CONCLUSIONS AND IMPLICATIONS: These findings contribute to evidence identifying the σ(1) receptor as a modulator of activity-induced spinal sensitization and pain hypersensitivity, and suggest σ(1) receptor antagonists as potential novel treatments for neuropathic pain.

Comparison of the novel subtype-selective GABAA receptor-positive allosteric modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] with diazepam, zolpidem, bretazenil, and gaboxadol in rat models of inflammatory and neuropathic pain.

Spinal administration of GABA(A) receptor modulators, such as the benzodiazepine drug diazepam, partially alleviates neuropathic hypersensitivity that manifests as spontaneous pain, allodynia, and hyperalgesia. However, benzodiazepines are hindered by sedative impairments and other side effect issues occurring mainly as a consequence of binding to GABA(A) receptors containing the alpha(1) subunit. Here, we report on the novel subtype-selective GABA(A) receptor-positive modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], which possesses a functional efficacy selectivity profile of alpha(5) > alpha(3) > alpha(2) > alpha(1) at GABA(A) alpha subunit-containing receptors. Oral administration of NS11394 (1-30 mg/kg) to rats attenuated spontaneous nociceptive behaviors in response to hindpaw injection of formalin and capsaicin, effects that were blocked by the benzodiazepine site antagonist flumazenil. Ongoing inflammatory nociception, observed as hindpaw weight-bearing deficits after Freund's adjuvant injection, was also completely reversed by NS11394. Likewise, hindpaw mechanical allodynia was fully reversed by NS11394 in two rat models of peripheral neuropathic pain. Importantly, NS11394-mediated antinociception occurred at doses 20 to 40-fold lower than those inducing minor sedative or ataxic impairments. In contrast, putative antinociception associated with administration of either diazepam, zolpidem, or gaboxadol only occurred at doses producing intolerable side effects, whereas bretazenil was completely inactive despite minor influences on motoric function. In electrophysiological studies, NS11394 selectively attenuated spinal nociceptive reflexes and C-fiber-mediated wind-up in vitro pointing to involvement of a spinal site of action. The robust therapeutic window seen with NS11394 in animals suggests that compounds with this in vitro selectivity profile could have potential benefit in clinical treatment of pain in humans.

Retigabine-induced population primary afferent hyperpolarisation in vitro.

Retigabine is a compound of potential interest in analgesia which acts as an M-channel opener to depress neuronal excitability. Here we study the effects of retigabine and its antagonist XE-991 on populations of primary afferents. Experiments were performed using a hemisected spinal cord preparation from rat pups maintained under in vitro conditions. Recording from dorsal roots were performed using tight fitting suction electrodes coupled to AC and DC amplifiers. The adjacent dorsal root was electrically stimulated at regular intervals. The effects of the modulators on basal potential, spontaneous potentials and dorsal root-dorsal root responses were studied. Superfusion of retigabine (10 microM) produced long lasting and robust hyperpolarisation of primary afferents which persisted during superfusion of picrotoxin (20 microM) and tetrodotoxin (0.5 microM). Other effects of retigabine were (1) increase in stimulation threshold; (2) increase in size of responses to suprathreshold stimuli; and (3) increase in amplitude and decrease in frequency of spontaneous dorsal root potentials. Superfusion of XE-991 had little effect on its own but blocked all the effects of retigabine. These results indicate the presence of functional M-currents in central terminals of primary afferents and in the interneurones that mediate dorsal root potentials. The depressant effects of retigabine on the excitability of these structures may contribute to its analgesic effects after pain-inducing treatments.

Effects of M-current modulators on the excitability of immature rat spinal sensory and motor neurones.

M-currents have been shown to control neuronal excitability in a variety of central and peripheral neurones. Here we studied the effects of specific M-current modulators on the excitability of spinal neurones and their response to synaptic activation. Experiments were performed in vitro using the hemisected spinal cord from 7- to 11-day-old rats. Intracellular recordings were obtained from lumbar deep dorsal horn and motor neurones. Neuronal excitability was assessed by applying outward current pulses and synaptic responses were elicited by activation of a lumbar dorsal root. The M-current antagonist 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) and the agonist retigabine were superfused at 10 microM. Retigabine produced hyperpolarization and a large decrease in the excitability of motor (7/7) and dorsal horn neurones (11/12). The effects of retigabine were fully reversed by XE-991. XE-991 induced depolarization of most neurones tested and a large increase in the excitability of motor neurones (7/7) but only a weak increase in the excitability of a proportion of dorsal horn neurones (4/10). The effects of XE-991 were partly reversed by retigabine. Consistent with their effects on neuronal excitability, retigabine showed a general depressant effect on synaptic transmission, whereas XE-991 showed the opposite tendency to potentiate responses to dorsal root stimulation, particularly in motor neurones. The results show that retigabine can depress spinal excitability and the transmission of nociceptive information. Results also indicate a post-synaptic expression of functional M-currents in most motor neurones and a considerable proportion of deep dorsal horn neurones.

Effects of propofol and sevoflurane on the excitability of rat spinal motoneurones and nociceptive reflexes in vitro.

BACKGROUND: Spinal actions of halogenated ethers are widely recognized, whereas spinal actions of intravenous anaesthetics like propofol are less clear. The aim of this study was to compare the spinal effects of propofol and sevoflurane. METHODS: We used an isolated spinal cord in vitro preparation from rat pups and superfused the anaesthetics at known concentrations. Responses of motoneurones to single and repetitive C-fibre intensity stimulation (trains of 20 stimuli at 1 Hz) of a lumbar dorsal root were recorded from the corresponding ventral root via a suction electrode. RESULTS: Stimulation trains produced a wind-up of action potentials in motoneurones. Both propofol and sevoflurane produced a significant concentration-dependent depression of the evoked wind-up, although at clinically relevant concentrations sevoflurane exhibited a larger intrinsic efficacy. Applied at anaesthetic concentrations, sevoflurane 250 micro M abolished action potentials whereas propofol 1 micro M only produced a reduction close to 50%. At these concentrations, sevoflurane produced a large depressant effect on the monosynaptic reflex whereas propofol was ineffective. CONCLUSIONS: Sevoflurane produces large inhibitory effects on nociceptive and non-nociceptive reflexes which are likely to contribute to immobility during surgery. Compared with sevoflurane, propofol appears to have much weaker effects on spinal reflexes such as those recorded in an isolated preparation.

M-current modulators alter rat spinal nociceptive transmission: an electrophysiological study in vitro.

M-currents constitute a unique effector system to control neuronal excitability due to their voltage and ligand sensitivities. Here we have used retigabine, an M-current agonist, and XE-991, an M-current antagonist, to study the possible involvement of these currents in the processing of spinal sensory and motor processing of nociceptive information in normal, untreated rats. Experiments were performed in a hemisected spinal cord preparation from rat pups using extracellular recordings. Responses to activation of nociceptive and non-nociceptive afferent fibres were recorded. M-current modulators were bath applied to the entire cord or applied locally by pressure ejection. Retigabine and XE-991 produced long-lasting and concentration-dependent effects on nociceptive reflexes showing only minor effects on non-nociceptive reflexes. Retigabine depressed responses to repetitive stimulation of the dorsal root recorded from motor neurones and dorsal horn neurones, whereas XE-991 showed the opposite potentiatory effect and reversed effects of retigabine. Local application of the modulators close by motor nuclei produced changes in reflex responses similar to those caused by bath application. These results constitute a clear indication of the existence of functional M-currents in dorsal and ventral horn elements of the mammalian spinal cord where they may serve to regulate early sensory and motor processing of nociceptive information. The weak effect of modulators on non-nociceptive reflexes suggest that M-currents constitute a promising novel target for analgesics.