Spinal mechanisms of NPY analgesia

We review previously published data, and present some new data, indicating that spinal application of neuropeptide Y (NPY) reduces behavioral and neurophysiological signs of acute and chronic pain. In models of acute pain, early behavioral studies showed that spinal (intrathecal) administration of NPY and Y2 receptor agonists decrease thermal nociception. Subsequent neurophysiological studies indicated that Y2-mediated inhibition of excitatory neurotransmitter release from primary afferent terminals in the substantia gelatinosa may contribute to the antinociceptive actions of NPY. As with acute pain, NPY reduced behavioral signs of inflammatory pain such as mechanical allodynia and thermal hyperalgesia; however, receptor antagonist studies indicate an important contribution of spinal Y1 rather than Y2 receptors. Interestingly, Y1 agonists suppress inhibitory synaptic events in dorsal horn neurons (indeed, well known mu-opioid analgesic drugs produce similar cellular actions). To resolve the behavioral and neurophysiological data, we propose that NPY/Y1 inhibits the spinal release of inhibitory neurotransmitters (GABA and glycine) onto inhibitory neurons, e.g. disinhibition of pain inhibition, resulting in hyporeflexia. The above mechanisms of Y1- and Y2-mediated analgesia may also operate in the setting of peripheral nerve injury, and new data indicate that NPY dose-dependently inhibits behavioral signs of neuropathic pain. Indeed, neurophysiological studies indicate that Y2-mediated inhibition of Ca(2+) channel currents in dorsal root ganglion neurons is actually increased after axotomy. We conclude that spinal delivery of Y1 agonists may be of use in the treatment of chronic inflammatory pain, and that the use of Y1 and Y2 agonists in neuropathic pain warrants further consideration.     

缓激肽B1受体与疼痛

摘要：缓激肽B1受体（bradykinin 1 receptors,B1Rs）是与Gq蛋白相偶联的受体。正常状态下,B1R除了在神经系统中（如脊髓背角浅层和感觉神经节）有少数表达外,其他机体组织中几乎不存在。在炎症或者神经受损的情况下,脊髓背角浅层和感觉神经节B1R表达量大大上升,参与炎性疼痛和神经病理性疼痛的产生和维持。近年来的研究表明,B1R在糖尿病性神经病理疼痛的发病中起着重要的作用。阻断B1R能有效抑制糖尿病诱发的热痛觉过敏和冷觉及触觉超敏。此外,B1R和癌症痛的发生也有密切关系,所以,对B1R的研究可能会为治疗这些临床顽症提供新的靶点。关键词：缓激肽B1受体;炎性痛;神经病理性痛中图分类号：Q189;Q42;R338文献标志码：A     

Complete sciatic nerve transection induces increase of neuropeptide Y-like immunoreactivity in primary sensory neurons and spinal cord of frogs

Neuropeptide Y (NPY) was immunohistochemically investigated in the frog spinal cord and dorsal root ganglia after axotomy. In normal ganglia, moderate NPY-like immunoreactivity (NPY-IR) prevailed in large and medium cells. In the spinal cord, the NPY-IR was densest in the dorsal part of the lateral funiculus. Other fibers and neurons NPY-IR were observed in the dorsal and ventral terminal fields and mediolateral band. NPY-IR fibers were also found in the ventral horn and in the ventral and lateral funiculi. The sciatic nerve transection increased the NPY-IR in large and medium neurons of the ipsilateral and contralateral dorsal root ganglia at 3 and 7 days, but no clear change was found at 15 days. In the spinal cord, there was a bilateral increase in the NPY-IR of the dorsal part of the lateral funiculus. In the ipsilateral side, the NPY-IR was increased at 3 and 7 days but was decreased at 15 days. In the contralateral side, a significant reduction at 15 days occurred. These findings seem to favor the role of NPY in the modulation of pain-related information in frogs, suggesting that this role of NPY may have appeared early in vertebrate evolution.     

Spinal neuropeptide expression and neuropathic behavior in the acute and chronic phases after spinal cord injury: Effects of progesterone administration

Patients with spinal cord injury (SCI) develop chronic pain that severely compromises their quality of life. We have previously reported that progesterone (PG), a neuroprotective steroid, could offer a promising therapeutic strategy for neuropathic pain. In the present study, we explored temporal changes in the expression of the neuropeptides galanin and tyrosine (NPY) and their receptors (GalR1 and GalR2; Y1R and Y2R, respectively) in the injured spinal cord and evaluated the impact of PG administration on both neuropeptide systems and neuropathic behavior. Male rats were subjected to spinal cord hemisection at T13 level, received daily subcutaneous injections of PG or vehicle, and were evaluated for signs of mechanical and thermal allodynia. Real time PCR was used to determine relative mRNA levels of neuropeptides and receptors, both in the acute (1 day) and chronic (28 days) phases after injury. A significant increase in Y1R and Y2R expression, as well as a significant downregulation in GalR2 mRNA levels, was observed 1 day after SCI. Interestingly, PG early treatment prevented Y1R upregulation and resulted in lower NPY, Y2R and GalR1 mRNA levels. In the chronic phase, injured rats showed well-established mechanical and cold allodynia and significant increases in galanin, NPY, GalR1 and Y1R mRNAs, while maintaining reduced GalR2 expression. Animals receiving PG treatment showed basal expression levels of galanin, NPY, GalR1 and Y1R, and reduced Y2R mRNA levels. Also, and in line with previously published observations, PG-treated animals did not develop mechanical allodynia and showed reduced sensitivity to cold stimulation. Altogether, we show that SCI leads to considerable changes in the spinal expression of galanin, NPY and their associated receptors, and that early and sustained PG administration prevents them. Moreover, our data suggest the participation of galaninergic and NPYergic systems in the plastic changes associated with SCI-induced neuropathic pain, and further supports the therapeutic potential of PG- or neuropeptide-based therapies to prevent and/or treat chronic pain after central injuries.     

Galanin and NPY, two peptides with multiple putative roles in the nervous system.

In the present brief overview we summarize results from several studies focusing on two neuropeptides, galanin and neuropeptide Y (NPY) in discrete neuronal systems, where they coexist with classic transmitters. On the basis of studies in different animal models we propose that these peptides may be involved in regulation of certain CNS functions and that drugs acting on their receptors may be of use in new therapeutic strategies. At the spinal level galanin and NPY are regulated in DRG neurons by nerve injury and in dorsal horn neurons by inflammation. It is possible that this leads to attenuation of pain sensitivity. Moreover, both peptides may exert trophic effects, for example to enhance regeneration. In the hypothalamic arcuate nucleus NPY and its receptors are part of the feeding circuitry, and we suggest that derangement of these NPY neurons may at least in part underlay the lethal phenotype of anorectic mice, which die 22 days postnatally after showing decreased food intake and growth retardation. Expression of NPY and NPY receptors is changed in the hippocampus of mice comparatively early after prion inoculation, indicating that this peptide system is affected in this spongiform degenerative disease in a region of importance for learning and memory. Finally, galanin is co-localized with classic monoamine transmitters in two central systems, the dorsal raphe serotonin neurons and the locus coeruleus noradrenergic neurons. In both cases galanin causes hyperpolarization (at high concentrations) and prolongs monoamine-induced outward currents (at low concentrations), thus modulating activity in two systems of importance for many brain functions including mood regulation. It may therefore be interesting to analyse to what extent drugs affecting galaninergic transmission also may be efficient in the treatment of, for example, depression.     

The role of spinal cord vanilloid (TRPV1) receptors in pain modulation

Transient receptor potential vanilloid 1 (TRPV1) receptor is a nonselective cation channel activated by capsaicin, a pungent substance from chili peppers. It is considered to act as an integrator of various physical and chemical nociceptive stimuli, as it can be gated by noxious heat (>43 oC), low pH (protons) and also by recently described endogenous lipids. The structure and function of TRPV1 receptors was vigorously studied, especially since its cloning in 1997. However, most of the research was pointed towards the role of TRPV1 receptors in the peripheral tissues. Mounting evidence now suggests that TRPV1 receptors on the central branches of dorsal root ganglion neurons in the spinal cord may play an important role in modulation of pain and nociceptive transmission. The aim of this short review was to summarize the knowledge about TRPV1 receptors in the spinal cord dorsal horn, preferentially from morphological and electrophysiological studies on spinal cord slices and from in vivo experiments.     

Identification and characterization of two G protein-coupled receptors for neuropeptide FF

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.     

Central neuropeptide Y induces proximal stomach relaxation via Y1 receptors in the dorsal vagal complex of the rat

Effects of neuropeptide Y (NPY) on motility of the proximal stomach was examined in anesthetized rats. Intragastric pressure was measured using a balloon situated in the proximal part of the stomach. Administration of NPY into the fourth ventricle induced relaxation of the proximal stomach in a dose-dependent manner. Administration of an Y1 receptor (Y1R) agonist [Leu31, Pro34]NPY induced a larger relaxation than NPY. The administration of an Y2 receptor agonist (NPY 13-36) did not induce significant changes in motility. Microinjections of [Leu31, Pro34]NPY into the caudal part of the dorsal vagal complex (DVC) induced relaxation of the proximal stomach. In contrast, similar injections into the intermediate part of the DVC increased IGP of the proximal stomach. Administration of NPY into the fourth ventricle did not induce relaxation after bilateral injections of the Y1R antagonist (1229U91) into the caudal DVC. These results indicate that NPY induces relaxation in the proximal stomach via Y1Rs situated in the DVC. Because bilateral vagotomy below the diaphragm abolished the relaxation induced by the administration of NPY into the fourth ventricle, relaxation induced by NPY is probably mediated by vagal preganglionic neurons. Intravenous injection of atropine methyl nitrate reduced relaxation induced by administration of NPY. Therefore, relaxation induced by NPY is likely mediated by peripheral cholinergic neurons.     

Regulation of increased glutamatergic input to spinal dorsal horn neurons by mGluR5 in diabetic neuropathic pain

Diabetic neuropathic pain is associated with increased glutamatergic input in the spinal dorsal horn. Group I metabotropic glutamate receptors (mGluRs) are involved in the control of neuronal excitability, but their role in the regulation of synaptic transmission in diabetic neuropathy remains poorly understood. Here we studied the role of spinal mGluR5 and mGluR1 in controlling glutamatergic input in a rat model of painful diabetic neuropathy induced by streptozotocin. Whole-cell patch-clamp recordings of lamina II neurons were performed in spinal cord slices. The amplitude of excitatory post-synaptic currents (EPSCs) evoked from the dorsal root and the frequency of spontaneous EPSCs (sEPSCs) were significantly higher in diabetic than in control rats. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) inhibited evoked EPSCs and sEPSCs more in diabetic than in control rats. Also, the percentage of neurons in which sEPSCs and evoked EPSCs were affected by MPEP or the group I mGluR agonist was significantly higher in diabetic than in control rats. However, blocking mGluR1 had no significant effect on evoked EPSCs and sEPSCs in either groups. The mGluR5 protein level in the dorsal root ganglion, but not in the dorsal spinal cord, was significantly increased in diabetic rats compared with that in control rats. Furthermore, intrathecal administration of MPEP significantly increased the nociceptive pressure threshold only in diabetic rats. These findings suggest that increased mGluR5 expression on primary afferent neurons contributes to increased glutamatergic input to spinal dorsal horn neurons and nociceptive transmission in diabetic neuropathic pain.     

Ionotropic Glutamate Receptors in Spinal Nociceptive Processing

Glutamate is the predominant excitatory transmitter used by primary afferent synapses and intrinsic neurons in the spinal cord dorsal horn. Accordingly, ionotropic glutamate receptors mediate basal spinal transmission of sensory, including nociceptive, information that is relayed to supraspinal centers. However, it has become gradually more evident that these receptors are also crucially involved in short- and long-term plasticity of spinal nociceptive transmission, and that such plasticity have an important role in the pain hypersensitivity that may result from tissue or nerve injury. This review will cover recent findings on pre- and postsynaptic regulation of synaptic function by ionotropic glutamate receptors in the dorsal horn and how such mechanisms contribute to acute and chronic pain.     

Distribution of neuropeptide Y in the spinal cords of cat, dog, rat, man and pig

Regional distribution of neuropeptide Y (NPY) in spinal cord, dorsal root ganglia (DRG) and peripheral nerves was quantitated in rat, cat, dog, pig, and man. Spinal cords were harvested post mortem and dissected into regions or individual segments. A further dissection into dorsal and ventral horns was carried out, and DRG were harvested in all species except rat. Tissues were extracted into boiling 0.1 M HCl, and NPY was measured by radioimmunoassay using a specific antibody and I125-labeled NPY. Highest concentrations of NPY were consistently found in the dorsal horn of the lumbo-sacral cord (200-800 ng/g). DRG concentrations, in contrast, were routinely low or undetectable. Sciatic nerve concentrations in rat and pig were considerable. High performance liquid chromatography (HPLC) confirmed that the NPY immunoreactivity measured in dorsal horns of each species coeluted with authentic NPY (1-36) as a single peak.     

Distribution of Y-Receptors in Murine Lingual Epithelia

Peptide hormones and their cognate receptors belonging to neuropeptide Y (NPY) family mediate diverse biological functions in a number of tissues. Recently, we discovered the presence of the gut satiation peptide YY (PYY) in saliva of mice and humans and defined its role in the regulation of food intake and body weight maintenance. Here we report the systematic analysis of expression patterns of all NPY receptors (Rs), Y1R, Y2R, Y4R, and Y5R in lingual epithelia in mice. Using four independent assays, immunohistochemistry, in situ hybridization, immunocytochemistry and RT PCR, we show that the morphologically different layers of the keratinized stratified epithelium of the dorsal layer of the tongue express Y receptors in a very distinctive yet overlapping pattern. In particular, the monolayer of basal progenitor cells expresses both Y1 and Y2 receptors. Y1Rs are present in the parabasal prickle cell layer and the granular layer, while differentiated keratinocytes display abundant Y5Rs. Y4Rs are expressed substantially in the neuronal fibers innervating the lamina propria and mechanoreceptors. Basal epithelial cells positive for Y2Rs respond robustly to PYY_3–36 by increasing intracellular Ca²⁺ suggesting their possible functional interaction with salivary PYY. In taste buds of the circumvallate papillae, some taste receptor cells (TRCs) express YRs localized primarily at the apical domain, indicative of their potential role in taste perception. Some of the YR-positive TRCs are co-localized with neuronal cell adhesion molecule (NCAM), suggesting that these TRCs may have synaptic contacts with nerve terminals. In summary, we show that all YRs are abundantly expressed in multiple lingual cell types, including epithelial progenitors, keratinocytes, neuronal dendrites and TRCs. These results suggest that these receptors may be involved in the mediation of a wide variety of functions, including proliferation, differentiation, motility, taste perception and satiation.     

Regulation of GABA(A) receptor dynamics by interaction with purinergic P2X(2) receptors

γ-Aminobutyric acid type A receptors (GABA(A)Rs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X(2) receptors (P2X(2)Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABA(A)Rs. Here, we investigated a possible "dynamic" interaction between GABA(A)Rs and P2X(2)Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X(2)Rs forms a transient complex with GABA(A)Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X(2)Rs and GABA(A)Rs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X(2)Rs results in a Ca(2+)-dependent as well as an apparently Ca(2+)-independent increase in the mobility and an enhanced degradation of GABA(A)Rs, whereas P2X(2)Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X(2)Rs and GABA(A)Rs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA(A)Rs.     

Calcitonin gene-related peptide 8-37 inhibits the evoked discharge frequency of wide dynamic range neurons in dorsal horn of the spinal cord in rats.

The present study was performed to explore the effect of calcitonin gene-related peptide 8-37 (CGRP8-37) on the electrical stimulation-evoked discharge frequency of wide dynamic range (WDR) neurons in the dorsal horn of the spinal cord in rats. The discharge frequencies of WDR neurons were evoked by transdermic electrical stimulation applied on the ipsilateral hindpaw. CGRP8-37 was applied directly on the dorsal surface of the L3 to L5 spinal cord. After the administration of 3 nmol of CGRP8-37, the evoked discharge frequency of WDR neurons decreased significantly, an effect lasting more than 30 min. The results indicate that CGRP receptors play an important role in the transmission of presumed nociceptive information in the dorsal horn of the spinal cord.     

The Y1 receptor for NPY: a key modulator of the adaptive immune system

Growing evidence suggests that the neuropeptide Y (NPY) system plays an important role in the immune system. Yet, little is known about the expression of NPY and receptors in the immune system. Moreover, original contradicting results have confused the picture and hampered a clear understanding of its role in the immune system. The use of Y(1) receptor-deficient mice, combined with advanced methods to investigate immune functions, have provided the solution to the problem raised by previous disparities. From results obtained using Y(1)-deficient mice (Y(1)(-/-)), we uncovered a bimodal role for Y(1) on immune cells. Y(1) expression on antigen-presenting cells (APC) is essential for their function as T cell priming elements. Conversely, Y(1) signaling in T cells plays a regulatory role without which T cells are hyper-responsive. The opposite role of Y(1) on APC and T cells has reconciled previous disparities by showing that signaling via Y(1) protects against inflammation by inhibiting T cell responses, whereas Y(1)(-/-) mice are protected in the same inflammatory models due to defective APCs.     

Bone and joint neuropathy in rats with type-2 diabetes

We have previously demonstrated that Goto-Kakizaki (GK) rats with spontaneous type-2 diabetes and peripheral neuropathy exhibit regional osteopathic changes. In the present study on 18 GK rats and 21 control Wistar rats, the occurrence of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the autonomic neuropeptide Y (NPY) was analysed in bone and joints, dorsal root ganglia and lumbar spinal cord by immunohistochemistry and radioimmunoassay (RIA). Immunohistochemistry disclosed a predominance of immunoreactivities in vessel-related nerve fibers, although some were also seen in free terminals. While SP, CGRP and NPY in periosteum, cortical bone and synovium was confined to neuronal tissue, the bone marrow in addition exhibited an abundance of NPY-positive megakaryocytes. Apart from this cellular source of NPY, the observations suggest that the three neuropeptides analysed in bone and joints are of neuronal origin. Quantification by RIA showed a significant decrease of NPY in cortical bone (-36%), bone marrow (-66%) and ankle (-29%) of GK rats. CGRP was decreased in the spinal cord (-19%) and dorsal root ganglia (-26%) but was unchanged in bone and joints, as with SP. Given the suggested anabolic role of NPY and CGRP on bone, neuropeptidergic deficit in diabetes may prove to be an important factor underlying the development of regional osteopenia.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Expression of nitric oxide synthase isoforms in the dorsal horn of monoarthritic rats: effects of competitive and uncompetitive <Emphasis Type="Italic">N</Emphasis>-methyl-D-aspartate antagonists

Chronic pain is associated with N-methyl-D-aspartate (NMDA) receptor activation and downstream production of nitric oxide, which has a pivotal role in multisynaptic local circuit nociceptive processing in the spinal cord. The formation of nitric oxide is catalyzed by three major nitric oxide synthase (NOS) isoforms (neuronal, nNOS; inducible, iNOS; endothelial, eNOS), which are increased in the spinal cord of rodents subjected to some tonic and chronic forms of experimental pain. Despite the important role of NOS in spinal cord nociceptive transmission, there have been no studies exploring the effect of NMDA receptor blockade on NOS expression in the dorsal horn during chronic pain. Furthermore, NOS isoforms have not been fully characterized in the dorsal horn of animals subjected to arthritic pain. The aim of this work was therefore to study the expression of nNOS, iNOS and eNOS in the dorsal horns of monoarthritic rats, and the modifications in NOS expression induced by pharmacological blockade of spinal cord NMDA receptors. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the right tibio-tarsal joint. At week 4, monoarthritic rats were given either the competitive NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or the uncompetitive NMDA antagonist ketamine. After 6 and 24 hours, animals were killed and posterior quadrants of the lumbar spinal cord were dissected. Sample tissues were homogenized and subjected to immunoblotting with anti-nNOS, anti-iNOS or anti-eNOS monoclonal antibodies. The nNOS isoform, but not the iNOS and eNOS isoforms, were detected in the dorsal horns of control rats. Monoarthritis increased the expression of nNOS, iNOS and eNOS in the dorsal horns ipsilateral and contralateral to the inflamed hindpaw. Intrathecal administration of CPP and ketamine reduced nNOS expression in monoarthritic rats but increased the expression of iNOS and eNOS. Results suggest that blockade of spinal cord NMDA receptors produces complex regulatory changes in the expression of NOS isoforms in monoarthritic rats that may be relevant for nitridergic neuronal/glial mechanisms involved in the pathophysiology of monoarthritis and in the pharmacological response to drugs interacting with NMDA receptors.     

Activation of the neurokinin-1 receptor by substance P triggers the release of substance P from cultured adult rat dorsal root ganglion neurons

Hypoxia and ischemia occur in the spinal cord when blood vessels of the spinal cord are compressed under pathological conditions such as spinal stenosis, tumors, and traumatic spinal injury. Here by using spinal cord slice preparations and patch-clamp recordings we investigated the influence of an ischemia-simulating medium on dorsal horn neurons in deep lamina, a region that plays a significant role in sensory hypersensitivity and pathological pain. We found that the ischemia-simulating medium induced large inward currents in dorsal horn neurons recorded. The onset of the ischemia-induced inward currents was age-dependent, being onset earlier in older animals. Increases of sensory input by the stimulation of afferent fibers with electrical impulses or by capsaicin significantly speeded up the onset of the ischemia-induced inward currents. The ischemia-induced inward currents were abolished by the glutamate receptor antagonists CNQX (20 μM) and APV (50 μM). The ischemia-induced inward currents were also substantially inhibited by the glutamate transporter inhibitor TBOA (100 μM). Our results suggest that ischemia caused reversal operation of glutamate transporters, leading to the release of glutamate via glutamate transporters and the subsequent activation of glutamate receptors in the spinal dorsal horn neurons.