Jennifer M.A. Laird

Altered nociception, analgesia and aggression in mice lacking the receptor for substance P

The peptide neurotransmitter substance P modulates sensitivity to pain by activating the neurokinin-1 (NK-1) receptor, which is expressed by discrete populations of neurons throughout the central nervous system. Substance P is synthesized by small-diameter sensory ‘pain’ fibres, and release of the peptide into the dorsal horn of the spinal cord following intense peripheral stimulation promotes central hyperexcitability and increased sensitivity to pain. However, despite the availability of specific NK-1 antagonists, the function of substance P in the perception of pain remains unclear. Here we investigate the effect of disrupting the gene encoding the NK-1 receptor in mice. We found that the mutant mice were healthy and fertile, but the characteristic amplification (‘wind up’) and intensity coding of nociceptive reflexes was absent. Although substance P did not mediate the signalling of acute pain or hyperalgesia, it was essential for the full development of stress-induced analgesia and for an aggressive response to territorial challenge, demonstrating that the peptide plays an unexpected role in the adaptive response to stress.

Wind-up of spinal cord neurones and pain sensation: much ado about something?

Wind-up is a frequency-dependent increase in the excitability of spinal cord neurones, evoked by electrical stimulation of afferent C-fibres. Although it has been studied over the past thirty years, there are still uncertainties about its physiological meaning. Glutamate (NMDA) and tachykinin NK1 receptors are required to generate wind-up and therefore a positive modulation between these two receptor types has been suggested by some authors. However, most drugs capable of reducing the excitability of spinal cord neurones, including opioids and NSAIDs, can also reduce or even abolish wind-up. Thus, other theories involving synaptic efficacy, potassium channels, calcium channels, etc. have also been proposed for the generation of this phenomenon. Whatever the mechanisms involved in its generation, wind-up has been interpreted as a system for the amplification in the spinal cord of the nociceptive message that arrives from peripheral nociceptors connected to C-fibres. This probably reflects the physiological system activated in the spinal cord after an intense or persistent barrage of afferent nociceptive impulses. On the other hand, wind-up, central sensitisation and hyperalgesia are not the same phenomena, although they may share common properties. Wind-up can be an important tool to study the processing of nociceptive information in the spinal cord, and the central effects of drugs that modulate the nociceptive system. This paper reviews the physiological and pharmacological data on wind-up of spinal cord neurones, and the perceptual correlates of wind-up in human subjects, in the context of its possible relation to the triggering of hyperalgesic states, and also the multiple factors which contribute to the generation of wind-up.

A new model of visceral pain and referred hyperalgesia in the mouse.

&NA; The generation of transgenic mice that lack or overexpress genes relevant to pain is becoming increasing common. However, only one visceral pain model, the writhing test, is widely used in mice. Here we describe a novel model, chemical stimulation of the colon, which we have developed in mice. Mice of either sex were injected i.v. with 30 mg/kg Evans Blue for subsequent determination of plasma extravasation. For behavioural testing, they were placed on a raised grid and 50 &mgr;l of saline, mustard oil (0.25–2.5%) or capsaicin (0.03–0.3%) was administered by inserting a fine cannula into the colon via the anus. Visceral pain‐related behaviours (licking abdomen, stretching, contractions of abdomen etc) were counted for 20 min. Before intracolonic administration, and 20 min after, the frequency of withdrawal responses to the application of von Frey probes to the abdomen was tested. The colon was removed post‐mortem and the Evans Blue content measured. Mustard oil and capsaicin administration evoked dose‐dependent visceral pain behaviours, referred hyperalgesia (significant increase in responses to von Frey hairs) and colon plasma extravasation. The peak behavioural responses were evoked by 0.1% capsaicin and by 1% mustard oil respectively. The nociceptive behavioural responses were dose‐dependently reversed by morphine (ED50=1.9±1 mg/kg s.c.). We conclude that this model represents a useful tool both for phenotyping mutant mice and for classical pharmacology since information on visceral pain, referred hyperalgesia and colon inflammation can all obtained from the same animal.

Mechanisms of touch-evoked pain (allodynia): a new model

&NA; In this paper we review the current neurophysiological models of touch‐evoked pain and present a new proposal that addresses the mechanisms of allodynia. The new model is based on the notion that A‐&bgr; mechanoreceptors can gain access to nociceptive neurones by means of a presynaptic link, at central level, between low threshold mechanoreceptors and nociceptors. We propose that the excitation of nociceptors provoked by a peripheral injury activates the spinal interneurones that mediate primary afferent depolarization (PAD) between low threshold mechanoreceptors and nociceptors. As a consequence of the increased and persistent barrage driving these neurones their excitability is increased such that, when activated by low threshold mechanoreceptors from areas surrounding the injury site, they produce a very intense PAD in the nociceptive afferents which is capable of generating spike activity. This activation would be conducted antidromically in the form of dorsal root reflexes (DRRs) but would also be conducted forward activating the second order neurones normally driven by nociceptors. The sensory consequence of this mechanism is pain evoked by the activation of low threshold mechanoreceptors from an area surrounding an injury site (allodynia).

Deficits in visceral pain and hyperalgesia of mice with a disruption of the tachykinin NK1 receptor gene

Studies in mice lacking genes encoding for substance P or its receptor (NK1), or with NK1 antagonists, have shown that this system contributes to nociception, but the data are complex. Here, we have further examined the role of NK1 receptors in pain and hyperalgesia by comparing nociceptive responses to mechanical and chemical stimulation of viscera and the resulting hyperalgesia and inflammation in NK1 knockout (-/-) and wild-type (+/+) mice. We concentrated on visceral nociception because substance P is expressed by a much greater proportion of visceral than cutaneous afferents. NK1 -/- mice showed normal responses to visceral mechanical stimuli, measured as behavioural responses to intraperitoneal acetylcholine or hypertonic saline or reflex responses to colon distension in anaesthetized mice, although -/- mice failed to encode the intensity of noxious colon distensions. In contrast, NK1 -/- mice showed profound deficits in spontaneous behavioural reactions to an acute visceral chemical stimulus (intracolonic capsaicin) and failed to develop referred hyperalgesia or tissue oedema. However, in an identical procedure, intracolonic mustard oil evoked normal spontaneous behaviour, referred hyperalgesia and oedema in -/- mice. The inflammatory effects of capsaicin were abolished by denervation of the extrinsic innervation of the colon in rats, whereas those of mustard oil were unchanged, showing that intracolonic capsaicin evokes neurogenic inflammation, but mustard oil does not. Tests of other neurogenic inflammatory stimuli in NK1 -/- mice revealed impaired behavioural responses to cyclophosphamide cystitis and no acute reflex responses or primary hyperalgesia to intracolonic acetic acid. We conclude that NK1 receptors have an essential role mediating central nociceptive and peripheral inflammatory responses to noxious stimuli that evoke neurogenic inflammation, and modulating responses to noxious mechanical stimuli. We propose that two separate hyperalgesia pathways exist, one of which is NK1 receptor dependent, whereas the other does not require intact substance P/NK1 signalling.

The Tetrodotoxin‐Resistant Na+ Channel Nav1.8 is Essential for the Expression of Spontaneous Activity in Damaged Sensory Axons of Mice

The tetrodotoxin‐resistant sodium channel α subunit, Nav1.8, is exclusively expressed in primary sensory neurons and is suggested to play a role in the generation of ectopic action potentials after axonal injury and thereby contribute to neuropathic pain. Here we investigated the involvement of Nav1.8 in ectopic impulse generation in damaged axons by examining spontaneous activity and mechanosensitivity in neuromas formed by section of the saphenous nerve in Nav1.8 null mice and in their wild‐type littermates. We recorded 522 identified units from 24 neuromas in vitro at two time points, 8–11 days (median 10 days) and 19–29 days (median 22 days) post‐operatively. At ≈10 days, neither genotype showed spontaneous activity, but a significantly higher proportion of fibres were mechanosensitive in wild‐type (54 %) compared to Nav1.8 null neuromas (18 %). At ≈22 days, 19 % of fibres recorded in wild‐type neuromas showed spontaneous activity, whereas only one fibre of the 238 (0.4 %) recorded in neuromas taken from null mice showed ongoing activity. In recordings at ≈22 days, a similar proportion of fibres were mechanosensitive in wild‐type and Nav1.8 null neuromas (51 and 46 %, respectively). We conclude that Nav1.8 is essential for the expression of spontaneous activity in damaged sensory axons, and may also contribute to the development of ectopic mechanosensitivity.

In vivo recruitment by painful stimuli of AMPA receptor subunits to the plasma membrane of spinal cord neurons

&NA; The persistent increase in pain sensitivity observed after injury, known as hyperalgesia, depends on synaptic plasticity in the pain pathway, particularly in the spinal cord. Several potential mechanisms have been proposed, including post‐synaptic exocytosis of the AMPA subclass of glutamate receptors (AMPA‐R), which is known to play a critical role in synaptic plasticity in the hippocampus. AMPA‐R trafficking has been described in spinal neurons in culture but it is unknown if it can also occur in spinal neurons in vivo, or if it can be induced by natural painful stimulation. Here we have induced referred mechanical hyperalgesia in vivo by intracolonic instillation of capsaicin in mice and have observed a recruitment of GluR1 AMPA‐R subunits to neuronal plasma membranes in the lumbar spinal cord. Intracolonic capsaicin induced a rapid (10 min) increase in GluR1, but not GluR2/3 in the synaptosomal membrane fraction which lasted at least 3 h and a decrease in GluR1 subunit in the cytosolic fraction. Capsaicin treatment also provoked CaMKII activation and pre‐treatment with a specific CaMKII inhibitor prevented the GluR1 trafficking. Brefeldin‐A, an antibiotic that inhibits exocytosis of proteins, not only prevented GluR1 trafficking to the membrane but also inhibited referred hyperalgesia in capsaicin‐treated mice. Our results show that delivery of GluR1 AMPA receptor subunits to the cell membrane through a CaMKII activity‐dependent exocytotic regulated pathway contributes to the development of hyperalgesia after a painful stimulus. We conclude that AMPA‐R trafficking contributes to the synaptic strengthening induced in the pain pathway by natural stimulation.

Secondary hyperalgesia and presynaptic inhibition: an update

One of the most prominent features of secondary hyperalgesia is touch‐evoked pain, i.e., pain evoked by dynamic tactile stimuli applied to areas adjacent or remote from the originating injury. It is generally accepted that the neurobiological mechanism of this sensory alteration involves the central nervous system (CNS) so that incoming impulses in low‐threshold mechanoreceptors from the area of secondary hyperalgesia can evoke painful sensations instead of touch. Some years ago we proposed a mechanistic model for this form of pain based on presynaptic interactions in the spinal dorsal horn between the terminals of low‐threshold mechanoreceptors and of nociceptors. Here we review the evidence gathered in support of this model in the intervening years with special reference to experimental studies of antidromic activity (Dorsal Root Reflexes – DRRs) in nociceptive afferents and on the acquisition of low‐threshold inputs by nociceptor‐specific neurons in the spinal dorsal horn. We also discuss and identify potential molecular mechanisms that may underlie the presynaptic interaction model and therefore that could be responsible for the development of secondary hyperalgesia.

Extracellular signaling-regulated kinase-1 and -2 (ERK 1/2) mediate referred hyperalgesia in a murine model of visceral pain

We have investigated the role of spinal extracellular signaling-regulated kinase-1 and -2 (ERK1/2) in a model of visceral pain and hyperalgesia induced by intracolonic instillation of irritants in adult mice. Instillation of either capsaicin or mustard oil induced a significant activation of lumbosacral spinal ERK1/2, measured by immunoblot, with a peak 2.4-fold increase over control levels between 45 and 90 min post-treatment. Intracolonic saline did not produce significant activation of lumbosacral spinal ERK1/2, and none of the treatments evoked ERK1/2 activation in thoracic or cervical spinal cord. These studies suggested a preferential nuclear localization, which was explored by subcellular fractionation. Both mustard oil and capsaicin produced a redistribution of phosphorylated ERK1/2 from cytosol into the nucleus that was statistically significant at 45 min after treatment. Spinal ERK1/2 activation with capsaicin treatment correlated with the development of prolonged referred hyperalgesia. The upstream inhibitor of ERK phosphorylation, U0126 (100-400 microg/kg, i.v., 10 min pre-capsaicin), dose-dependently inhibited referred hyperalgesia 3-6 h after capsaicin. Treatment with U0126 did not affect spontaneous pain behavior or colon inflammation. Our data show that ERK activation plays a specific role in maintaining prolonged referred (secondary) hyperalgesia in visceral pain. The time course and subcellular localization of the effects observed suggest that ERK is involved in transcriptional events underlying the maintenance of secondary hyperalgesia.

Differential effects of N-Methyl-d-aspartate receptor blockade on nociceptive somatic and visceral reflexes

N-methyl-D-aspartate (NMDA) receptors appear to play little part in nociceptive responses evoked by acute stimulation of normal somatic tissues, but rather are involved in hyperalgesic responses after peripheral injury and inflammation. Previous studies from this laboratory have shown important differences in the neural organization of somatic and visceral nociceptive pathways. Here, we have explored the role of NMDA receptors in processing acute visceral noxious input, compared with somatic noxious input. The left ureter was cannulated close to the bladder in adult female Wistar rats anaesthetized with pentobarbitone (50 mg/kg i.p). Graded distentions of the ureter (30 s, 25-80 mmHg) evoked increases in blood pressure. These responses were dose-dependently inhibited by the NMDA receptor ion channel blockers ketamine and memantine (ID50 = 2.4+/-1.6 and 14.5+/-1.3 mg/kg, i.v.), and by the Merz glycine site antagonist Mrz 2/ 576 (ID50 = 0.2+/-0.2 mg/kg). Graded pinch stimuli (30 s, 2-4 N) of one hind-paw evoked similar pressor responses which were not affected by ketamine (up to 10 mg/kg). Similarly, Mrz 2/576 did not affect responses to noxious pinch, whereas memantine (ID50 = 17+/-12 mg/kg) did inhibit responses to pinch stimuli. However, in the dose range used neither ketamine nor Mrz 2/576 inhibited a pressor response of non-nociceptive origin (produced by bilateral carotid occlusion) whereas memantine did. Thus the effects of memantine are likely due to a non-specific cardiovascular effect. These results show that NMDA receptor antagonists inhibit nociceptive reflexes evoked from the normal ureter, and suggest that NMDA receptors are involved in the processing of acute nociceptive inputs from viscera. We conclude that acute stimulation of normal visceral tissue provokes intense responses that recruit neural mechanisms mediated by NMDA receptors. However, in somatic pathways, these mechanisms are recruited only by an enhanced peripheral input such as that produced after injury or inflammation.