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.

PKCgamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain.

In previous studies we provided evidence that the gamma isoform of protein kinase C (PKCgamma) is an important contributor to the increased pain sensitivity that occurs after injury. Here we combined electrophysiological and behavioral approaches in wild-type and PKCgamma-null mice to compare the hyperexcitability of wide dynamic range neurons in lamina V of the spinal cord dorsal horn with the behavioral hyperexcitability produced by the same injury [application of a C-fiber irritant, mustard oil (MO), to the hindpaw]. Wild-type and null mice did not differ in their response to mechanical or thermal stimuli before tissue injury, and the magnitude of the response to the MO stimuli was comparable. In wild-type mice, MO produced a dramatic and progressive enhancement of the response of lamina V neurons to innocuous mechanical and thermal stimuli. The time course of the neuronal hyperexcitability paralleled the time course of the MO-induced behavioral allodynia (nocifensive behavior in response to a previously innocuous mechanical stimulus). Neuronal hyperexcitability was also manifest in the PKCgamma-null mice, but it lasted <30 min. By contrast, the behavioral allodynia produced by MO in the PKCgamma-null mice, although reduced to approximately half that of the wild-type mice, persisted long after the lamina V hyperexcitability had subsided. Because the MO-induced behavioral allodynia was completely blocked by an NMDA receptor antagonist, we conclude that PKCgamma mediates the transition from short- to long-term hyperexcitability of lamina V nociresponsive neurons but that the persistence of injury-induced pain must involve activity within multiple NMDA-dependent spinal cord circuits.

Contribution of alpha(2) 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 alpha adrenergic receptors (AR) may be involved. In addition to pro-nociceptive actions of AR stimulation, alpha(2) AR agonists produce antinociceptive effects. Here we studied the contribution of the alpha(2) AR subtypes, alpha(2A), alpha(2B) and alpha(2C) to the development of neuropathic pain. We also examined the antinociceptive effect produced by the alpha(2) AR agonist dexmedetomidine in nerve-injured mice. The studies were performed in mice that carry either a point (alpha(2A)) or a null (alpha(2B) and alpha(2C)) mutation in the gene encoding the alpha(2) 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 (3 microg kg(-1), s.c.) and produced antinociceptive effects at higher doses (10 - 30 microg kg(-1)) in all groups except in alpha(2A) AR mutant mice. The effect of dexmedetomidine was reversed by intrathecal, but not systemic, injection of the alpha(2) AR antagonist RS 42206. These results suggest that neither alpha(2A), alpha(2B) nor alpha(2C) AR is required for the development of neuropathic pain after peripheral nerve injury, however, the spinal alpha(2A) AR is essential for the antinociceptive effects of dexmedetomidine.

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice.

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Impaired nociception and pain sensation in mice lacking the capsaicin receptor.

The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the "pain" pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43 degrees C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Here, we demonstrate that sensory neurons from mice lacking VR1 are severely deficient in their responses to each of these noxious stimuli. VR1-/- mice showed normal responses to noxious mechanical stimuli but exhibited no vanilloid-evoked pain behavior, were impaired in the detection of painful heat, and showed little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia.

Elevated substance-P-like immunoreactivity levels in spinal dialysates during the formalin test in normal and diabetic rats.

Pharmacologic studies implicate the involvement of substance P in spinal nociceptive processing during the formalin test. However, no direct measurement of the temporal changes in substance P levels within the spinal cord of conscious animals has been reported. Further, dissociation between substance P levels and formalin-evoked nocifensive behavior may exist in diabetic rats, as exaggerated hyperalgesic behavior coexists with reduced peripheral nerve substance P levels. The present study was performed to directly measure the appearance of substance-P-like immunoreactivity (SP-LI) in spinal CSF of conscious, unrestrained rats using microdialysis techniques following injection of formalin into the hindpaw. The effect of diabetes upon formalin-evoked SP-LI levels in spinal CSF dialysates was also determined. In control rats, SP-LI increased in spinal dialysates following formalin injection and levels were maximal 20-30 min after injection, rising to 325% of basal values (p<0.02). Diabetic rats exhibited reduced (p<0.05) SP-LI in their spinal roots, while basal levels in spinal CSF were not different from controls. Formalin-evoked nocifensive behavior was increased in diabetic rats but SP-LI levels in spinal CSF dialysates after paw formalin injection were significantly (p<0.05) attenuated, reaching a maximum of only 161% of basal levels. This was accompanied by attenuated swelling at the formalin injection site and increased thermal response latencies. While increased SP-LI in spinal CSF coincides with phase 2 behavior in the formalin test and may contribute to spinal nociceptive processing during this period, exaggerated spinal substance P release is unlikely to underlie the increased nocifensive behavior seen in diabetic rats.

Inflammation-induced up-regulation of protein kinase Cgamma 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 Freund's adjuvant in the plantar surface of the rat hindpaw. Complete Freund's 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.

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.

Pain: nocistatin spells relief.

Tissue or nerve injury can dramatically alter the transmission of sensory stimuli by spinal cord neurons, so that a light touch produces pain. The discovery that peptide products of prepronociceptin processing either facilitate or inhibit these mechanisms suggests novel approaches to treating these conditions.

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

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.

Preserved acute pain and reduced neuropathic pain in mice lacking PKCgamma.

In normal animals, peripheral nerve injury produces a persistent, neuropathic pain state in which pain is exaggerated and can be produced by nonpainful stimuli. Here, mice that lack protein kinase C gamma (PKCgamma) displayed normal responses to acute pain stimuli, but they almost completely failed to develop a neuropathic pain syndrome after partial sciatic nerve section, and the neurochemical changes that occurred in the spinal cord after nerve injury were blunted. Also, PKCgamma was shown to be restricted to a small subset of dorsal horn neurons, thus identifying a potential biochemical target for the prevention and therapy of persistent pain.

Diminished inflammation and nociceptive pain with preservation of neuropathic pain in mice with a targeted mutation of the type I regulatory subunit of cAMP-dependent protein kinase.

To assess the contribution of PKA to injury-induced inflammation and pain, we evaluated nociceptive responses in mice that carry a null mutation in the gene that encodes the neuronal-specific isoform of the type I regulatory subunit (RIbeta) of PKA. Acute pain indices did not differ in the RIbeta PKA mutant mice compared with wild-type controls. However, tissue injury-evoked persistent pain behavior, inflammation of the hindpaw, and ipsilateral dorsal horn Fos immunoreactivity was significantly reduced in the mutant mice, as was plasma extravasation induced by intradermal injection of capsaicin into the paw. The enhanced thermal sensitivity observed in wild-type mice after intraplantar or intrathecal (spinal) administration of prostaglandin E2 was also reduced in mutant mice. In contrast, indices of pain behavior produced by nerve injury were not altered in the mutant mice. Thus, RIbeta PKA is necessary for the full expression of tissue injury-evoked (nociceptive) pain but is not required for nerve injury-evoked (neuropathic) pain. Because the RIbeta subunit is only present in the nervous system, including small diameter trkA receptor-positive dorsal root ganglion cells, we suggest that in inflammatory conditions, RIbeta PKA is specifically required for nociceptive processing in the terminals of small-diameter primary afferent fibers.

Neonatal capsaicin treatment abolishes formalin-induced spinal PGE2 release.

We investigated the effect of neonatal capsaicin treatment on formalin-evoked pain behavior and spinal levels of nociceptive neuromodulators using in vivo intrathecal microdialysis in conscious adult rats and age-matched controls. Capsaicin-treated rats displayed thermal hypoalgesia and a significant decrease in tissue content of calcitonin gene-related peptide. Paw swelling, flinching and release of spinal prostaglandin E2 induced by injection of formalin into the hindpaw were also reduced in capsaicin-treated rats compared with controls, whereas glutamate, aspartate and taurine release was unaffected. These data suggest that formalin-induced inflammation, pain behavior and spinal prostaglandin E2 release are mediated by mechanisms sensitive to neonatal capsaicin while the formalin-evoked release of amino acids in the spinal cord is not.

Capsaicin-evoked release of pituitary adenylate cyclase activating peptide (PACAP) and calcitonin gene-related peptide (CGRP) from rat spinal cord in vivo.

Capsaicin-evoked release of pituitary adenylate cyclase activating peptide (PACAP)-like immunoreactivity (LI) from rat spinal cord was examined in vivo. In anaesthetized rats, a catheter was inserted through the atlanto-occipital membrane into the subarachnoid space at the level of the sacral spinal cord for infusion of artificial cerebrospinal fluid. Another catheter was placed in the cisternal opening for outflow. Blood pressure was monitored and kept stable during the experiment. Perfusion samples were analyzed for PACAP and calcitonin gene-related peptide (CGRP) by radioimmunoassay. The addition of capsaicin (10 microM) to the perfusate elevated the concentrations of PACAP-27-LI in the artificial cerebrospinal fluid by 177%, PACAP-38-LI by 93% and CGRP-LI by 692%. In view of the presence of PACAP-immunoreactive nerve fibres in the superficial layers of the dorsal horn and the expression of PACAP in the small sized neurons in the dorsal root ganglia, the findings suggest that PACAP is released into the artificial cerebrospinal fluid from C-fibres in the spinal cord. PACAP conceivably plays a modulating role in nociception.

Efficacy of spinal NMDA receptor antagonism in formalin hyperalgesia and nerve injury evoked allodynia in the rat.

Neuropathic pain remains a significant clinical problem. Current understanding implicates the spinal cord dorsal horn N-methyl-d-aspartate (NMDA) receptor apparatus in its pathogenesis. Previous reports have described NMDA antagonist reduction of nerve injury-induced thermal hyperalgesia and formalin injection-related electrical activity. We examined a panel of spinally administered NMDA antagonists in two models: allodynia evoked by tight ligation of the fifth and sixth lumbar spinal nerves (a model of chronic nerve injury pain), and the formalin paw test (a model wherein pretreatment with drug may preempt the development of a pain state). A wide range of efficacies was observed. In the nerve injury model, order of efficacy (expressed as percent of maximum possible effect +/- S.E.), at the maximum dose not yielding motor impairment, was memantine (96 +/- 5%) = AP5 (91 +/- 7%) > dextrorphan (64 +/- 11%) = dextromethorphan (65 +/- 22%) > MK801 (34 +/- 8%) > ketamine (18 +/- 6%). For the formalin test, the order of efficacy was AP5 (86 +/- 9%) > memantine (74 +/- 5%) > or = MK801 (67 +/- 16%) > dextrorphan (47 +/- 16%) > dextromethorphan (31 +/- 12%) > ketamine (17 +/- 15%). In the nerve injury model, no supraspinal action was seen after intracerebroventricular injections of dextromethorphan and ketamine. NMDA antagonists by the spinal route appear to be useful therapeutic agents for chemically induced facilitated pain as well as nerve injury induced tactile allodynia. It is not known what accounts for the wide range of efficacies.

The effect of alpha-trinositol (D-myo-inositol 1,2,6-trisphosphate) on formalin-evoked spinal amino acid and prostaglandin E2 levels.

The effect of the inositol trisphosphate analog alpha-trinositol on noxious-evoked behavior, amino acid and prostaglandin E2 (PGE2) release was examined in unanesthetized rats using intrathecal microdialysis probes. Subcutaneous injection of 50 microliters 5% formalin solution produced two phases of pain-like behavior and significant elevation of glutamate, aspartate, glycine, taurine and serine during phase 1. PGE2 concentrations were increased during both phases 1 and 2. Intraperitoneal delivery of 300 mg/kg alpha-trinositol significantly suppressed both phases 1 and 2 of formalin-induced behavior and the associated elevation of amino acids and PGE2. These data demonstrate that the antinociceptive effect of alpha-trinositol corresponds to suppression of noxious-evoked release of amino acids and PGE2 from the spinal cord.

The effect of pituitary adenylate cyclase activating peptide (PACAP) on the nociceptive formalin test.

Recent studies have suggested that pituitary adenylate cyclase activating peptide (PACAP) may be involved in nociceptive transmission. The present study examined the effect of low-dose PACAP-27 on nociceptive behavior using the formalin test. PACAP-27 was administered intrathecally. Twenty minutes later, formalin (50 microliters, 5%) was injected subcutaneously into the dorsal surface of the right hind paw. Intrathecal PACAP-27 at 0.6 pmol suppressed the second phase response to formalin, while 5 pmol depressed both phases. PACAP-27 at 5 pmol did not impair motor function. Hence, the data suggest that the effect of PACAP-27 on formalin-induced pain-related behavior is specific. The findings may lead to a better understanding of the role of PACAP in nociceptive transmission.

Antinociceptive effect of alpha-trinositol, a novel D-myo-inositol phosphate derivative, in the formalin test in rats.

The antinociceptive effect of alpha-trinositol was examined in rats using the formalin test following systemic, spinal and local subcutaneous administration. Injection of formalin into the paw evoked two phases (phase 1: 0-9 min; phase 2: 10-60 min) of flinching behavior of the injected paw. Intrathecal administration of alpha-trinositol resulted in a dose-dependent suppression of the first (ED50: 8 microg) and second (ED50: 9 microg) phase of formalin-evoked behavioral response. Similarly, intraperitoneal delivery showed a dose-dependent reduction of the first (ED50: 83 mg/kg) and second (ED50: 56 mg/kg) phase of the formalin test. Subcutaneous injection of 100 microg, but not 10 mu g, alpha-trinositol into the rat paw together with the formalin solution, had no effect on the first phase, but reduced by 20% the second phase of behavior. These data show that alpha-trinositol produces a suppression of acute and prolonged nociceptive behaviors with a central mechanism of action, although some peripheral component may contribute to the reduction of the late phase following systemic administration.