Annika B. Malmberg

The Cloned Capsaicin Receptor Integrates Multiple Pain-Producing Stimuli

Capsaicin, the main pungent ingredient in hot chili peppers, elicits buming pain by activating specific (vanilloid) receptors on sensory nerve endings. The cloned vanilloid receptor (VR1) is a cation channel that is also activated by noxious heat. Here, analysis of heat-evoked single channel currents in excised membrane patches suggests that heat gates VR1 directly. We also show that protons decrease the temperature threshold for VR1 activation such that even moderately acidic conditions (pH < or = 5.9) activate VR1 at room temperature. VR1 can therefore be viewed as a molecular integrator of chemical and physical stimuli that elicit pain. Immunocytochemical analysis indicates that the receptor is located in a neurochemically heterogeneous population of small diameter primary afferent fibers. A role for VR1 in injury-induced hypersensitivity at the level of the sensory neuron is presented.

Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates

&NA; The generation of knock‐out and transgenic mice offers a promising approach to the identification of novel biochemical factors that contribute to persistent pain conditions. To take advantage of these mice, however, it is important to demonstrate that the traditional models of persistent pain, which were largely developed for studies in the rat, can be used in the mouse. Here, we combined behavioral and anatomical methods to characterize the pathophysiology of a partial nerve injury‐evoked pain condition in the ‘normal’ mouse. In male C57BL6 mice we tied a tight ligature around 1/3 to 1/2 of the diameter of the sciatic nerve and evaluated the time‐course and magnitude of the ensuing mechanical and thermal allodynia. We also used immunocytochemistry to analyze nerve injury‐induced changes in substance P (SP) and NK‐1 (SP) receptor expression in the spinal cord. As in the rat, partial nerve injury markedly decreased paw withdrawal thresholds to both mechanical and thermal stimuli on the injured side. We detected threshold changes one day after the injury. The thermal allodynia resolved by 49 days, but the mechanical allodynia persisted for the duration of the study (70 days). We found no changes contralateral to the nerve injury. Sympatholytic treatment with guanethidine significantly reduced both the thermal and mechanical allodynia. We observed a reduction of SP immunoreactivity in the superficial dorsal horn on the injured side at 7 and 14, but not at 3 or 70 days after the nerve injury, and we observed an increase of NK‐1 receptor expression at 3, 7, 14 and 42, but not at 70 days after the injury. We conclude that partial injury to the sciatic nerve produces a comparable allodynia and neurochemical plasticity in the rat and mouse. These results establish a valuable model for future studies of the biochemical basis of neuropathic pain in mice with specific gene modifications.

The 5-HT3 Subtype of Serotonin Receptor Contributes to Nociceptive Processing via a Novel Subset of Myelinated and Unmyelinated Nociceptors

Serotonin is a major component of the inflammatory chemical milieu and contributes to the pain of tissue injury via an action on multiple receptor subtypes. Here we studied mice after genetic or pharmacological disruption of the 5-HT3 receptor, an excitatory serotonin-gated ion channel. We demonstrate that tissue injury-induced persistent, but not acute, nociception is significantly reduced after functional elimination of this receptor subtype. Specifically, in the setting of tissue injury, the 5-HT3receptor mediates activation of nociceptors but does not contribute to injury-associated edema. This result is explained by the localization of 5-HT3 receptor transcripts to a previously uncharacterized subset of myelinated and unmyelinated afferents, few of which express the proinflammatory neuropeptide substance P. Finally, we provide evidence that central serotonergic circuits modulate nociceptive transmission via a facilitatory action at spinal 5-HT3 receptors. We conclude that activation of both peripheral and central 5-HT3 receptors is pronociceptive and that the contribution of peripheral 5-HT3 receptors involves a novel complement of primary afferent nociceptors.

The Paired Homeodomain Protein DRG11 Is Required for the Projection of Cutaneous Sensory Afferent Fibers to the Dorsal Spinal Cord

Cutaneous sensory neurons that detect noxious stimuli project to the dorsal horn of the spinal cord, while those innervating muscle stretch receptors project to the ventral horn. DRG11, a paired homeodomain transcription factor, is expressed in both the developing dorsal horn and in sensory neurons, but not in the ventral spinal cord. Mouse embryos deficient in DRG11 display abnormalities in the spatio-temporal patterning of cutaneous sensory afferent fiber projections to the dorsal, but not the ventral spinal cord, as well as defects in dorsal horn morphogenesis. These early developmental abnormalities lead, in adults, to significantly attenuated sensitivity to noxious stimuli. In contrast, locomotion and sensori-motor functions appear normal. Drg11 is thus required for the formation of spatio-temporally appropriate projections from nociceptive sensory neurons to their central targets in the dorsal horn of the spinal cord.

Inflammation-induced up-regulation of protein kinase Cγ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing

Activation of various second messengers contributes to long-term changes in the excitability of dorsal horn neurons and to persistent pain conditions produced by injury. Here, we compared the time-course of decreased mechanical nociceptive thresholds and the density of protein kinase Cgamma immunoreactivity in the dorsal horn after injections of complete Freunds adjuvant in the plantar surface of the rat hindpaw. Complete Freunds adjuvant significantly increased paw diameter and mechanical sensitivity ipsilateral to the inflammation. The changes peaked one day post-injury, but endured for at least two weeks. In these rats, we recorded a 75-100% increase in protein kinase Cgamma immunoreactivity in the ipsilateral superficial dorsal horn of the L4 and L5 segments at all time-points. Electron microscopy revealed that the up-regulation was associated with a significant translocation of protein kinase Cgamma immunoreactivity to the plasma membrane. In double-label cytochemical studies, we found that about 20% of the protein kinase Cgamma-immunoreactive neurons, which are concentrated in inner lamina II, contain glutamate decarboxylase-67 messenger RNA, but none stain for parvalbumin or nitric oxide synthase. These results indicate that persistent changes in protein kinase Cgamma immunoreactivity parallel the time-course of mechanical allodynia and suggest that protein kinase Cgamma contributes to the maintenance of the allodynia produced by peripheral inflammation. The minimal expression of protein kinase Cgamma in presumed inhibitory neurons suggests that protein kinase Cgamma-mediated regulation of excitatory interneurons underlies the changes in spinal cord activity during persistent nociception.

Reduced development of tolerance to the analgesic effects of morphine and clonidine in PKCγ mutant mice

&NA; A variety of second messenger systems have been implicated in the intracellular mechanisms of tolerance development to the analgesic actions of morphine, a mu opioid, and clonidine, an alpha‐2 adrenergic receptor agonist. Here, we studied mice that carry a null mutation in the gene encoding a neuronal specific isoform of protein kinase C (PKC), namely, PKC&ggr;. We used the tail‐flick test to construct dose–response curves before and 4 days after chronic morphine (75‐mg pellets, subcutaneously (s.c.)) or clonidine treatment (0.3 mg/kg, s.c., twice daily). Baseline tail‐flick latencies did not differ in PKC&ggr; mutant and wild‐type mice (3–4 s). Both morphine and clonidine produced a dose‐dependent suppression of the tail‐flick response with an ED50 (effective dose resulting in a 50% reduction of the control response) value (2.0 mg/kg for morphine and 0.1 mg/kg for clonidine) that was similar for naive mutant and wild‐type mice. In contrast, after 4 days of drug delivery, mutant mice showed significantly less rightward shift in the dose–response curve to morphine (six‐fold for wild‐type and three‐fold for mutant mice) and to clonidine (five‐fold for wild‐type and no shift for the mutant mice). These results indicate that PKC&ggr; contributes to the development of tolerance to the analgesic effects of both morphine and clonidine. Chronic morphine treatment can also result in sensitization of spinal cord neurons and increased pain behaviors following a noxious insult. To assess the contribution of PKC&ggr; to this process, we studied the responses of wild‐type and mutant mice to an intraplantar injection of formalin (a model of persistent pain) following chronic morphine treatment. Although morphine tolerance increased formalin‐evoked persistent pain behavior and Fos‐LI in wild‐type mice, there was no difference between placebo‐ and morphine‐treated mutant mice, suggesting that PKC&ggr; also contributes to chronic morphine‐induced changes in nociceptive processing.

Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T.

&NA; Subunit non‐selective N‐methyl‐d‐aspartate (NMDA) receptor antagonists reduce injury‐induced pain behavior, but generally produce unacceptable side effects. In this study, we examined the antinociceptive and motor effects of cone snail venom‐derived peptides, conantokins G and T (conG and conT), which are selective inhibitors of the NR2B or NR2A and NR2B subtypes of the NMDA receptor, respectively. We tested the effects of conG and conT in models of tissue (formalin test), nerve injury (partial sciatic nerve ligation) and inflammation‐induced (intraplantar Complete Freunds Adjuvant; CFA) pain in mice. In the formalin test, intrathecal (i.t.) conG or conT suppressed the ongoing pain behavior (ED50 and 95% confidence intervals (CI), 11 (7–19) and 19 (11–33), respectively) at doses that were 17–27 times lower than those required to impair motor function (accelerating rotarod treadmill test: ED50 and 95% CI, 300 (120–730) and 320 (190–540) pmol, respectively). By comparison, SNX‐111, an N‐type voltage‐sensitive calcium channel antagonist that is also derived from cone snail venom, produced significant motor impairment at a dose (3.0 pmol, i.t.) that was only partially efficacious in the formalin test. Furthermore, conG reversed the allodynia produced by nerve injury, with greater potency on thermal (ED50 and 95% CI, 24 (10–55) pmol) than on mechanical allodynia (59 (33–105) pmol). Finally, a single dose of conG (100 pmol, i.t.) also reduced CFA‐evoked thermal and mechanical allodynia. Taken together, these results demonstrate that conantokins exhibit potent antinociceptive effects in several models of injury‐induced pain. The study supports the notion that drugs directed against subtypes of the NMDA receptor, by virtue of their reduced side‐effect profile, hold promise as novel therapeutic agents for the control of pain.

Contribution of α2 receptor subtypes to nerve injury-induced pain and its regulation by dexmedetomidine

There is evidence that noradrenaline contributes to the development and maintenance of neuropathic pain produced by trauma to a peripheral nerve. It is, however, unclear which subtype(s) of α adrenergic receptors (AR) may be involved. In addition to pro‐nociceptive actions of AR stimulation, α2 AR agonists produce antinociceptive effects. Here we studied the contribution of the α2 AR subtypes, α2A, α2B and α2C to the development of neuropathic pain. We also examined the antinociceptive effect produced by the α2 AR agonist dexmedetomidine in nerve‐injured mice. The studies were performed in mice that carry either a point (α2A) or a null (α2B and α2C) mutation in the gene encoding the α2 AR. To induce a neuropathic pain condition, we partially ligated the sciatic nerve and measured changes in thermal and mechanical sensitivity. Baseline mechanical and thermal withdrawal thresholds were similar in all mutant and wild‐type mice; and, after peripheral nerve injury, all mice developed comparable hypersensitivity (allodynia) to thermal and mechanical stimulation. Dexmedetomidine reversed the allodynia at a low dose (3u2003μgu2003kg−1, s.c.) and produced antinociceptive effects at higher doses (10u2003–u200330u2003μgu2003kg−1) in all groups except in α2A AR mutant mice. The effect of dexmedetomidine was reversed by intrathecal, but not systemic, injection of the α2 AR antagonist RS 42206. These results suggest that neither α2A, α2B nor α2C AR is required for the development of neuropathic pain after peripheral nerve injury, however, the spinal α2A AR is essential for the antinociceptive effects of dexmedetomidine.

Molecular pain, a new era of pain research and medicine

Molecular pain is a relatively new and rapidly expanding research field that represents an advanced step from conventional pain research. Molecular pain research addresses physiological and pathological pain at the cellular, subcellular and molecular levels. These studies integrate pain research with molecular biology, genomics, proteomics, modern electrophysiology and neurobiology. The field of molecular pain research has been rapidly expanding in the recent years, and has great promise for the identification of highly specific and effective targets for the treatment of intractable pain. Although several existing journals publish articles on classical pain research, none are specifically dedicated to molecular pain research. Therefore, a new journal focused on molecular pain research is needed. Molecular Pain, an Open Access, peer-reviewed, online journal, will provide a forum for molecular pain scientists to communicate their research findings in a targeted manner to others in this important and growing field.

Pharmacological, Pharmacokinetic, and Primate Analgesic Efficacy Profile of the Novel Bradykinin B1 Receptor Antagonist ELN441958

The bradykinin B1 receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B1 receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described. ELN441958 competitively inhibited the binding of the B1 agonist ligand [3H]desArg10-kallidin ([3H]DAKD) to IMR-90 human fibroblast membranes with high affinity (Ki = 0.26 ± 0.02 nM). ELN441958 potently antagonized DAKD (but not bradykinin)-induced calcium mobilization in IMR-90 cells, indicating that it is highly selective for B1 over B2 receptors. Antagonism of agonist-induced calcium responses at B1 receptors from different species indicated that ELN441958 is selective for primate over rodent B1 receptors with a rank order potency (KB, nanomolar) of human (0.12 ± 0.02) ∼ rhesus monkey (0.24 ± 0.01) > rat (1.5 ± 0.4) > mouse (14 ± 4). ELN441958 had good permeability and metabolic stability in vitro consistent with high oral exposure and moderate plasma half-lives in rats and rhesus monkeys. Because ELN441958 is up to 120-fold more potent at primate than at rodent B1 receptors, it was evaluated in a primate pain model. ELN441958 dose-dependently reduced carrageenan-induced thermal hyperalgesia in a rhesus monkey tail-withdrawal model, with an ED50 ∼3 mg/kg s.c. Naltrexone had no effect on the antihyperalgesia produced by ELN441958, indicating a lack of involvement of opioid receptors. ELN441958 is a novel small molecule bradykinin B1 receptor antagonist exhibiting high oral bioavailability and potent systemic efficacy in rhesus monkey inflammatory pain.