Deep Dorsal Horn Research Articles

Mustard oil enhances spinal neuronal responses to noxious heat but not cooling

The TRPA1 agonist mustard oil (allyl isothiocyanate = AITC) induces heat hyperalgesia and mechanical allodynia in human skin and sensitizes rat spinal wide dynamic range (WDR) neuronal responses to noxious skin heating. We presently used electrophysiological methods to investigate if AITC affects the responsiveness of individual spinal WDR neurons to intense skin cooling. Recordings were made from cold-sensitive WDR neurons in lamina I and deeper dorsal horn; 21/23 also responded to noxious skin heating. Topical application of AITC excited 8/18 units and significantly enhanced their responses to noxious heat while not significantly affecting responses to the cold stimulus. Vehicle (mineral oil) had no effect on thermal responses. The data confirm a role for the TRPA1 agonist AITC in enhancing heat nociception without significantly affecting cold sensitivity.

C-Fos or pERK, Which is a Better Marker for Neuronal Activation and Central Sensitization After Noxious Stimulation and Tissue Injury?

c-Fos, the protein of the protooncogene c-fos, has been extensively used as a marker for the activation of nociceptive neurons in the spinal cord for more than twenty years since Hunt et al. first reported that peripheral noxious stimulation to a hind paw of rats leads to a marked induction of c-Fos in superficial and deep dorsal horn neurons in 1987. In 1999, Ji et al. reported that phosphorylated extracellular signal-regulated kinase (pERK) is specifically induced by noxious stimulation in superficial dorsal horn neurons. Accumulating evidence indicates that pERK induction or ERK activation in dorsal horn neurons is essential for the development of central sensitization, increased sensitivity of dorsal horn neurons that is responsible for the generation of persistent pain. Further, molecular mechanisms underlying ERK-mediated central sensitization have been revealed. In contrast, direct evidence for c-Fos-mediated central sensitization is not sufficient. After a noxious stimulus (e.g., capsaicin injection) or tissue injury, c-Fos begins to be induced after 30-60 minutes, whereas pERK can be induced within a minute, which can correlate well with the development of pain hypersensitivity. While c-Fos is often induced in the nuclei of neurons, pERK can be induced in different subcellular structures of neurons such as nuclei, cytoplasma, axons, and dendrites. pERK can even be induced in spinal cord microglia and astrocytes after nerve injury. In summary, both c-Fos and pERK can be used as markers for neuronal activation following noxious stimulation and tissue injury, but pERK is much more dynamic and appears to be a better marker for central sensitization.

Spinal cord compression injury in adult rats initiates changes in dorsal horn remodeling that may correlate with development of neuropathic pain

Spinal cord injury commonly causes chronic, neuropathic pain. The mechanisms are poorly understood but may include structural plasticity within spinal and supraspinal circuits. Our aim was to determine whether structural remodeling within the dorsal horn rostral to an incomplete injury differs from a complete spinal cord transection. Four immunohistochemical populations of primary afferent C-fibers, and descending catecholamine and serotonergic projections, were examined in segments T9-T12 at 2 and 12 weeks after a T13 clip-compression injury in adult male rats. Dorsal root ganglia were also examined. Two weeks after injury, fibers immunoreactive for calcitonin gene-related peptide (CGRP) or GDNF-family receptors (GFRalpha1, GFRalpha2, GFRalpha3) showed distinct injury responses within the superficial dorsal horn. CGRP fibers decreased, but GFRalpha1, GFRalpha2 and GFRalpha3 fibers did not change. In contrast, all groups were decreased by 12 weeks after injury. Catecholamine fibers showed a decrease at 2 weeks followed by an increase in density at 12 weeks, whereas serotonergic fibers showed a decrease (restricted to deep dorsal horn) at 12 weeks. These results show that the dorsal horn of the spinal cord undergoes substantial structural plasticity rostral to a compression injury, with the most profound effect being a prolonged and possibly permanent loss of primary afferent fibers. This loss was more extensive and more prolonged than the loss that follows spinal cord transection. Our results provide further evidence that anatomical reorganization of sensory and nociceptive dorsal horn circuits rostral to an injury could factor in the development or maintenance of spinal cord injury pain.

Transient receptor potential vanilloid type 1 receptor regulates glutamatergic synaptic inputs to the spinothalamic tract neurons of the spinal cord deep dorsal horn

The spinothalamic tract (STT) neurons in the spinal dorsal horn play an important role in transmission and processing of nociceptive sensory information. Although transient receptor potential vanilloid type 1 (TRPV1) receptors are present in the spinal cord dorsal horn, their physiological function is not fully elucidated. In this study, we examined the role of TRPV1 in modulating neuronal activity of the STT neurons through excitatory and inhibitory synaptic inputs. Whole-cell patch-clamp recordings were performed on STT neurons labeled by a retrograde fluorescent tracer injected into the ventral posterior lateral (VPL) nucleus of the thalamus. Capsaicin (1 μM) increased the frequency of miniature excitatory postsynaptic currents (mEPSC) without changing the amplitude or decay time constant of mEPSC. In contrast, capsaicin had no distinct effect on GABAergic miniature inhibitory postsynaptic currents (mIPSC). Capsazepine (10 μM), a TRPV1 receptor antagonist, abolished the effect of capsaicin on mEPSCs. Capsazepine itself did not affect the baseline amplitude and frequency of mEPSC. The effect of capsaicin on mEPSC was also abolished by removal of external Ca 2+, but not by treatment with Cd 2+. Furthermore, capsaicin increased the firing activity of the STT neurons and this increase in neuronal activity by capsaicin was abolished in the presence of non– N-methyl- d-aspartic acid (NMDA) and NMDA receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and (R)-amino-5-phosphonovaleric acid (APV). These data suggest that activation of TRPV1 potentiates the glutamate release from excitatory terminals of primary afferent fibers and subsequently increases the neural activity of STT neurons of the rat spinal cord deep dorsal horn.

Propagation of Sinusoidal Electrical Waves along the Spinal Cord during a Fictive Motor Task

We present for the first time direct electrophysiological evidence of the phenomenon of traveling electrical waves produced by populations of interneurons within the spinal cord. We show that, during a fictive rhythmic motor task, scratching, an electrical field potential of spinal interneurons takes the shape of a sinuous wave, "sweeping" the lumbosacral spinal cord rostrocaudally with a mean speed of approximately 0.3 m/s. We observed that traveling waves and scratching have the same cycle duration and that duration of the flexor phase, but not of the extensor phase, is highly correlated with the cycle duration of the traveling waves. Furthermore, we found that the interneurons from the deep dorsal horn and the intermediate nucleus can generate the spinal traveling waves, even in the absence of motoneuronal activity. These findings show that the sinusoidal field potentials generated during fictive scratching could be a powerful tool to disclose the organization of central pattern generator networks.

Different Forms of Glycine- and GABAA-Receptor Mediated Inhibitory Synaptic Transmission in Mouse Superficial and Deep Dorsal Horn Neurons

BackgroundNeurons in superficial (SDH) and deep (DDH) laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR) and GABAA-receptors (GABAARs) contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits) in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood.Methods and ResultsHere we compare fast inhibitory synaptic transmission in mouse (P17-37) SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23°C). GlyR-mediated mIPSCs were detected in 74% (25/34) and 94% (25/27) of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18). Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 ± 3.9 vs. 64.7 ± 5.0 pA; n = 25 each), decay time was slower (8.5 ± 0.8 vs. 5.5 ± 0.3 ms), and frequency was lower (0.15 ± 0.03 vs. 0.72 ± 0.13 Hz) in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 ± 2.4, n = 14 vs. 25. ± 2.0 pA, n = 18) and frequencies (0.21 ± 0.08 vs. 0.18 ± 0.04 Hz) in both regions; however, decay times were slower (23.0 ± 3.2 vs. 18.9 ± 1.8 ms) in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 ± 1.6 pS, n = 11 vs. 55.7 ± 1.8, n = 8) and GABAARs (22.7 ± 1.7 pS, n = 10 vs. 22.4 ± 2.0 pS, n = 11) in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA), had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 μM) reduced GlyR-mediated mIPSC frequency in SDH and DDH, but did not affect other properties. Similar results were observed for GABAAR mediated mIPSCs, however, rise time was slowed by methAEA in SDH neurons.ConclusionTogether these data show that Gly- and GABAARs with clearly differing physiological properties and cannabinoid-sensitivity contribute to fast synaptic inhibition in mouse SDH and DDH.

Loss of GABA-B inhibition in the dorsal spinal cord of a neuropathic rat model

Event Abstract Back to Event Loss of GABA-B inhibition in the dorsal spinal cord of a neuropathic rat model Sophie Laffray1, Marie-Amelie Papon1, Rabia Bouali-Benazzouz1, Alexandre Favereaux1, Yves Le Feuvre1, Ulo Langel1, Frédéric Nagy1 and Marc Landry1* 1 University Bordeaux 2, Pathophysiology of spinal neuronal networks, France Pathological states of neuropathic pain are related to sensitization of deep dorsal horn neurons that are likely to amplify nociceptive transmission, causing allodynia and hyperalgesia. The efficiency of GABAb agonists in the treatment of chronic pain is controversial. The present project aims to investigate possible dysfunction of GABAb inhibitory control of pain sensitization in the spinal cord of animal models. Our working hypothesis is that functional alteration of heterodimeric GABAb receptor depends on its dissociation, possibly triggered by intracellular partner proteins such as 14.3.3. We investigated the co-distribution of GABAb1 and 14.3.3 with microscopy techniques. The physical interactions between the various partner proteins have been studied with FRET / FLIM imaging and with co-immunoprecipitation approaches. The functional outcome of these interactions has been assessed in vitro with patch-clamp techniques, and in vivo with behavioural tests. Our results showed that in a rat neuropathic pain model, 14.3.3 expression is up regulated in the dorsal horn. GABAb receptor and 14.3.3 are codistributed in the same spinal neurons. The relative distribution of GABAb and 14.3.3 in spinal cord, investigated by electron microscopy, showed that the subcellular colocalization between GABAb1 and 14-3-3 is increased after spinal nerve ligation. Moreover, in cultures of spinal neuron, 14.3.3 overexpression resulted in a partial loss of colocalization between GABAb1 and b2 subunits, suggesting that 14.3.3 induced the dissociation of the GABAb receptor. A FRET-based imaging approach further confirmed the dynamic of 14.3.3-GABAb1 interactions in neurons. In COS cell culture; immunoprecipitation experiments demonstrated the physical association between 14.3.3 and GABAb1 subunit. Furthermore, it ensured the effects of 14.3.3 overexpression in disrupting the GABAb heterodimer. To reverse GABAb dysfunction in the dorsal horn, we designed different strategies based on siRNA-mediated 14.3.3 knockdown and on the use of synthetic peptides that block 14.3.3-binding site on GABAb1 subunit. The behavioural assessment of mechanical allodynia revealed that both strategies are efficient in potentiating the inhibitory effect of intrathecally injected Baclofen in neuropathic rats. By targeting associated proteins, it may thus be possible to improve the pharmacological efficiency of GABAb agonists in the treatment of neuropathic pain. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Symposium 04 – Dis-inhibition processes in pain sensitization Citation: Laffray S, Papon M, Bouali-Benazzouz R, Favereaux A, Le Feuvre Y, Langel U, Nagy F and Landry M (2009). Loss of GABA-B inhibition in the dorsal spinal cord of a neuropathic rat model. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.022 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Nov 2009; Published Online: 19 Nov 2009. * Correspondence: Marc Landry, University Bordeaux 2, Pathophysiology of spinal neuronal networks, Bordeaux, France, marc.landry@u-bordeaux.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sophie Laffray Marie-Amelie Papon Rabia Bouali-Benazzouz Alexandre Favereaux Yves Le Feuvre Ulo Langel Frédéric Nagy Marc Landry Google Sophie Laffray Marie-Amelie Papon Rabia Bouali-Benazzouz Alexandre Favereaux Yves Le Feuvre Ulo Langel Frédéric Nagy Marc Landry Google Scholar Sophie Laffray Marie-Amelie Papon Rabia Bouali-Benazzouz Alexandre Favereaux Yves Le Feuvre Ulo Langel Frédéric Nagy Marc Landry PubMed Sophie Laffray Marie-Amelie Papon Rabia Bouali-Benazzouz Alexandre Favereaux Yves Le Feuvre Ulo Langel Frédéric Nagy Marc Landry Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

L1 cell adhesion molecule is essential for the maintenance of hyperalgesia after spinal cord injury

Spinal cord injury (SCI) results in a loss of normal motor and sensory function, leading to severe disability and reduced quality of life. A large proportion of individuals with SCI also suffer from neuropathic pain symptoms. The causes of abnormal pain sensations are not well understood, but can include aberrant sprouting and reorganization of injured or spared sensory afferent fibers. L1 is a cell adhesion molecule that contributes to axonal outgrowth, guidance and fasciculation in development as well as synapse formation and plasticity throughout life. In the present study, we used L1 knockout (KO) mice to determine whether this adhesion molecule contributes to sensory dysfunction after SCI. Both wild-type (WT) and KO mice developed heat hyperalgesia following contusion injury, but the KO mice recovered normal response latencies beginning at 4 weeks post-injury. Histological analyses confirmed increased sprouting of sensory fibers containing calcitonin-gene related peptide (CGRP) in the deep dorsal horn of the lumbar spinal cord and increased numbers of interneurons expressing protein kinase C gamma (PKCγ) in WT mice 6 weeks after injury. In contrast, L1 KO mice had less CGRP + fiber sprouting, but even greater numbers of PKCγ + interneurons at the 6 week time point. These data demonstrate that L1 plays a role in maintenance of thermal hyperalgesia after SCI in mice, and implicate CGRP + fiber sprouting and the upregulation of PKCγ expression as potential contributors to this response.

The Antinociceptive Response to Nicotinic Agonists in a Mouse Model of Postoperative Pain

Nicotine, the prototypical broad spectrum agonist at central nicotinic receptors, has analgesic action after surgery. Various subtype-specific nicotinic agonists have antinociceptive effects in animal models, but the response is highly dependent on the model tested. In an effort to determine what nicotinic subtypes might be targeted in future clinical studies, we tested agonists selective for alpha 4 beta 2 and alpha 7 containing nicotinic receptors in a mouse model of postoperative pain. After paw incision, mice were tested for heat latency and pressure threshold before and after treatment with a dose range of ligands selective for alpha 4 beta 2 and alpha 7 containing nicotinic receptors. To demonstrate that nicotine reduced nociceptive input in this model, the lumbar spinal cords of a subgroup of these mice were stained for the phosphorylated form if CREB. Nicotine and metanicotine (alpha 4 beta 2 selective) were fully effective as an analgesic in heat and pressure testing. The alpha 7 partial agonist GTS-21 significantly increased the heat latency after surgery, but did not alter pressure threshold. The alpha 7 selective antagonist methyllicaconitine decreased the efficacy of nicotine to increase heat latency but did not affect pressure threshold. The number of cells in the superficial dorsal horn with nuclei that stained for pCREB was double on the surgical side and the ratio was reduced by nicotine in a dose-dependent manner. Our findings suggest that nicotine reduced nociceptive input to the superficial and deep dorsal horn. It also provides support for alpha 4 beta 2 and alpha 7 nicotinic-mediated antinociceptive actions.

Termination Zones of Functionally Characterized Spinothalamic Tract Neurons Within the Primate Posterior Thalamus

The primate posterior thalamus has been proposed to contribute to pain sensation, but its precise role is unclear. This is in part because spinothalamic tract (STT) neurons that project to the posterior thalamus have received little attention. In this study, antidromic mapping was used to identify individual STT neurons with axons that projected specifically to the posterior thalamus in Macaca fascicularis. Each axon was located by antidromic activation at low stimulus amplitudes (<30 microA) and was then surrounded distally by a grid of stimulating points in which 500-microA stimuli were unable to activate the axon antidromically, thereby indicating the termination zone. Several nuclei within the posterior thalamus were targets of STT neurons: the posterior nucleus, suprageniculate nucleus, magnocellular part of the medial geniculate nucleus, and limitans nucleus. STT neurons projecting to the ventral posterior inferior nucleus were also studied. Twenty-five posterior thalamus-projecting STT neurons recorded in lumbar spinal cord were characterized by their responses to mechanical, thermal, and chemical stimuli. Sixteen of 25 neurons were recorded in the marginal zone and the balance was located within the deep dorsal horn. Thirteen neurons were classified as wide dynamic range and 12 as high threshold. One-third of STT neurons projecting to posterior thalamus responded to noxious heat (50 degrees C). Two-thirds of those tested responded to cooling. Seventy-one percent responded to an intradermal injection of capsaicin. These data indicate that the primate STT transmits noxious and innocuous mechanical, thermal, and chemical information to multiple posterior thalamic nuclei.

A Molecular Program for Contralateral Trajectory: Rig-1 Control by LIM Homeodomain Transcription Factors

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.

Involvement of spinal nitric oxide (NO) in rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karsch

In the present study, we investigated the role of spinal nitric oxide (NO) in rat pain-related behaviors induced by the venom of scorpion Buthus martensi Karsch (BmK). The results showed that the number of neuronal NO synthase (nNOS) positive neurons significantly increased in superficial (I–II), deep (V–VI) dorsal horn laminae and the ventral gray laminae (VII–X), but not in the nucleus proprius (III and IV) of bilateral L4–L5 lumbar spinal cord after unilateral intraplantar injection of BmK venom from 2 h to 7 d. This increase on the ipsilateral side to BmK venom injection was always greater than that on the contralateral side. Western blotting analysis confirmed that spinal nNOS expression was significantly up-regulated following BmK venom administration. In addition, intrathecal delivery of N ω-nitro- l-arginine methyl ester hydrochloride ( l-NAME; a NOS inhibitor) before intraplantar injection of BmK venom by 10 min significantly attenuated spontaneous nociceptive responses and prevented the development of primary thermal hyperalgesia as well as bilateral mechanical hyperalgesia. Intrathecal injection of l-NAME could also partially inhibit BmK venom-induced c-Fos expression in lumbar spinal cord at 2 h. Thus, the results suggest that spinal NO as a critical mediator is involved in various pain-related behaviors and c-Fos expression induced by BmK venom in rats.

Long-lasting enhancement in the intrinsic excitability of deep dorsal horn neurons

Intrinsic excitability (IE) can be defined as an output of action potentials from a given input signal. Changes to the IE of a neuron are an important aspect of the cellular plasticity that underlies learning and memory process. In this study, long-term plastic change in IE of deep dorsal horn neurons (DHNs) was investigated. Associative spike pairing stimulation (PS) induced a long-lasting increase in IE. Buffering intracellular calcium with BAPTA (10 mM) prevented the induction of a long-lasting increase in IE. PS failed to induce a long-lasting increase in IE in the presence of either D-APV (50 μM) or cadmium chloride (100 μM). Apamin (100 nM) partially blocked the induction of a long-lasting increase in IE. This intrinsic plasticity requires a rise in postsynaptic Ca 2+ and NMDA receptor activation during the induction period, and this process might be mediated by the down-regulation of small-conductance calcium-dependent potassium (SK) channels. In deep DHNs, PS induced excitatory postsynaptic potential (EPSP)-spike (E-S) potentiation, which increases the firing probability and the number of spikes, by consistent dorsal rootlet stimulation. Under bath application of bicuculline (10 μM) and strychnine (1 μM), PS induced E-S potentiation and long-lasting increases in IE. These results suggest that an increase in IE might underlie E-S potentiation, while a reduction in inhibitory transmission does not contribute to E-S potentiation and long-lasting increases in IE. We conclude that PS enhances the IE of deep DHNs, which may play an important role in spinal processing of nociceptive information.

Involvement of group I metabotropic glutamate receptors and glutamate transporters in the slow excitatory synaptic transmission in the spinal cord dorsal horn

Our experiments demonstrate a novel role for group I metabotropic glutamate receptor (mGluR) subtypes 1 and 5 in generating a long-lasting synaptic excitation in the substantia gelatinosa (SG) and deep dorsal horn (DH) neurons of the rat spinal cord. In the present study we have investigated a slow excitatory postsynaptic current (EPSC), elicited by a brief high intensity (at Aδ/C fiber strength) and high frequency (20 or 100 Hz) stimulation of primary afferent fibers (PAFs) using whole-cell patch-clamp recordings from neurons located in the DH (laminae II–V) in spinal cord slices of young rats and wild-type and gene-targeted mice lacking mGluR1 subtype. The results shown here suggest that the activation of both mGluR1 and mGluR5 along with NK1 receptors, may be involved in the generation of the slow EPSC in the spinal cord DH. Inhibition of glial and neuronal glutamate transporters by dl-threo-β-benzyloxyaspartate (TBOA) enhanced the group I mGluR-dependent slow EPSC about eightfold. Therefore, we conclude, that glutamate transporters strongly influence the group I mGluR activation by PAFs possibly at sensory synapses in the DH. Overall these data indicate that stimulus trains can generate a sustained and widespread glutamate signal that can further elicit prolonged EPSCs predominantly mediated by the group I mGluRs. These slow excitatory synaptic currents may have important functional implications for DH cell firing and synaptic plasticity of sensory transmission, including nociception.

Ketamine blocks enhancement of spinal long‐term potentiation in chronic opioid treated rats

Long-term opioid treatment is associated with the development of hyperalgesia. In a rat model we wanted to study if chronic opioid treatment changed the induction and maintenance of spinal long-term potentiation (LTP), a form of hyperexcitability in the spinal cord. We also wanted to investigate if the clinically available NMDA receptor antagonist ketamine inhibited the effect of chronic opioid treatment on LTP. The animals were randomized into four groups (saline, morphine 20 mg/kg/day, ketamine 20 mg/kg/day, morphine 20 mg/kg/day and ketamine 20 mg/kg/day). Drugs were given as continuous subcutaneous infusions by means of osmotic minipumps. After 7 days of treatment and during ongoing treatment single unit extracellular recordings were made from the lumbar deep dorsal horn under urethane anesthesia. Single electrical stimuli were applied to the sciatic nerve, and the C-fiber evoked responses of WDR neurons were recorded before and during 3 h following low frequency (3 Hz) electrical conditioning stimulation. The potentiation of C-fiber evoked responses by conditioning stimulation was significantly increased in the morphine-treated group compared to the saline group, while there was no significant difference between the saline, the ketamine and the morphine/ketamine groups. The potentiated responses in the morphine/ketamine group were significantly reduced compared to the morphine group (P=0.01). Our results indicate that animals treated with long-term opioid show amplification of stimulus-induced central sensitisation compared to opioid naïve animals. Ketamine inhibited the morphine-induced enhancement of LTP, supporting the role of ketamine in prevention of opioid induced hyperalgesia.

Selective ablation of dorsal horn NK 1 expressing cells reveals a modulation of spinal alpha2-adrenergic inhibition of dorsal horn neurones

Activity in descending systems from the brainstem modulates nociceptive transmission through the dorsal horn. Intrathecal injection of the neurotoxin saporin conjugated to SP (SP–SAP) into the lumbar spinal cord results in the selective ablation of NK 1 receptor expressing (NK 1+ve) neurones in the superficial dorsal horn (lamina I/III). Loss of these NK 1+ve neurones attenuates excitability of deep dorsal horn neurones due to a disruption of both intrinsic spinal circuits and a spino-bulbo-spinal loop, which activates a descending excitatory drive, mediated through spinal 5HT 3 receptors. Descending inhibitory pathways also modulate spinal activity and hence control the level of nociceptive transmission relayed to higher centres. To ascertain the spinal origins of the major descending noradrenergic inhibitory pathway we studied the effects of a selective alpha2-adrenoceptor antagonist, atipamezole, on neuronal activity in animals pre-treated with SP–SAP. Intrathecal application of atipamezole dose dependently facilitated the mechanically evoked neuronal responses of deep dorsal horn neurones to low intensity von Frey hairs (5–15 g) and noxious thermal (45–50 °C) evoked responses in SAP control animals indicating a physiological alpha2-adrenoceptor control. This facilitatory effect of atipamezole was lost in the SP–SAP treated group. These data suggest that activity within noradrenergic pathways have a dependence on dorsal horn NK 1+ve cells. Further, noradrenergic descending inhibition may in part be driven by lamina I/III (NK 1+ve) cells, and mediated via spinal alpha2-adrenoceptor activation. Since the same neuronal population drives descending facilitation and inhibition, the reduced excitability of lamina V/VI WDR neurones seen after loss of these NK 1+ve neurones indicates a dominant role of descending facilitation.

The pattern of Fos expression in the spinal dorsal horn following acute noxious mechanical stimulation of bone

Little is known of the spinal mechanisms that mediate bone nociception. The aim of this study was to determine the pattern of neuronal activation in the spinal dorsal horn following acute noxious mechanical stimulation of bone. This was achieved by examining Fos expression in the spinal dorsal horn following acute, noxious mechanical stimulation of the rat tibia. Noxious mechanical stimuli were applied by bone drilling and raising tibial intra-osseous pressure. Control experiments consisted of surgery to expose the tibia. There was a significant increase in the number of Fos-like immunoreactive (Fos-LI) nuclei in the superficial, ipsilateral dorsal horn of animals in the bone drilling and pressure groups relative to animals of the control group at spinal cord segments L3 and L4 ( P < 0.05). The number of Fos-LI nuclei in the deep dorsal horn was always lower than the number in the superficial dorsal horn (significant at L3 but not L4; P < 0.05). Whilst there appeared to be a small increase in the number of Fos-LI nuclei in the ipsilateral deep dorsal horn of bone drilling and pressure groups relative to the ipsilateral deep dorsal horn control group at both L3 and L4 segments, no significant effect was observed ( P > 0.05). The present study implicates the superficial dorsal horn of the spinal cord as a region of interest in studies of acute bone pain, and highlights the notion that spinal mechanisms that mediate bone nociception may be different to those that mediate nociception of cutaneous and visceral origin.

Anti-nociceptive effects induced by intrathecal injection of BmK AS, a polypeptide from the venom of Chinese-scorpion Buthus martensi Karsch, in rat formalin test

Aim of the study Asian scorpion Buthus martensi Karsch (BmK) is widely used to treat neurological symptoms, especially chronic pain, in traditional Chinese medicine for thousands of years. BmK AS, a polypeptide from BmK venom, could produce peripheral potent anti-nociceptive effects in rats. In the present study, spinal anti-nociceptive effects of BmK AS were investigated in rat formalin test. Materials and Methods Spinal anti-nociceptive activity of BmK AS was studied using formalin test in rats. BmK AS in doses of 0.02, 0.1 and 0.5 μg was administered intrathecally before formalin injection 10 min. The suppression by intrathecal injection of BmK AS on formalin-induced spontaneous nociceptive behaviors and spinal c-Fos expression were investigated. Results Intrathecal injection of BmK AS markedly reduced formalin-evoked biphasic spontaneous nociceptive behaviors in a dose-dependent manner. Formalin-induced c-Fos expression could be dose-dependently inhibited by BmK AS in superficial (I–II), the nucleus proprius (III and IV) and deep (V–VI) dorsal horn laminae, but not in the ventral gray laminae (VII–X) of lumbar spinal cord. The suppression by BmK AS on c-Fos expression in superficial laminaes was much stronger than that in deep laminaes. Conclusion The present study demonstrates that BmK AS is capable of producing remarkable anti-nociceptive effects not only in periphery but also in spinal cord.

Spread of excitation across modality borders in spinal dorsal horn of neuropathic rats

Under physiological conditions, nociceptive information is mainly processed in superficial laminae of the spinal dorsal horn, whereas non-nociceptive information is processed in deeper laminae. Neuropathic pain patients often suffer from touch-evoked pain (allodynia), suggesting that modality borders are disrupted in their nervous system. We studied whether excitation evoked in deep dorsal horn neurons either via stimulation of primary afferent Aβ-fibres, by direct electrical stimulation or via glutamate microinjection leads to activation of neurons in the superficial dorsal horn. We used Ca 2+-imaging in transversal spinal cord slices of neuropathic and control animals to monitor spread of excitation from the deep to the superficial spinal dorsal horn. In neuropathic but not control animals, a spread of excitation occurred from the deep to the superficial dorsal horn. The spread of excitation was synaptically mediated as it was blocked by the AMPA receptor antagonist CNQX. In contrast, block of NMDA receptors was ineffective. In control animals, the violation of modality borders could be reproduced by bath application of GABA A and glycine receptor antagonists. Furthermore, we could show that neuropathic animals were more prone to synchronous network activity than control animals. Thus, following peripheral nerve injury, excitation generated in dorsal horn areas which process non-nociceptive information can invade superficial dorsal horn areas which normally receive nociceptive input. This may be a spinal mechanism of touch-evoked pain.

Peripheral Nerve Injury–Induced Changes in Spinal α2-Adrenoceptor–Mediated Modulation of Mechanically Evoked Dorsal Horn Neuronal Responses

Peripheral nerve injury has been associated with changes in the modulatory action of noradrenergic pathways on nociceptive traffic through the spinal cord. Thus, the purpose of this study was to assess whether endogenous noradrenergic descending inhibition, acting via spinal alpha(2)-receptors, is altered after peripheral nerve damage. We investigated the effects of spinal administration of a selective alpha(2)-adrenoceptor antagonist, atipamezole, on the evoked activity of deep dorsal horn neurons in animals with selective spinal nerve ligation (SNL) compared with a sham-operated group. Intrathecal administration of atipamezole (1, 10, and 100 microg) did not produce any significant effects on the electrically evoked neuronal responses in either animal group, with the exception of a small but significant enhancement of the postdischarge in the sham control group only. Similarly, no significant effects were observed with the heat-evoked neuronal responses in either group. Interestingly, atipamezole significantly increased the evoked responses of neurons to low-intensity mechanical stimuli in the sham control group but was without effect in the SNL group. Thus, our findings suggest that peripheral nerve injury can result in the suppression of noradrenergic spinal alpha(2)-adrenoceptor-mediated inhibition of spinal dorsal horn neuronal activity evoked by low-intensity mechanical stimuli. These results suggest that a tonically active noradrenergic inhibition of mechanically evoked spinal dorsal horn neuronal responses is lost after nerve injury. This shift in the balance of noradrenergic controls may be one of the many underlying mechanisms by which behavioral symptoms of hypersensitivity develop after nerve damage.