Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

Transient receptor potential ankyrin 1 in the knee is involved in osteoarthritis pain.

Transient receptor potential families play important roles in the pathology of osteoarthritis (OA) of the knee. While transient receptor potential ankyrin 1 (TRPA1) is also an essential component of the pathogenesis of various arthritic conditions, its association with pain is controversial. Thus, we researched whether TRPA1 is involved in knee OA pain by in vivo patch-clamp recordings and evaluated the behavioral responses using CatWalk gait analysis and pressure application measurement (PAM). Injection of the Trpa1 agonist, allyl isothiocyanate (AITC), into the knee joint significantly increased spontaneous excitatory synaptic current (sEPSC) frequency in the substantia gelatinosa of rats with knee OA, while injection of the Trpa1 antagonist, HC-030031, significantly decreased the sEPSC. Meanwhile, AITC did not affect the sEPSC in sham rats. In the CatWalk and PAM behavioral tests, AITC significantly decreased pain thresholds, but no difference between HC-030031 and saline injections was observed. Our results indicate that Trpa1 mediates knee OA-induced pain. We demonstrated that Trpa1 is activated in the knee joints of rats with OA, and Trpa1 activity enhanced the pain caused by knee OA.

Anti-allodynic and promotive effect on inhibitory synaptic transmission of riluzole in rat spinal dorsal horn.

Riluzole (2-amino-6-(trifluoromethoxy)benzothiazole) is a drug known for its inhibitory effect on glutamatergic transmission and its anti-nociceptive and anti-allodynic effects in neuropathic pain rat models. Riluzole also has an enhancing effect on GABAergic synaptic transmission. However, the effect on the spinal dorsal horn, which plays an important role in modulating nociceptive transmission, remains unknown. We investigated the ameliorating effect of riluzole on mechanical allodynia using the von Frey test in a rat model of neuropathic pain and analyzed the synaptic action of riluzole on inhibitory synaptic transmission in substantia gelatinosa (SG) neurons using whole-cell patch clamp recordings. We found that single-dose intraperitoneal riluzole (4 mg/kg) administration effectively attenuated mechanical allodynia in the short term in a rat model of neuropathic pain. Moreover, 300 µM riluzole induced an outward current in rat SG neurons. The outward current induced by riluzole was not suppressed in the presence of tetrodotoxin. Furthermore, we found that the outward current was suppressed by simultaneous bicuculline and strychnine application, but not by strychnine alone. Altogether, these results suggest that riluzole enhances inhibitory synaptic transmission monosynaptically by potentiating GABAergic synaptic transmission in the rat spinal dorsal horn.

Inhibition by O-desmethyltramadol of glutamatergic excitatory transmission in adult rat spinal substantia gelatinosa neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a µ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating µ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Molecular mechanisms of the antispasticity effects of baclofen on spinal ventral horn neurons.

BACKGROUND: Baclofen is a lipophilic γ-aminobutyric acid (GABA) derivative that exhibits strong intrinsic activity and a high affinity for GABAB receptors. Intrathecal baclofen therapy has been used as an antispasticity and muscle relaxant drug in the clinical treatment of patients with severe spasticity. However, the cellular mechanisms of the antispasticity effects of baclofen on the ventral horn neurons of the spinal cord are unknown. OBJECTIVE: We examined the action of baclofen on excitatory synaptic transmission in ventral horn neurons in the rat spinal cord by whole-cell patch-clamp recordings. RESULTS: Baclofen significantly reduced the frequency and amplitude of miniature excitatory postsynaptic currents. The reduction in miniature excitatory postsynaptic current frequency was particularly strong, indicating presynaptic inhibition by baclofen. Moreover, baclofen-induced outward currents in all neurons tested. The baclofen-induced outward currents persisted in the presence of tetrodotoxin and glutamate receptor antagonists and were diminished in the presence of the postsynaptic intracellular K channel blocker cesium sulfate and the G-protein inhibitor guanosine 5'-(ß-thio)diphosphate trilithium salt. These results indicate direct postsynaptic depression mediated by G-protein-activated K channels by GABAB receptors on ventral horn neurons. The baclofen-induced outward currents and the inhibitory effects on spontaneous excitatory postsynaptic currents were blocked by the selective GABAB receptor antagonist CGP35348. CONCLUSION: Baclofen may have both presynaptic and postsynaptic capacity to inhibit synaptic transmission in ventral horn neurons by GABAB receptors. These cellular mechanisms may induce the antispasticity effects of intrathecal baclofen therapy in the spinal cord.

Top-down descending facilitation of spinal sensory excitatory transmission from the anterior cingulate cortex.

Spinal sensory transmission is under descending biphasic modulation, and descending facilitation is believed to contribute to chronic pain. Descending modulation from the brainstem rostral ventromedial medulla (RVM) has been the most studied, whereas little is known about direct corticospinal modulation. Here, we found that stimulation in the anterior cingulate cortex (ACC) potentiated spinal excitatory synaptic transmission and this modulation is independent of the RVM. Peripheral nerve injury enhanced the spinal synaptic transmission and occluded the ACC-spinal cord facilitation. Inhibition of ACC reduced the enhanced spinal synaptic transmission caused by nerve injury. Finally, using optogenetics, we showed that selective activation of ACC-spinal cord projecting neurons caused behavioral pain sensitization, while inhibiting the projection induced analgesic effects. Our results provide strong evidence that ACC stimulation facilitates spinal sensory excitatory transmission by a RVM-independent manner, and that such top-down facilitation may contribute to the process of chronic neuropathic pain.

Interferon-gamma potentiates NMDA receptor signaling in spinal dorsal horn neurons via microglia-neuron interaction.

BACKGROUND: Glia-neuron interactions play an important role in the development of neuropathic pain. Expression of the pro-inflammatory cytokne →cytokine Interferon-gamma (IFNγ) is upregulated in the dorsal horn after peripheral nerve injury, and intrathecal IFNγ administration induces mechanical allodynia in rats. A growing body of evidence suggests that IFNγ might be involved in the mechanisms of neuropathic pain, but its effects on the spinal dorsal horn are unclear. We performed blind whole-cell patch-clamp recording to investigate the effect of IFNγ on postsynaptic glutamate-induced currents in the substantia gelatinosa neurons of spinal cord slices from adult male rats. RESULTS: IFNγ perfusion significantly enhanced the amplitude of NMDA-induced inward currents in substantia gelatinosa neurons, but did not affect AMPA-induced currents. The facilitation of NMDA-induced current by IFNγ was inhibited by bath application of an IFNγ receptor-selective antagonist. Adding the Janus activated kinase inhibitor tofacitinib to the pipette solution did not affect the IFNγ-induced facilitation of NMDA-induced currents. However, the facilitatory effect of IFNγ on NMDA-induced currents was inhibited by perfusion of the microglial inhibitor minocycline. These results suggest that IFNγ binds the microglial IFNγ receptor and enhances NMDA receptor activity in substantia gelatinosa neurons. Next, to identify the effector of signal transmission from microglia to dorsal horn neurons, we added an inhibitor of G proteins, GDP-ß-S, to the pipette solution. In a GDP-ß-S-containing pipette solution, IFNγ-induced potentiation of the NMDA current was significantly suppressed after 30 min. In addition, IFNγ-induced potentiation of NMDA currents was blocked by application of a selective antagonist of CCR2, and its ligand CCL2 increased NMDA-induced currents. CONCLUSION: Our findings suggest that IFNγ enhance the amplitude of NMDA-induced inward currents in substantia gelatinosa neurons via microglial IFNγ receptors and CCL2/CCR2 signaling. This mechanism might be partially responsible for the development of persistent neuropathic pain.

Leukotriene enhances NMDA-induced inward currents in dorsal horn neurons of the rat spinal cord after peripheral nerve injury.

BACKGROUND: LTB4 is classified as a leukotriene (LT), a group of lipid mediators that are derived from arachidonic acid. It is recognized that leukotrienes are involved in the pathogenesis of many diseases, including peripheral inflammatory pain. However, little is known about the effects of leukotrienes on the spinal dorsal horn during neuropathic pain. Previously, we reported that there was increased expression of 5-lipoxygenase (5-LO) at spinal microglia, and the leukotriene B4 receptor 1 (BLT1), a high affinity receptor of LTB4, in spinal neurons in spared nerve injury (SNI) model rats. In the present study, we examined the effects of LTB4 on spinal dorsal horn neurons in both naïve and SNI model rats using patch-clamp methods. RESULTS: Bath application of LTB4 did not change AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) or membrane potentials. However, we found that LTB4 enhanced the amplitude of NMDA receptor-mediated sEPSCs and significantly increased exogenous NMDA-induced inward currents in SNI model rats. This increase of inward currents could be inhibited by a selective LTB4 antagonist, U75302, as well as a GDP-ß-S, a G-protein inhibitor. These results indicate that both increased LTB4 from spinal microglia or increased BLT1 in spinal neurons after peripheral nerve injury can enhance the activity of NMDA receptors through intracellular G-proteins in spinal dorsal horn neurons. CONCLUSION: Our findings showed that LTB4, which may originate from microglia, can activate BLT1 receptors which are expressed on the membrane of spinal dorsal horn neurons during neuropathic pain. This glia-neuron interaction induces the enhancement of NMDA currents through intracellular G-proteins. The enhancement of NMDA receptor sensitivity of dorsal horn neurons may lead to central sensitization, leading to mechanical pain hypersensitivity.

In vivo patch-clamp analysis of the antinociceptive actions of TRPA1 activation in the spinal dorsal horn.

BACKGROUND: Transient receptor potential (TRP) channels are nonselective cation channels expressed in a variety of sensory structures, and are important molecular mediators of thermal, mechanical, cellular and chemical signals. We investigated the function of one key member of the TRP superfamily, TRPA1, in the spinal dorsal horn using in vivo patch-clamp recordings. RESULTS: The application of allyl isothiocyanate (AITC), a TRPA1 agonist, significantly increased the frequency and amplitude of inhibitory postsynaptic currents (IPSCs; holding potential (VH) = 0 mV) as well as excitatory postsynaptic currents (EPSCs; VH = -70 mV) in substantia gelatinosa (SG) neurons. The AITC-induced increases in EPSC frequency and amplitude were resistant to the Na(+) channel blocker tetrodotoxin (TTX). In the presence of the glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activity. The AITC-induced increases in IPSC frequency and amplitude were abolished by TTX or glutamate receptor antagonists. Moreover, the duration of IPSCs enhanced by TRPA1 activation were significantly longer than those of EPSCs enhanced by activation of this channel in the spinal dorsal horn. AITC induced hyperpolarization of the membrane potential of SG neurons in the spinal cord but depolarized the membrane potential in the presence of TTX. Furthermore, we examined the effects of mechanical stimuli to the skin during TRPA1 activation in the spinal dorsal horn in normal rats in both voltage-clamp and current-clamp modes. In the peripheral tissue stimuli test, AITC significantly suppressed EPSCs evoked by pinch or air puff stimulation of the skin. In current-clamp mode, AITC significantly suppressed excitatory postsynaptic potentials (EPSPs) evoked by pinch stimuli. CONCLUSIONS: TRPA1 appears to be localized not only at presynaptic terminals on SG neurons, enhancing glutamate release, but also in the terminals of primary afferents innervating spinal inhibitory interneurons, which have synaptic interactions with SG neurons. This study offers further insight into the mechanisms underlying the possible antinociceptive actions of TRPA1 activation in the spinal dorsal horn. Our findings suggest that pharmacological activation of spinal TRPA1 channels may have therapeutic potential for the treatment of pain.

In vivo two-photon imaging of structural dynamics in the spinal dorsal horn in an inflammatory pain model.

Two-photon microscopy imaging has recently been applied to the brain to clarify functional and structural synaptic plasticity in adult neural circuits. Whereas the pain system in the spinal cord is phylogenetically primitive and easily exhibits behavioral changes such as hyperalgesia in response to inflammation, the structural dynamics of dendrites has not been analysed in the spinal cord mainly due to tissue movements associated with breathing and heart beats. Here we present experimental procedures to prepare the spinal cord sufficiently to follow morphological changes of neuronal processes in vivo by using two-photon microscopy and transgenic mice expressing fluorescent protein specific to the nervous system. Structural changes such as the formation of spine-like structures and swelling of dendrites were observed in the spinal dorsal horn within 30 min after the multiple-site injections of complete Freund's adjuvant (a chemical irritant) to a leg, and these changes continued for 5 h. Both AMPA and N-methyl-D-aspartate receptor antagonists, and gabapentin, a presynaptic Ca(2+) channel blocker, completely suppressed the inflammation-induced structural changes in the dendrites in the spinal dorsal horn. The present study first demonstrated by in vivo two-photon microscopy imaging that structural synaptic plasticity occurred in the spinal dorsal horn immediately after the injection of complete Freund's adjuvant and may be involved in inflammatory pain. Furthermore, acute inflammation-associated structural changes in the spinal dorsal horn were shown to be mediated by glutamate receptor activation.

Early transcutaneous electrical nerve stimulation reduces hyperalgesia and decreases activation of spinal glial cells in mice with neuropathic pain.

Although transcutaneous electrical nerve stimulation (TENS) is widely used for the treatment of neuropathic pain, its effectiveness and mechanism of action in reducing neuropathic pain remain uncertain. We investigated the effects of early TENS (starting from the day after surgery) in mice with neuropathic pain, on hyperalgesia, glial cell activation, pain transmission neuron sensitization, expression of proinflammatory cytokines, and opioid receptors in the spinal dorsal horn. Following nerve injury, TENS and behavioral tests were performed every day. Immunohistochemical, immunoblot, and flow cytometric analysis of the lumbar spinal cord were performed after 8 days. Early TENS reduced mechanical and thermal hyperalgesia and decreased the activation of microglia and astrocytes (P<0.05). In contrast, the application of TENS at 1 week (TENS-1w) or 2 weeks (TENS-2w) after injury was ineffective in reducing hyperalgesia (mechanical and thermal) or activation of microglia and astrocytes. Early TENS decreased p-p38 within microglia (P<0.05), the expression levels of protein kinase C (PKC-γ), and phosphorylated anti-phospho-cyclic AMP response element-binding protein (p-CREB) in the superficial spinal dorsal horn neurons (P<0.05), mitogen-activated protein (MAP) kinases, and proinflammatory cytokines, and increased the expression levels of opioid receptors (P<0.05). The results suggested that the application of early TENS relieved hyperalgesia in our mouse model of neuropathic pain by inhibiting glial activation, MAP kinase activation, PKC-γ, and p-CREB expression, and proinflammatory cytokines expression, as well as maintenance of spinal opioid receptors. The findings indicate that TENS treatment is more effective when applied as early after nerve injury as possible.

Involvement of Tyr1472 phosphorylation of NMDA receptor NR2B subunit in postherpetic neuralgia in model mice.

BACKGROUND: Postherpetic neuralgia is spontaneous pain and allodynia that persist long after the disappearance of the cutaneous lesions caused by herpes zoster. Inoculation of mice with herpes simplex virus-1 causes herpes zoster-like skin lesions and herpetic and postherpetic pain. Although NMDA receptors have been suggested to be involved in postherpetic pain as in other types of neuropathic pain, the neural mechanism remains unclear. NMDA receptor NR2B subunit is the most tyrosine-phosphorylated protein in the brain, and Tyr1472 is the major phosphorylation site of this subunit. RESULTS: To elucidate the role of Tyr1472 phosphorylation of the NR2B subunit in herpetic and postherpetic allodynia, we inoculated herpes simplex virus-1 into the unilateral hind paw of knock-in mice with a mutation of Tyr1472 of the NR2B subunit to Phe (Y1472F-KI). On day 7 post-inoculation, acute herpetic allodynia was observed in more than 80% of the inoculated wild-type and Y1472F-KI mice. Y1472F-KI mice showed significantly reduced intensity and incidence of postherpetic allodynia on days 45-50 post-inoculation as compared with wild-type mice. The innervation in the skin at the postherpetic neuralgia phase was retained to a greater extent in the Y1472F-KI mice. The level of activating transcription factor-3 mRNA, a marker of axonal damage, increased much less in the dorsal root ganglia (DRGs) of Y1472F-KI mice than in those of wild-type mice; and the level of nerve growth factor mRNA significantly increased in wild-type mice, but not at all in Y1472F-KI mice on day 7 post-inoculation. Production of nerve growth factor was at the basal level in the skin of both groups of mice on day 50 post-inoculation. Nerve growth factor and glial cell-derived neurotrophic factor stimulated neurite outgrowth of cultured DRG neurons from Y1472F-KI mice, similarly or less so as they did the outgrowth of those from wild-type mice. Wild-type DRG neurons were more susceptible to glutamate neurotoxicity than Y1472F-KI ones. CONCLUSIONS: Taken together, the present data suggest that phosphorylation of the NR2B subunit at its Tyr1472 is involved in the development of postherpetic allodynia due to nerve damage and that the nerve damage at the acute herpetic phase is correlated with the incidence of postherpetic pain.

Distinct degree of radiculopathy at different levels of peripheral nerve injury.

BACKGROUND: Lumbar radiculopathy is a common clinical problem, characterized by dorsal root ganglion (DRG) injury and neural hyperactivity causing intense pain. However, the mechanisms involved in DRG injury have not been fully elucidated. Furthermore, little is known about the degree of radiculopathy at the various levels of nerve injury. The purpose of this study is to compare the degree of radiculopathy injury at the DRG and radiculopathy injury proximal or distal to the DRG. RESULTS: The lumbar radiculopathy rat model was created by ligating the L5 nerve root 2 mm proximal to the DRG or 2 mm distal to the DRG with 6.0 silk. We examined the degree of the radiculopathy using different points of mechanical sensitivity, immunohistochemistry and in vivo patch-clamp recordings, 7 days after surgery. The rats injured distal to the DRG were more sensitive than those rats injured proximal to the DRG in the behavioral study. The number of activated microglia in laminas I-II of the L5 segmental level was significantly increased in rats injured distal to the DRG when compared with rats injured proximal to the DRG. The amplitudes and frequencies of EPSC in the rats injured distal to the DRG were higher than those injured proximal to the DRG. The results indicated that there is a different degree of radiculopathy at the distal level of nerve injury. CONCLUSIONS: Our study examined the degree of radiculopathy at different levels of nerve injury. Severe radiculopathy occurred in rats injured distal to the DRG when compared with rats injured proximal to the DRG. This finding helps to correctly diagnose a radiculopathy.

Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, and Ser831-GluR1 in neuropathic pain.

Previously we demonstrated that phosphorylation of NR2B subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor at Tyr1472 is increased in a neuropathic-pain model and that this phosphorylation is required for the maintenance of neuropathic pain by L5-spinal nerve transection. We obtained these results by using a selective NR2B antagonist and mice deficient in Fyn, which is an Src-family tyrosine protein kinase. However, how Tyr1472 phosphorylation of NR2B is involved in the maintenance of neuropathic pain was unclear. Here, we demonstrated that neuropathic pain was markedly attenuated in the spared nerve injury model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of NR2B (Y1472F-KI). While phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) at its Thr286 and that of the GluR1 subunit of the AMPA receptor at its Ser831 was enhanced in the spinal dorsal horn after spared nerve injury in wild-type mice, such phosphorylation was markedly impaired in Y1472F-KI mice. Inhibition of CaMKII by intrathecal injection of KN93, an inhibitor of CaMKII, reduced mechanical allodynia and phosphorylation of CaMKII at its Thr286 and that of GluR1 at its Ser831 in the spinal cord 7 days after spared nerve injury. These results demonstrate that the phosphorylation of CaMKII and GluR1 occurs downstream of the Tyr1472 phosphorylation of NR2B subunits in the spinal cord and give the first suggestion that activation of CaMKII and GluR1-AMPA receptors may be involved in mechanical allodynia caused by peripheral nerve injury.

In vivo patch-clamp analysis of dopaminergic antinociceptive actions on substantia gelatinosa neurons in the spinal cord.

To elucidate the mechanisms of antinociception mediated by the dopaminergic descending pathway in the spinal cord, we investigated the actions of dopamine (DA) on substantia gelatinosa (SG) neurons by in vivo whole-cell patch-clamp methods. In the voltage-clamp mode (V(H)=-70mV), the application of DA induced outward currents in about 70% of SG neurons tested. DA-induced outward current was observed in the presence of either Na(+) channel blocker, tetrodotoxin (TTX) or a non-NMDA receptor antagonist, CNQX, and was inhibited by either GDP-ß-S in the pipette solution or by perfusion of a non-selective K(+) channel blocker, Ba(2+). The DA-induced outward currents were mimicked by a selective D2-like receptor agonist, quinpirole and attenuated by a selective D2-like receptor antagonist, sulpiride, indicating that the DA-induced outward current is mediated by G-protein-activated K(+) channels through D2-like receptors. DA significantly suppressed the frequency and amplitude of glutamatergic spontaneous excitatory postsynaptic currents (EPSCs). DA also significantly decreased the frequency of miniature EPSCs in the presence of TTX. These results suggest that DA has both presynaptic and postsynaptic inhibitory actions on synaptic transmission in SG neurons. We showed that DA produced direct inhibitory effects in SG neurons to both noxious and innocuous stimuli to the skin. Furthermore, electrical stimulation of dopaminergic diencephalic spinal neurons (A11), which project to the spinal cord, induced outward current and suppressed the frequency and amplitude of EPSCs. We conclude that the dopaminergic descending pathway has an antinociceptive effect via D2-like receptors on SG neurons in the spinal cord.

Excitation of rat spinal ventral horn neurons by purinergic P2X and P2Y receptor activation.

ATPgammaS, a nonhydrolyzable ATP analog, was found to dose-dependently generate an inward current at a holding potential of -70 mV (EC(50)=43 microM) in lamina IX neurons of rat spinal cord slices using the whole-cell patch-clamp technique. This inward current had an extrapolated reversal potential of -9 mV and was resistant to the Na(+)-channel blocker tetrodotoxin, glutamate-receptor antagonists or nominally Ca(2+)-free medium. ATP gamma S also increased the frequency and amplitude of glutamatergic spontaneous excitatory postsynaptic current (sEPSC); this action was dose-dependent and sensitive to tetrodotoxin. Unlike ATP gamma S, the P2X-receptor agonist, BzATP or alpha,beta-methylene ATP, did not change holding currents, but the current response produced by ATP gamma S disappeared in the presence of the P2-receptor antagonist PPADS. The sEPSC frequency and amplitude increase was observed with alpha,beta-methylene ATP, but not with the P2Y-receptor agonist, 2-methylthio ADP, UTP or UDP. The current response by ATP gamma S was suppressed by the addition of GDP beta S into the patch-pipette solution. As for ATP gamma S, 2-methylthio ADP produced an inward current, while UTP and UDP had no effect on holding currents. The P2Y(1)-receptor antagonist MRS2179 inhibited the ATP gamma S-induced inward current, but did not affect the sEPSC frequency and amplitude increase produced by ATP gamma S. These data indicate that extracellular ATP increases the excitability of lamina IX neurons by membrane depolarization (probably through non-selective cation-channel activation) and spontaneous excitatory transmission enhancement, which may be mediated by P2Y(1) and P2X receptors, respectively. This finding supports the idea that purinergic receptor antagonists provide a therapy for spinal cord injury.

TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase.

We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na(+)-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of L-glutamate.

Inhibitory effects of opioids on compound action potentials in frog sciatic nerves and their chemical structures.

An opioid tramadol more effectively inhibits compound action potentials (CAPs) than its metabolite mono-O-demethyl-tramadol (M1). To address further this issue, we examined the effects of opioids (morphine, codeine, ethylmorphine and dihydrocodeine) and cocaine on CAPs by applying the air-gap method to the frog sciatic nerve. All of the opioids at concentrations less than 10 mM reduced the peak amplitude of the CAP in a reversible and dose-dependent manner. The sequence of the CAP peak amplitude reductions was ethylmorphine>codeine>dihydrocodeine> or = morphine; the effective concentration for half-maximal inhibition (IC(50)) of ethylmorphine was 4.6 mM. All of the CAP inhibitions by opioids were resistant to a non-specific opioid-receptor antagonist naloxone. The CAP peak amplitude reductions produced by morphine, codeine and ethylmorphine were related to their chemical structures in such that this extent enhanced with an increase in the number of -CH(2) in a benzene ring, as seen in the inhibitory actions of tramadol and M1. Cocaine reduced CAP peak amplitudes with an IC(50) value of 0.80 mM. It is concluded that opioids reduce CAP peak amplitudes in a manner being independent of opioid-receptor activation and with an efficacy being much less than that of cocaine. It is suggested that the substituted groups of -OH bound to the benzene ring of morphine, codeine and ethylmorphine as well as of tramadol and M1, the structures of which are quite different from those of the opioids, may play an important role in producing nerve conduction block.

Adenosine modulates excitatory synaptic transmission and suppresses neuronal death induced by ischaemia in rat spinal motoneurones.

Although adenosine is an important neuromodulator, its role in modulating motor functions at the level of the spinal cord is poorly understood. In the present study, we investigated the effects of adenosine on excitatory synaptic transmission and neuronal death induced by experimental ischaemia by using whole-cell patch-clamp recordings from lamina IX neurones in spinal cord slices. Adenosine significantly decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in almost all neurones examined that could be mimicked by an A(1) receptor agonist, N (6)-cyclopentyladenosine (CPA), and inhibited by an A(1) receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). Interestingly, adenosine increased mEPSC frequency in the presence of DPCPX in a subpopulation of neurones. In these neurones, an A(2A) receptor agonist, 2-[4-(2-carbonylethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), increased mEPSC frequency. Adenosine also induced an outward current that was blocked by the addition of Cs(+) and tetraethylammonium into the patch-pipette solution and inhibited in the presence of Ba(2+). The adenosine-induced outward current was mimicked by CPA, but not CGS21680, and inhibited by DPCPX. Moreover, superfusing with ischaemia simulating medium (ISM) generated an agonal inward current in all of the neurones tested. The latencies of the inward currents induced by ISM were significantly prolonged by adenosine or CPA, but not by CGS21680. These results suggest that adenosine receptors are functionally expressed in both the pre- and postsynaptic sites of lamina IX neurones and that their activation may exert multiple effects on motor function. Moreover, this study has provided a cellular basis for an involvement of A(1) receptors in the neuroprotective actions of adenosine.

Activation of GIRK channels in substantia gelatinosa neurones of the adult rat spinal cord: a possible involvement of somatostatin.

Recent studies have suggested that spinal G-protein-coupled, inwardly rectifying K(+) (GIRK) channels play an important role in thermal nociception and the analgesic actions of morphine and other agents. In this study, we show that spinal GIRK channels are activated by an endogenous neurotransmitter using whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in adult rat spinal cord slices. Although repetitive stimuli applied to the dorsal root did not induce any slow responses, ones focally applied to the spinal dorsal horn produced slow inhibitory postsynaptic currents (IPSCs) at a holding potential of -50 mV in about 30% of the SG neurones recorded. The amplitude and duration of slow IPSCs increased with the number of stimuli and decreased with removal of Ca(2+) from the external Krebs solution. Slow IPSCs were associated with an increase in membrane conductance; their polarity was reversed at a potential close to the equilibrium potential for K(+), calculated from the Nernst equation. Slow IPSCs were blocked by addition of GDP-beta-S into the patch-pipette solution, reduced in amplitude in the presence of Ba(2+), and significantly suppressed in the presence of an antagonist of GIRK channels, tertiapin-Q. Somatostatin produced an outward current in a subpopulation of SG neurones and the slow IPSC was occluded during the somatostatin-induced outward current. Moreover, slow IPSCs were significantly inhibited by the somatostatin receptor antagonist cyclo-somatostatin. These results suggest that endogenously released somatostatin may induce slow IPSCs through the activation of GIRK channels in SG neurones; this slow synaptic transmission might play an important role in spinal antinociception.