Megumu Yoshimura

Anti-hyperalgesic effects of calcitonin on neuropathic pain interacting with its peripheral receptors

BackgroundThe polypeptide hormone calcitonin is clinically well known for its ability to relieve neuropathic pain such as spinal canal stenosis, diabetic neuropathy and complex regional pain syndrome. Mechanisms for its analgesic effect, however, remain unclear. Here we investigated the mechanism of anti-hyperalgesic action of calcitonin in a neuropathic pain model in rats.ResultsSubcutaneous injection of elcatonin, a synthetic derivative of eel calcitonin, relieved hyperalgesia induced by chronic constriction injury (CCI). Real-time reverse transcriptase-polymerase chain reaction analysis revealed that the CCI provoked the upregulation of tetrodotoxin (TTX)-sensitive Nav.1.3 mRNA and downregulation of TTX-resistant Nav1.8 and Nav1.9 mRNA on the ipsilateral dorsal root ganglion (DRG), which would consequently increase the excitability of peripheral nerves. These changes were reversed by elcatonin. In addition, the gene expression of the calcitonin receptor and binding site of 125I-calcitonin was increased at the constricted peripheral nerve tissue but not at the DRG. The anti-hyperalgesic effect and normalization of sodium channel mRNA by elcatonin was parallel to the change of the calcitonin receptor expression. Elcatonin, however, did not affect the sensitivity of nociception or gene expression of sodium channel, while it suppressed calcitonin receptor mRNA under normal conditions.ConclusionsThese results suggest that the anti-hyperalgesic action of calcitonin on CCI rats could be attributable to the normalization of the sodium channel expression, which might be exerted by an unknown signal produced at the peripheral nerve tissue but not by DRG neurons through the activation of the calcitonin receptor. Calcitonin signals were silent in the normal condition and nerve injury may be one of triggers for conversion of a silent to an active signal.

Effects of baclofen on mechanical noxious and innocuous transmission in the spinal dorsal horn of the adult rat: in vivo patch-clamp analysis.

The effects of a GABAB agonist, baclofen, on mechanical noxious and innocuous synaptic transmission in the substantia gelatinosa (SG) were investigated in adult rats with the in vivo patch‐clamp technique. Under current‐clamp conditions, perfusion with baclofen (10 μm) on the surface of the spinal cord caused hyperpolarisation of SG neurons and a decrease in the number of action potentials elicited by pinch and touch stimuli applied to the receptive field of the ipsilateral hindlimb. The suppression of action potentials was preserved under blockade of postsynaptic G‐proteins, although baclofen‐induced hyperpolarisation was completely blocked. These findings suggest presynaptic effects of baclofen on the induced action potentials. Under voltage‐clamp conditions, application of baclofen reduced the frequency, but not the amplitude, of miniature excitatory postsynaptic currents (mEPSCs), whereas the GABAB receptor antagonist CGP55845 increased the frequency of mEPSCs without affecting the amplitude. Furthermore, application of a GABA uptake inhibitor, nipecotic acid, decreased the frequency of mEPSCs; this effect was blocked by CGP55845, but not by the GABAA antagonist bicuculline. Both the frequency and the amplitude of the pinch‐evoked barrage of excitatory postsynaptic currents (EPSCs) were suppressed by baclofen in a dose‐dependent manner. The frequency and amplitude of touch‐evoked EPSCs was also suppressed by baclofen, but the suppression was significantly smaller than that of pinch‐evoked EPSCs. We conclude that mechanical noxious transmission is presynaptically blocked through GABAB receptors in the SG, and is more effectively suppressed than innocuous transmission, which may account for a part of the mechanism of the efficient analgesic effects of baclofen.

Enhancement of GABAergic tonic currents by midazolam and noradrenaline in rat substantia gelatinosa neurons in vitro

Background:Substantia gelatinosa of the spinal dorsal horn is crucial for transmission and modification of noxious stimuli. Previous studies have demonstrated that intrathecal midazolam, a benzodiazepine agonist, enhanced perioperative analgesia. Not only synaptic but also extrasynaptic inhibitory currents contribute to modification of noxious stimuli. Thus, the effects of midazolam on extrasynaptic &ggr;-aminobutyric acid (GABA) type A receptors in substantia gelatinosa neurons and interaction with noradrenaline, a transmitter of the descending inhibitory systems, were investigated. Methods:Using whole cell patch-clamp technique in the adult rat spinal cord slices, extrasynaptic GABAergic currents were recorded in substantia gelatinosa neurons in the presence of gabazine (1 &mgr;m), which blocked synaptic GABAergic currents, and then midazolam (5 &mgr;m) and noradrenaline (20 &mgr;m) were applied. Results:Bath application of midazolam induced tonic outward currents in the presence of gabazine. Although the decay time of synaptic current was prolonged, neither frequency nor amplitude was affected by midazolam. In contrast, the application of noradrenaline markedly increased both frequency and amplitude of synaptic currents with a slight enhancement of tonic currents. Coapplication of noradrenaline and midazolam markedly increased tonic currents, and the increase was much greater than the sum of currents induced by noradrenaline and midazolam. Conclusions:Midazolam had much larger effects on extrasynaptic GABA type A receptors than the synaptic receptors, suggesting a role of the enhancement of GABAergic extrasynaptic currents in the midazolam-induced analgesia. Because noradrenaline is shown to increase extrasynaptic GABA concentration, simultaneous administration of noradrenaline and midazolam may enhance the increased GABA action by midazolam, thereby resulting in an increase in tonic extrasynaptic currents.

Attenuation of inflammatory and neuropathic pain behaviors in mice through activation of free fatty acid receptor GPR40

BackgroundThe G-protein-coupled receptor 40 (GPR40) is suggested to function as a transmembrane receptor for medium- to long-chain free fatty acids and is implicated to play a role in free fatty acids-mediated enhancement of glucose-stimulated insulin secretion from pancreas. However, the functional role of GPR40 in nervous system including somatosensory pain signaling has not been fully examined yet.ResultsIntrathecal injection of GPR40 agonist (MEDICA16 or GW9508) dose-dependently reduced ipsilateral mechanical allodynia in CFA and SNL models and thermal hyperalgesia in carrageenan model. These anti-allodynic and anti-hyperalgesic effects were almost completely reversed by a GPR40 antagonist, GW1100. Immunohistochemical analysis revealed that GPR40 is expressed in spinal dorsal horn and dorsal root ganglion neurons, and immunoblot analysis showed that carrageenan or CFA inflammation or spinal nerve injury resulted in increased expression of GPR40 in these areas. Patch-clamp recordings from spinal cord slices exhibited that bath-application of either MEDICA16 or GW9508 significantly decreased the frequency of spontaneous excitatory postsynaptic currents in the substantia gelatinosa neurons of the three pain models.ConclusionsOur results indicate that GPR40 signaling pathway plays an important suppressive role in spinal nociceptive processing after inflammation or nerve injury, and that GPR40 agonists might serve as a new class of analgesics for treating inflammatory and neuropathic pain.

Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors

BackgroundThe phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund’s adjuvant (CFA) models.ResultsWe have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1-100 pmol) or IC261 (0.1-1xa0nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1xa0μM) or IC261 (1xa0μM) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models.ConclusionsThese results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.

Direct Effect of Remifentanil and Glycine Contained in Ultiva® on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses.

Background Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord. Methods We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli. Results Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine. Conclusions We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous administrations.

Excitatory effect of Neurotropin® on noradrenergic neurons in rat locus coeruleus

AIMSnAlthough the clinical use of Neurotropin® as an analgesic for chronic pain has been firmly established, its analgesic mechanism is still unclear. In this study, we investigate the direct effects of Neurotropin using an electrophysiological method.nnnMAIN METHODSnBlind patch-clamp recordings were made from rat locus coeruleus (LC) and periaqueductal gray (PAG) neurons in brainstem slices of normal rats. The effects of intracerebroventricular (icv) injection of Neurotropin on nociceptive transmission were recorded from spinal substantia gelatinosa (SG) neurons in fifth lumbar spinal nerve-ligated (L5-SNL) rats using an in vivo patch-clamp method.nnnKEY FINDINGSnNeurotropin (0.2–1.0 NU/mL) dose-dependently increased the firing rate in noradrenergic LC neurons of normal rats. Under the voltage-clamp condition, Neurotropin induced an inward current in 90% of LC neurons thatwas not affected by tetrodotoxin or an injection of GDP-β-S (G protein inhibitor) through recording pipettes. In contrast, Neurotropin had no effects on all PAG neurons tested. Using in vivo patch-clamp recordings, the icv injection of Neurotropin inhibited both frequency and amplitude of pinch-evoked excitatory postsynaptic currents of SG neurons in L5-SNL rats. These results suggest that Neurotropin directly excites the descending noradrenergic LC neurons and inhibits nociceptive transmission in the spinal dorsal horn.nnnSIGNIFICANCEnThis study is the first direct demonstration that Neurotropin activates the noradrenergic descending pain inhibitory systems, and this would reinforce the usefulness of Neurotropin in the treatment of human neuropathic pain.

Functional role of the TRPV1 receptor expressed at the terminals of primary afferent C fibers in the spinal dorsal horn

Cbln1 is a newly identified synaptic organizer belonging to the C1q family. In the cerebellum, Cbln1 is released from granule cells and plays an essential role in the formation and maintenance of excitatory synapses between granule cells and Purkinje cells. Cbln1 binds to neurexin (NRX) expressed on the presynaptic site and the 2 glutamate receptor (GluD2) at the postsynaptic site, acting on both preand post-synaptic sites by forming the NRX/Cbln1/GluD2 tripartite complex at the synaptic cleft. Although Cbln1 and its family members Cbln2 and Cbln4 are expressed in brain regions other than the cerebellum, whether and how they regulate synapse formation in these brain regions remains unclear. In addition, how Cbln family proteins are released from presynaptic neurons is uncharacterized. In this study, we show that Cbln1 and Cbln2, but not Cbln4, specifically bind to its presynaptic receptor NRXs and postsynaptic receptor the 1 glutamate receptor (GluD1), and induced synaptogenesis in hippocampal and cortical neurons in vitro. Cbln1 competed with synaptogenesis mediated by neuroligin 1, which lacks the splice sites A and B, but not leucine-rich repeat transmembrane protein 2, possibly by sharing the presynaptic receptor NRXs. Interestingly, unlike neurexins/neuroligins or neurexins/leucine-rich repeat transmembrane proteins, the interaction between NRX and Cbln1 was insensitive to extracellular Ca2+ concentrations. These findings revealed the unique and general roles of Cbln family proteins in mediating the formation and maintenance of synapses not only in the cerebellum but also in various other brain regions. Research fund: CREST.

In vivo analysis of 5-HT-inudced pruritic response in rat DRG C neurons

Itching is a common symptom in dermatologic diseases and causes restless scratching of the skin, which aggravates the condition. The mechanism of the itch sensation is, however, enigmatic. The present study included behavioral tests and electrophysiological recordings from rat dorsal root ganglion (DRG) neurons in vivo to analyze the response to pruritic stimuli induced by topical application of 5-HT to the skin. Topically applied 5-HT to the rostral back evoked scratching, which was decreased by injection of the opioid receptor antagonist naloxone, suggesting that the 5-HT-mediated scratching was mediated by itch but not pain sensation. To elucidate the firing properties of DRG neurons in response to topically applied 5-HT, intracellular recordings were made from DRG neurons in vivo. None of the A-beta and A-delta neurons responded to 5-HT; in contrast, 27% of C neurons exhibited repetitive firing to 5-HT. The responses could be classified into two firing patterns. One was a transient type, characterized by low firing frequency that decreased within 5 min. The other was a long-lasting type, having high firing frequency that continued increased after 5 min. The time course of the firing pattern of long-lasting C neurons was consistent with the scratching behavior. Intriguingly, the long-lasting type neurons has a significantly smaller fast afterhyperpolarization than that of the 5-HT-insensitive neurons. These observations suggest that the long lasting firing C neurons in rat DRG sensitive to 5-HT are responsible for conveying pruritic information to the spinal cord.