Anthony H. Dickenson

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel α subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

EVIDENCE FOR SPINAL N-METHYL-D-ASPARTATE RECEPTOR INVOLVEMENT IN PROLONGED CHEMICAL NOCICEPTION IN THE RAT

We used in vivo electrophysiology and a model of more persistent nociceptive inputs to monitor spinal cord neuronal activity in anaesthetised rats to reveal the pharmacology of enhanced pain signalling. The study showed that all responses were blocked by non-selective antagonism of glutamate receptors but a selective and preferential role of the N-methyl-d-aspartate (NMDA) receptor in the prolonged plastic responses was clearly seen. The work lead to many publications, initially preclinical but increasingly from patient studies, showing the importance of the NMDA receptor in central sensitisation within the spinal cord and how this could relate to persistent pain states. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Subcutaneous formalin-induced activity of dorsal horn neurones in the rat: differential response to an intrathecal opiate administered pre or post formalin

&NA; Many studies of pain and nociception use short‐lasting acute stimuli which may have limited relevance to prolonged or chronic pain states. Using extracellular single‐unit recording in the dorsal horn of the rat lumbar spinal cord the present study examines the response of neurones to a long‐lasting nociceptive stimulus i.e., 50 &mgr;l 5% formalin injected into the corresponding receptive field in the ipsilateral hind paw, and modulation of this response by an opioid. Formalin produced a distinct biphasic excitatory response in all convergent neurones tested; an immediate acute or phasic peak of neuronal firing (mean maximum 22 spikes/sec) 0–10 min post injection, and a second more prolonged tonic excitatory response (mean maximum 12 spikes/sec) over a period 20–65 min after formalin. Cells only activated by innocuous stimuli were not excited by formalin indicating the involvement of C fibre afferents in the excitatory response of convergent neurones to formalin. Both the biphasic nature and the time course of the neuronal response are similar to those observed in behavioural studies. Intrathecal DAGO (Tyr‐D‐AlaGlyMePheGly‐ol), a potent and selective mu opioid receptor agonist, applied 20 min prior to formalin completely inhibited both peaks of excitation. Co‐administration of intrathecal naloxone with the agonist restored the biphasic response. By contrast, when the administration of naloxone was delayed to 2 min post formalin so that inhibition of the first peak by DAGO pretreatment occurred, there was no subsequent second peak of activity although antagonism of the opioid would have occurred. When DAGO was applied 2 min post formalin so the initial acute response occurred, the inhibitory effect of the agonist on the second peak was far less. Thus the relative ability of DAGO to modulate the biphasic excitatory response of cells to formalin depends on whether the agonist is administered prior to or after the formalin and the appearance of the second peak may depend on the presence of the first. These results are discussed in light of the role of these neurones in nociception, opioid effects and changes in neural systems following peripheral stimuli

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X2/3 heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious ‘warming’ stimuli.

Superficial NK1-expressing neurons control spinal excitability through activation of descending pathways

The increase in pain sensitivity that follows injury is regulated by superficially located projection neurons in the dorsal horn of the spinal cord that express the neurokinin-1 (NK1) receptor. After selective ablation of these neurons in rats, we identified changes in receptive field size, mechanical and thermal coding and central sensitization of deeper dorsal horn neurons that are important for both pain sensations and reflexes. We were able to reproduce these changes by pharmacological block of descending serotonergic facilitatory pathways. Using Fos histochemistry, we found changes in the activation of serotonergic neurons in the brainstem as well as evidence for a loss of descending control of spinal excitability. We conclude that NK1-positive spinal projection neurons, activated by primary afferent input, project to higher brain areas that control spinal excitability—and therefore pain sensitivity—primarily through descending pathways from the brainstem.

The cell and molecular basis of mechanical, cold, and inflammatory pain.

Peripheral pain pathways are activated by a range of stimuli. We used diphtheria toxin to kill all mouse postmitotic sensory neurons expressing the sodium channel Nav1.8. Mice showed normal motor activity and low-threshold mechanical and acute noxious heat responses but did not respond to noxious mechanical pressure or cold. They also showed a loss of enhanced pain responses and spontaneous pain behavior upon treatment with inflammatory insults. In contrast, nerve injury led to heightened pain sensitivity to thermal and mechanical stimuli indistinguishable from that seen with normal littermates. Pain behavior correlates well with central input from sensory neurons measured electrophysiologically in vivo. These data demonstrate that Nav1.8-expressing neurons are essential for mechanical, cold, and inflammatory pain but not for neuropathic pain or heat sensing.

Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat

The effects of two excitatory amino acid receptor antagonists gamma-D-glutamylglycine (DGG) and 2-amino-5-phosphonovaleric acid (APS), applied onto the spinal cord surface, were tested on the responses of dorsal horn nociceptive neurones in the anaesthetized rat. DGG is a non-selective antagonist at both the N-methyl-D-aspartate (NMDA) and non-NMDA receptors, whereas AP5 acid is selective for the NMDA receptor. DGG dose-dependently reduced the A and C fibre-evoked responses of neurones in all laminae of the dorsal horn and also inhibited the post-discharges of intermediate and deep neurones resulting from repeated C fibre stimulation. There was little difference in the effects of the antagonist on the intermediate neuronal population compared to superficial or deep cells in the dorsal horn. AP5 has little effect on C fibre-evoked activity in superficial cells but produced slight inhibitions of the C fibre-evoked responses and clear reductions in the post-discharge of the deep neurones. This contrasts with the excitatory effects of the antagonist on both types of responses in the intermediate cells. A fibre-evoked responses were unaffected by AP5. Taking into account the results with the two antagonists it appears that both A and C fibre-evoked responses of dorsal horn nociceptive neurones are mediated by non-NMDA receptors whilst the C fibre-evoked wind-up of deep dorsal horn cells involves the NMDA receptor which also seems to mediate, in a complex manner, C fibre responses of intermediate, presumed substantia gelatinosa neurones. The results are discussed with regard to nociceptive mechanisms in the dorsal horn.

Electrophysiological evidence for a role of nitric oxide in prolonged chemical nociception in the rat

The role of nitric oxide in the periphery and the spinal cord, during acute electrically-evoked and prolonged chemically-evoked nociceptive stimulation, was investigated in rats anaesthetised with halothane. The responses of single dorsal horn neurones to electrically-evoked A beta fibre and C fibre inputs were reduced by topical application (directly onto the spinal cord) of both the nitric oxide inhibitor, nitro-L-arginine methyl ester (L-NAME; 500-1500 micrograms) and the precursor of nitric oxide, L-arginine (4500 micrograms). Administration of L-NAME, either directly into the receptive field (500-1500 micrograms) or intravenously (10-100 mg/kg) had little or no effect on the acute electrically-evoked activity. Intravenous injection of L-NAME, administered 40 min prior to injection of formalin, significantly reduced the prolonged second peak of firing, with only a small effect on the short-duration first peak. Administration of L-NAME, directly into the site of injection of formalin, as a 10 min pretreatment, significantly reduced the second but not the first peak of the response. Topical application of L-NAME onto the spinal cord, as a 30 min pretreatment, significantly reduced both the first and second peaks of the response. This inhibition was not reversed by the coadministration of L-arginine, which was inhibitory by itself. Thus, nitric oxide may be involved, in a complex way, in nociceptive events both in the periphery and within the spinal cord.

Electrophysiological studies on the effects of intrathecal morphine on nociceptive neurones in the rat dorsal horn

&NA; We have studied the effects of intrathecal morphine on the responses of 38 dorsal horn neurones in the intact rat under halothane anaesthesia to A and C fibre electrical stimulation and to natural stimuli applied to their receptive fields. Morphine selectively reduced the C fibre and pinch evoked activity in a dose‐dependent naloxone‐reversible manner with an ED50 of 7 nmoles. The ‘wind‐up’ of neurones to repetitive stimulation was little altered except with the highest doses (50–150 nmoles) tested. By contrast, the A fibre evoked responses of the neurones were only slightly reduced by morphine and both the tactile responses and receptive field size to innocuous stimuli enhanced for certain cells. The results are discussed in relation to the spinal actions of opiates and their clinical applications.

Peripheral origins and central modulation of subcutaneous formalin-induced activity of rat dorsal horn neurones

Extracellular recordings of single convergent dorsal horn neurones in the spinal cord region L1-L3 were made in rats anaesthetised with halothane in a gaseous mix of N2O and O2. Subcutaneous formalin (5%, 50 microliters) has previously been found to produce a prolonged distinct biphasic response of dorsal horn convergent neurones in the same preparation. The present study demonstrates that the second period of neuronal excitation which occurs at about 20 min and lasts for at least another 40 min, could be abolished by lignocaine (2%, 50 microliters) injected into the site of the formalin injection (n = 5). The inhibition of formalin-evoked activity lasted for about 10-20 min and was followed by complete recovery of the neuronal response. The same dose of i.v. lignocaine had no effect on the formalin-induced neuronal activity (n = 4). Profound inhibitions of the second phase were also produced by tactile segmental stimulation and noxious stimuli applied to widespread areas of the body (diffuse noxious inhibitory controls). These findings are discussed with regard to the peripheral and central consequences of prolonged noxious stimuli.