Donna L. Hammond

Chloride regulation in the pain pathway

Melzack and Walls Gate Control Theory of Pain laid the theoretical groundwork for a role of spinal inhibition in endogenous pain control. While the Gate Control Theory was based on the notion that spinal inhibition is dynamically regulated, mechanisms underlying the regulation of inhibition have turned out to be far more complex than Melzack and Wall could have ever imagined. Recent evidence indicates that an exquisitely sensitive form of regulation involves changes in anion equilibrium potential (E(anion)), which subsequently impacts fast synaptic inhibition mediated by GABA(A), and to a lesser extent, glycine receptor activation, the prototypic ligand gated anion channels. The cation-chloride co-transporters (in particular NKCC1 and KCC2) have emerged as proteins that play a critical role in the dynamic regulation of E(anion) which in turn appears to play a critical role in hyperalgesia and allodynia following peripheral inflammation or nerve injury. This review summarizes the current state of knowledge in this area with particular attention to how such findings relate to endogenous mechanisms of hyperalgesia and allodynia and potential applications for therapeutics based on modulation of intracellular Cl(-) gradients or pharmacological interventions targeting GABA(A) receptors.

Efflux of 5‐hydroxytryptamine and noradrenaline into spinal cord superfusates during stimulation of the rat medulla.

High pressure liquid chromatography with electrochemical detection was used to quantify the efflux, in the same sample, of endogenous 5‐hydroxytryptamine (5‐HT), noradrenaline (NA), and 5‐hydroxyindoleacetic acid (5‐HIAA) into superfusates of the rat spinal cord in vivo. The efflux of these three agents was measured prior to, and during, electrical stimulation of the nucleus raphe magnus (n.r.m.) and nucleus reticularis paragigantocellularis (n.r.p.g.), two medullary nuclei implicated in antinociception. In untreated rats, basal efflux of 5‐HT and NA was 0.21 and 0.12 ng/ml of superfusate respectively; the basal efflux of 5‐HIAA was 18.17 ng/ml. Stimulation of the n.r.m. and n.r.p.g. in these animals increased the efflux of 5‐HT and 5‐HIAA, but did not alter the efflux of NA. 60 min after administration of fluoxetine (10 mg/kg, I.P.), a 5‐HT uptake inhibitor, basal efflux of 5‐HT and NA was unaltered, but the basal efflux of 5‐HIAA was decreased. In these rats, stimulation of the n.r.m. and n.r.p.g. increased the efflux of 5‐HT and of NA. The efflux of 5‐HIAA was not altered. In rats pre‐treated with both fluoxetine and desipramine (10 mg/kg, I.P.), the basal efflux of NA was increased while that of 5‐HIAA was decreased; the basal efflux of 5‐HT was not affected. The efflux of NA, but not of 5‐HT, was increased in these animals during stimulation of the n.r.m. and n.r.p.g. The efflux of 5‐HIAA was not changed by stimulation. Addition of fluoxetine alone or with desipramine to the superfusate in high concentrations greatly increased basal efflux of 5‐HT. Failure of stimulation of the ventromedial medulla to increase the efflux of 5‐HT in these animals may be related to feed‐back inhibition of release by the high concentration of 5‐HT initially present in the superfusate. These results indicate that electrical stimulation of the n.r.m. and n.r.p.g. increases the efflux of endogenous 5‐HT and NA from the spinal cord. These stimulation sites are coincident with brain‐stem sites at which stimulation produces antinociception by activation of spinal serotonergic and noradrenergic receptors. Thus, the ability of stimulation at these sites to evoke the spinal release of the probable neurotransmitters further supports the hypothesis that the antinociceptive effect is mediated by activation of serotonergic and noradrenergic neurones projecting to the spinal cord.

Antagonism of stimulation-produced antinociception by intrathecal administration of methysergide or phentolamine

This study examined whether the antinociception produced by electrical stimulation of medullary raphe nuclei is mediated by activation of monoaminergic neurons projecting to the spinal cord. Ninety-four sites distributed about the midline of the medulla were stimulated and their ability to produce antinociception was determined using two different analgesiometric tests. Stimulation of sites in the nuclei raphe pallidus and raphe obscurus did not produce antinociception, but rather, produced tremor and, on occasion, extensor rigidity. In contrast, stimulation at sites located in the nucleus raphe magnus and the adjacent nucleus reticularis paragigantocellularis produced antinociception, as indicated by increased tail flick latencies and decreased responsiveness to noxious pinch applied to the extremities. Intrathecal administration of saline prior to electrical stimulation of these sites did not attenuate either the elevation of tail flick latencies or the decreased responsiveness to pinch. However, intrathecal administration of 30 micrograms of either methysergide or phentolamine prior to stimulation at these same sites significantly attenuated the increase in tail flick latency and restored responsiveness to pinch. Naloxone (1 mg/kg, s.c.) did not attenuate the stimulation-produced antinociception evoked from any of these sites. These data support the postulate that the antinociceptive effect of electrical stimulation of the nucleus raphe magnus and the nucleus reticularis paragigantocellularis is mediated by activation of serotonergic and noradrenergic neurons projecting to the spinal cord. The inability of either methysergide or phentolamine alone to completely antagonize the elevation of tail flick latency further suggests that the serotonergic and noradrenergic bulbospinal systems are coactivated by electrical stimulation of these sites.

Alterations in nociceptirve threshold and morphine-induced analgesia produced by intrathecally administered amine antagonists ☆

Intrathecal administration of either methysergide or phentolamine hyperalgesia. This suggests that tonically active serotonergic and noradrenergic neuronal systems modulate sensitivity to nociceptive stimuli at the level of the spinal cord. Methysergide did not attenuate the analgesia induced by either 2.0 or 7.5 mg/kg morphine (s.c.), while phentolamine attenuated the analgesia induced by 2.0, but not 7.5 mg/kg morphine. These findings suggest that bulbospinal serotonergic neurons are not integral components of the neuronal circuitry which mediates opiate-induced analgesia. Noradrenergic neurons, however, appear to mediate a portion of such analgesia.

Gabapentin and Pregabalin Can Interact Synergistically with Naproxen to Produce Antihyperalgesia

Background Gabapentin and pregabalin are anticonvulsants with antihyperalgesic effects in animal models of neuropathic and inflammatory nociception. This study characterized the manner in which gabapentin or pregabalin interacts with naproxen to suppress thermal hyperalgesia and inflammation in the carrageenan model of peripheral inflammation. Methods Gabapentin, pregabalin, naproxen, or a fixed-dose ratio of gabapentin + naproxen or pregabalin + naproxen was administered orally to rats after the induction of inflammation by intraplantar injection of &lgr;-carrageenan in one hind paw. Nociceptive thresholds were determined by the radiant heat paw-withdrawal test. Paw edema was measured by plethysmometry. Drug plasma concentrations were determined by a liquid chromatography–mass spectroscopy–mass spectroscopy method. Results Gabapentin, pregabalin, and naproxen alone reversed thermal hyperalgesia with ED50 values of 19.2, 6.0, and 0.5 mg/kg, respectively. Mixtures of gabapentin + naproxen in fixed-dose ratios of 50:1, 10:1, or 1:1 interacted synergistically to reverse carrageenan-induced thermal hyperalgesia. However, 1:50 gabapentin + naproxen produced only additive effects. No combination of gabapentin + naproxen decreased paw edema in a manner greater than additive. Plasma concentrations of gabapentin and naproxen were unaltered by the addition of the other drug. The mixture of 10:1 of pregabalin + naproxen interacted synergistically to reverse thermal hyperalgesia on the inflamed hind paw, whereas mixtures of 1:1 or 1:10 produced additive effects. Conclusions These data suggest that gabapentin + naproxen and pregabalin + naproxen can interact synergistically or additively to reverse thermal hyperalgesia associated with peripheral inflammation. Therefore, the use of gabapentin or pregabalin in low-dose combinations with naproxen may afford therapeutic advantages for clinical treatment of persistent inflammatory pain.

Pharmacological antagonism of the antinociceptive effects of serotonin in the rat spinal cord.

Serotonin creatinine sulfate (5-HT) administered via chronically implanted intrathecal catheters produced a dose-dependent increase in the rat hot plate and tail flick response latencies. To characterize the nature of the spinal receptor system through which this effect is mediated, putative serotonin antagonists were co-administered with 200 μg of 5-HT. In these experiments, methysergide (12–48 nmol), 2-bromolysergic acid diethylamide (BOL 6–48 nmol) and ketanserin (12–73 nmol) produced a dose-dependent antagonism of the effects of intrathecally administered 5-HT on the tail flick; with the IC50 values being: methysergide 17.5±2.5 nmol; BOL 26.5±6.5 nmol; ketanserin 34.5±5.1 nmol The effects of spiroperidol (12–44 nmol) and metergoline (6–32 nmol) were not dose-dependent, although a measurable antagonism did occur at the highest dose. In contrast to the results obtained with the tail flick test, none of the 5-HT antagonists attenuated the elevation of hot plate latency produced by 5-HT. With the exception of spiroperidol, none of these agents showed any effect on the baseline response latencies. Sacrifice and dissection of rat brain and cord at 5, 15 and 45 min after intrathecal [14C]5-HT revealed that significant quantities of radioactivity appeared in brain only at the longest interval, at a time when the magnitude of the analgesia was significantly diminished, indicating that these spinal drug effects were likely produced by a local action on spinal receptors. The ability of methysergide and BOL (agents with measurable 5-HT1 binding activity) to readily antagonize the effects of intrathecal 5-HT versus the relative inactivity of spiroperidol and ketanserin, used to characterize the 5-HT2 binding site, suggests the likely role of 5-HT1 receptors in mediating the spinnal effects of 5-HT in the tail flick, but not the hot plate test of nociception.

Inhibitory actions of delta 1-, delta 2-, and mu-opioid receptor agonists on excitatory transmission in lamina II neurons of adult rat spinal cord

This study examined the electrophysiological consequences of selective activation of delta 1-, delta 2-, or mu-opioid receptors using whole- cell recordings made from visually identified lamina II neurons in thin transverse slices of young adult rat lumbar spinal cord. Excitatory postsynaptic currents (EPSCs) or potentials (EPSPs) were evoked electrically at the ipsilateral dorsal root entry zone after blocking inhibitory inputs with bicuculline and strychnine, and NMDA receptors with D-2-amino-5-phosphonopentanoic acid. Bath application of the mu receptor agonist [D-Ala2, N-MePhe4, Gly5-ol]enkephalin (DAMGO) or the delta 1 receptor agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) produced a long-linear, concentration-dependent reduction in the amplitude of the evoked EPSP/EPSC. By comparison, the delta 2 receptor agonist [D- Ala2,Glu4]deltorphin (DELT) was unable to reduce the evoked EPSP/EPSC by more than 50% at 100 microM, the highest concentration tested. At concentrations that reduced evoked EPSP/EPSCs by 40–60%, neither DAMGO, DPDPE, nor DELT decreased the amplitude of the postsynaptic current produced by brief pressure ejection of (S)-alpha-amino-3-hydroxy-5- methyl-4-isoxazole-propionic acid, suggesting a presynaptic site of action of these opioid receptor agonists. Bath application of 200 nM naltriben (NTB), a delta 2 receptor antagonist, competitively increased the EC75 of DELT by 15.3-fold, but did not antagonize either DPDPE or DAMGO. The EC75 of DELT was further increased by 169.7-fold in the presence of 1 microM NTB. However, this high concentration of NTB also increased the EC50 of DPDPE by about threefold in a noncompetitive manner and antagonized DAMGO in a noncompetitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intrathecally administered methysergide and yohimbine on microstimulation-produced antinociception in the rat

This study examined whether intrathecal (i.t.) administration of the serotonergic antagonist methysergide, of the alpha 2 noradrenergic antagonist yohimbine, or of both drugs antagonized stimulation-produced antinociception (SPA) evoked from the nucleus raphe magnus (NRM) and the nucleus reticularis paragigantocellularis (NRPG) of lightly anesthetized rats. The increase in tail flick latency (TFL), but not the increase in paw pinch withdrawal threshold (PWT), evoked from NRM sites was antagonized by i.t. administration of methysergide. Intrathecal administration of yohimbine antagonized both the increase in TFL and the increase in PWT produced by stimulation of NRM sites. Stimulation of sites in the NRPG also increased TFL and PWT; these increases were not antagonized by i.t. administration of methysergide. Although i.t. administration of yohimbine antagonized the increase in TFL evoked from the NRPG, the increase in PWT was not antagonized. When coadministered intrathecally, methysergide and yohimbine antagonized the increases in TFL and PWT produced by stimulation of NRM and of NRPG sites. In contrast, i.v. administration of the same doses of methysergide and yohimbine did not antagonize either the increase in TFL or the increase in PWT evoked from either set of sites. These results support the concept that activation of serotonergic and noradrenergic bulbospinal neurons mediates SPA and additionally suggest that the noradrenergic component involves an alpha 2 noradrenergic receptor.

Effects of intrathecally administered thip, baclofen and muscimol on nociceptive threshold

This study examined whether the antinociceptive activity of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), a GABA agonist, is mediated through an action exerted at the level of the spinal cord. Intrathecal injection of doses of THIP devoid of motor effects (1-2 micrograms) did not increase tail flick or hot plate latencies in the rat. Although hot plate latency was transiently increased by intrathecal injection of 5 micrograms THIP, slight motor impairment was observed at this dose. Higher doses of THIP (15-50 micrograms) produced flaccidity of the hindlimbs. Intrathecal injection of low doses of muscimol (0.25 microgram) that did not produce motor impairment increased tail flick, but not hot plate, latencies. Baclofen produced motor incoordination following intrathecal injection of 10 micrograms; however, intrathecal injection of 1 microgram significantly increased both tail flick and hot plate latencies without attendant motor effects. Thus, baclofen was the only compound in which the antinociceptive effect was clearly distinguished from the motor effect. These results additionally indicate that the spinal cord does not mediate the antinociceptive activity of THIP.

CHANGES IN EXPRESSION OF SENSORY ORGAN-SPECIFIC microRNAs IN RAT DORSAL ROOT GANGLIA IN ASSOCIATION WITH MECHANICAL HYPERSENSITIVITY INDUCED BY SPINAL NERVE LIGATION

Chronic neuropathic pain caused by peripheral nerve injury is associated with global changes in gene expression in damaged neurons. To understand the molecular mechanisms underlying neuropathic pain, it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain. MicroRNAs are non-protein-coding RNA molecules that regulate gene expression in a wide variety of biological processes mainly at the level of translation. This study investigated the possible involvement of microRNAs in gene regulation relevant to neuropathic pain. The analyses focused on a sensory organ-specific cluster of microRNAs that includes miR-96, -182, and -183. Quantitative real-time polymerase chain reaction (qPCR) analyses confirmed that these microRNAs were highly enriched in the dorsal root ganglion (DRG) of adult rats. Using the L5 spinal nerve ligation (SNL) model of chronic neuropathic pain, we observed a significant reduction in expression of these microRNAs in injured DRG neurons compared to controls. In situ hybridization and immunohistochemical analyses revealed that these microRNAs are expressed in both myelinated (N52 positive) and unmyelinated (IB4 positive) primary afferent neurons. They also revealed that the intracellular distributions of the microRNAs in DRG neurons were dramatically altered in animals with mechanical hypersensitivity. Whereas microRNAs were uniformly distributed within the DRG soma of non-allodynic animals, they were preferentially localized to the periphery of neurons in allodynic animals. The redistribution of microRNAs was associated with changes in the distribution of the stress granule (SG) protein, T-cell intracellular antigen 1 (TIA-1). These data demonstrate that SNL induces changes in expression levels and patterns of miR-96, -182, and -183, implying their possible contribution to chronic neuropathic pain through translational regulation of pain-relevant genes. Moreover, SGs were suggested to be assembled and associated with microRNAs after SNL, which may play a role in modification of microRNA-mediated gene regulation in DRG neurons.