L-bupivacaine Inhibition of Nociceptive Transmission in Rat Peripheral and Dorsal Horn Neurons.

BACKGROUND: Although the widely used single L-enantiomers of local anesthetics have less toxic effects on the cardiovascular and central nervous systems, the mechanisms mediating their antinociceptive actions are not well understood. The authors hypothesized that significant differences in the ion channel blocking abilities of the enantiomers of bupivacaine would be identified. METHODS: The authors performed electrophysiologic analysis on rat dorsal root ganglion neurons in vitro and on spinal transmissions in vivo. RESULTS: In the dorsal root ganglion, these anesthetics decreased the amplitudes of action potentials. The half-maximum inhibitory concentrations of D-enantiomer D-bupivacaine were almost equal for Aß (29.5 µM), Aδ (29.7µM), and C (29.8 µM) neurons. However, the half-maximum inhibitory concentrations of L-bupivacaine was lower for Aδ (19.35 µM) and C (19.5 µM) neurons than for A ß (79.4 µM) neurons. Moreover, D-bupivacaine almost equally inhibited tetrodotoxin-resistant (mean ± SD: 15.8 ± 10.9% of the control, n = 14, P < 0.001) and tetrodotoxin-sensitive (15.4 ± 15.6% of the control, n = 11, P = 0.004) sodium currents. In contrast, L-bupivacaine suppressed tetrodotoxin-resistant sodium currents (26.1 ± 19.5% of the control, n = 18, P < 0.001) but not tetrodotoxin-sensitive sodium currents (74.5 ± 18.2% of the control, n = 11, P = 0.477). In the spinal dorsal horn, L-bupivacaine decreased the area of pinch-evoked excitatory postsynaptic currents (39.4 ± 11.3% of the control, n = 7, P < 0.001) but not touch-evoked responses (84.2 ± 14.5% of the control, n = 6, P = 0.826). In contrast, D-bupivacaine equally decreased pinch- and touch-evoked responses (38.8 ± 9.5% of the control, n = 6, P = 0.001, 42.9 ± 11.8% of the control, n = 6, P = 0.013, respectively). CONCLUSIONS: These results suggest that the L-enantiomer of bupivacaine (L-bupivacaine) effectively inhibits noxious transmission to the spinal dorsal horn by blocking action potential conduction through C and Aδ afferent fibers.

Analyses of the Mode of Action of an Alpha-Adrenoceptor Blocker in Substantia Gelatinosa Neurons in Rats.

To elucidate why naftopidil increases the frequency of spontaneous synaptic currents in only some substantia gelatinosa (SG) neurons, post-hoc analyses were performed. Blind patch-clamp recording was performed using slice preparations of SG neurons from the spinal cords of adult rats. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were recorded. The ratios of the frequency and amplitude of the sIPSCs and sEPSCs following the introduction of naftopidil compared with baseline, and after the application of naftopidil, serotonin (5-HT), and prazosin, compared with noradrenaline (NA) were evaluated. First, the sIPSC analysis indicated that SG neurons reached their full response ratio for NA at 50 µM. Second, they responded to 5-HT (50 µM) with a response ratio similar to that for NA, but prazosin (10 µM) did not change the sEPSCs and sIPSCs. Third, the highest concentration of naftopidil (100 µM) led to two types of response in the SG neurons, which corresponded with the reactions to 5-HT and prazosin. These results indicate that not all neurons were necessarily activated by naftopidil, and that the micturition reflex may be regulated in a sophisticated manner by inhibitory mechanisms in these interneurons.

Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo.

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem. The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood. Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group. Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Mechanisms of the analgesic effect of calcitonin on chronic pain by alteration of receptor or channel expression.

The polypeptide hormone calcitonin is well known clinically for its ability to relieve osteoporotic back pain and neuropathic pain such as spinal canal stenosis, diabetic neuropathy, chemotherapy-induced neuropathy, and complex regional pain syndrome. Because the analgesic effects of calcitonin have a broad range, the underlying mechanisms of pain relief by calcitonin are largely unknown. However, recent studies using several types of chronic pain models combined with various methods have been gradually clarifying the mechanism. Here, we review the mechanisms of the analgesic action of calcitonin on ovariectomy-induced osteoporotic and neuropathic pain. The analgesic action of calcitonin may be mediated by restoration of serotonin receptors that control selective glutamate release from C-afferent fibers in ovariectomized rats and by normalization of sodium channel expression in damaged peripheral nerves. Serotonin receptors are reduced or eliminated by the relatively rapid reduction in estrogen during the postmenopausal period, and damaged nerves exhibit hyperexcitability due to abnormal expression of Na+ channel subtypes. In addition, in chemotherapy-induced peripheral neuropathy, inhibition of signals related to transient receptor potential ankyrin-1 and melastatin-8 is proposed to participate in the anti-allodynic action of calcitonin. Further, an unknown calcitonin-dependent signal appears to be present in peripheral nervous tissues and may be activated by nerve injury, resulting in regulation of the excitability of primary afferents by control of sodium channel transcription in dorsal root ganglion neurons. The calcitonin signal in normal conditions may be non-functional because no target is present, and ovariectomy or nerve injury may induce a target. Moreover, it has been reported that calcitonin reduces serotonin transporter but increases serotonin receptor expression in the thalamus in ovariectomized rats. These data suggest that calcitonin could alleviate lower back pain in patients with osteoporosis or neuropathic pain by the alteration in receptor or channel expression.

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

BACKGROUND: The 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. RESULTS: Intrathecal 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. CONCLUSIONS: Our 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.

BACKGROUND: The 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. RESULTS: We 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 (0.1-1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 µM) or IC261 (1 µM) 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. CONCLUSIONS: These 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.

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.

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

BACKGROUND: The 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. RESULTS: Subcutaneous 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. CONCLUSIONS: These 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.

Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal dorsal horn in vitro.

BACKGROUND: 5-hydroxytryptamine (5-HT) is one of the major neurotransmitters widely distributed in the CNS. Several 5-HT receptor subtypes have been identified in the spinal dorsal horn which act on both pre- and postsynaptic sites of excitatory and inhibitory neurons. However, the receptor subtypes and sites of actions as well as underlying mechanism are not clarified rigorously. Several electrophysiological studies have been performed to investigate the effects of 5-HT on excitatory transmission in substantia gelatinosa (SG) of the spinal cord. In the present study, to understand the effects of 5-HT on the inhibitory synaptic transmission and to identify receptor subtypes, the blind whole cell recordings were performed from SG neurons of rat spinal cord slices. RESULTS: Bath applied 5-HT (50 µM) increased the frequency but not amplitudes of spontaneous inhibitory postsynaptic currents (sIPSCs) in 58% of neurons, and both amplitude and frequency in 23% of neurons. The frequencies of GABAergic and glycinergic mIPSCs were both enhanced. TTX (0.5 µM) had no effect on the increasing frequency, while the enhancement of amplitude of IPSCs was eliminated. Evoked-IPSCs (eIPSCs) induced by focal stimulation near the recording neurons in the presence of CNQX and APV were enhanced in amplitude by 5-HT. In the presence of Ba(2+) (1 mM), a potassium channel blocker, 5-HT had no effect on both frequency and amplitude. A 5-HT(2A) receptor agonist, TCB-2 mimicked the 5-HT effect, and ketanserin, an antagonist of 5-HT(2A) receptor, inhibited the effect of 5-HT partially and TCB-2 almost completely. A 5-HT(2C) receptor agonist WAY 161503 mimicked the 5-HT effect and this effect was blocked by a 5-HT(2C) receptor antagonist, N-desmethylclozapine. The amplitudes of sIPSCs were unaffected by 5-HT(2A) or 5-HT(2C) agonists. A 5-HT(3) receptor agonist mCPBG enhanced both amplitude and frequency of sIPSCs. This effect was blocked by a 5-HT(3) receptor antagonist ICS-205,930. The perfusion of 5-HT(2B) receptor agonist had no effect on sIPSCs. CONCLUSIONS: Our results demonstrated that 5-HT modulated the inhibitory transmission in SG by the activation of 5-HT(2A) and 5-HT(2C) receptors subtypes located predominantly at inhibitory interneuron terminals, and 5-HT(3) receptors located at inhibitory interneuron terminals and soma-dendrites, consequently enhanced both frequency and amplitude of IPSCs.

Organization of intralaminar and translaminar neuronal connectivity in the superficial spinal dorsal horn.

The spinal dorsal horn exhibits a high degree of intrinsic connectivity that is critical to its role in the processing of nociceptive information. To examine the spatial organization of this intrinsic connectivity, we used laser-scanning photostimulation in parasagittal and transverse slices of lumbar spinal cord to stimulate presynaptic neurons by glutamate uncaging, and mapped the location of sites that provide excitatory and inhibitory synaptic input to neurons of the superficial laminae. Excitatory interneuronal connectivity within lamina II exhibited a pronounced sagittal orientation, in keeping with the somatotopic organization present in the pattern of primary afferent projections. Excitatory inputs to all classes of lamina II neurons arose from a wider rostrocaudal area than inhibitory inputs, whereas both excitatory and inhibitory input zones were restricted mediolaterally. Lamina I-II neurons exhibited cell type-specific patterns in the laminar distribution of their excitatory inputs that were related to their dorsoventral dendritic expanse. All cell types received excitatory input predominantly from positions ventral to that of their soma, but in lamina I neurons and lamina II vertical cells this ventral displacement of the excitatory input zone was greater than in the other cell types, resulting in a more pronounced translaminar input pattern. A previously unknown excitatory input to the superficial dorsal horn from lamina III-IV was identified in a subset of the vertical cell population. These results reveal a specific three-dimensional organization in the local patterns of excitatory and inhibitory connectivity that has implications for the processing of information related to both somatotopy and sensory modality.

Responsiveness of C neurons in rat dorsal root ganglion to 5-hydroxytryptamine-induced pruritic stimuli in vivo.

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, however, is 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-hydroxytryptamine (5-HT) to the skin. Topically applied 5-HT to the rostral back evoked scratching, whereas application of the vehicle did not. Following subcutaneous injection of the opioid receptor antagonist naloxone, the number of scratches decreased, suggesting that the scratching was preferentially 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 Abeta and Adelta neurons responded to 5-HT; in contrast, 25 of 91 C neurons (27%) exhibited repetitive firing in response to 5-HT, which 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 increasing after 5 min. The time course of the firing pattern of long-lasting C neurons was comparable to the scratching behavior. Intriguingly, the long-lasting-type neurons had 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.

Bone cancer induces a unique central sensitization through synaptic changes in a wide area of the spinal cord.

BACKGROUND: Chronic bone cancer pain is thought to be partly due to central sensitization. Although murine models of bone cancer pain revealed significant neurochemical changes in the spinal cord, it is not known whether this produces functional alterations in spinal sensory synaptic transmission. In this study, we examined excitatory synaptic responses evoked in substantia gelatinosa (SG, lamina II) neurons in spinal cord slices of adult mice bearing bone cancer, using whole-cell voltage-clamp recording techniques. RESULTS: Mice at 14 to 21 days after sarcoma implantation into the femur exhibited hyperalgesia to mechanical stimuli applied to the skin of the ipsilateral hind paw, as well as showing spontaneous and movement evoked pain-related behaviors. SG neurons exhibited spontaneous excitatory postsynaptic currents (EPSCs). The amplitudes of spontaneous EPSCs were significantly larger in cancer-bearing than control mice without any changes in passive membrane properties of SG neurons. In the presence of TTX, the amplitude of miniature EPSCs in SG neurons was increased in cancer-bearing mice and this was observed for cells sampled across a wide range of lumbar segmental levels. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor- and N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs evoked by focal stimulation were also enhanced in cancer-bearing mice. Dorsal root stimulation elicited mono- and/or polysynaptic EPSCs that were caused by the activation of Adelta and/or C afferent fibers in SG neurons from both groups of animals. The number of cells receiving monosynaptic inputs from Adelta and C fibers was not different between the two groups. However, the amplitude of the monosynaptic C fiber-evoked EPSCs and the number of SG neurons receiving polysynaptic inputs from Adelta and C fibers were increased in cancer-bearing mice. CONCLUSIONS: These results show that spinal synaptic transmission mediated through Adelta and C fibers is enhanced in the SG across a wide area of lumbar levels following sarcoma implantation in the femur. This widespread spinal sensitization may be one of the underlying mechanisms for the development of chronic bone cancer pain.

TRPA1-expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord.

The TRPA1 channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using whole-cell patch-clamp recordings and morphological analyses in adult rat spinal cord slices. Cinnamaldehyde (TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons that responded to allyl isothiocyanate (less specific TRPA1 agonist) and capsaicin (TRPV1 agonist). Cinnamaldehyde evoked EPSCs in vertical and radial but not islet or central SG cells. Notably, cinnamaldehyde produced no change in inhibitory postsynaptic currents and nor did it produce direct postsynaptic effects. In the presence of tetrodotoxin, cinnamaldehyde increased the frequency but not amplitude of miniature EPSCs. Intriguingly, cinnamaldehyde had a selective inhibitory action on monosynaptic C- (but not Adelta-) fiber-evoked EPSCs. These results indicate that activation of spinal TRPA1 presynaptically facilitates miniature excitatory synaptic transmission from primary afferents onto vertical and radial cells to initiate action potentials. The presence of TRPA1 channels on the central terminals raises the possibility of bidirectional modulatory action in morphologically identified subclasses of SG neurons.

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 gamma-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 microm), which blocked synaptic GABAergic currents, and then midazolam (5 microm) and noradrenaline (20 microm) 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.

Comparative study on the actions of toxin extracts from two different puffer fishes on I(Na) and respiratory N-M transmission in the rat.

We performed a comparative study on the effects of toxin extracts prepared from muscle and liver of two different puffer fishes on voltage dependent sodium current (I(Na)), and compared the results with that of tetrodotoxin (TTX). The amount of toxin contained in the muscle or liver expressed as an amount of equipotent TTX differed in the two species (0.11-57.98 microg TTX/g tissue). In addition, we observed the effects of TTX or toxin extracts on the twitch contraction evoked by direct muscle stimulation of the rat hemidiaphragm or indirect phrenic nerve stimulations, in an attempt to understand the mechanisms involved in the transmission failure in the respiratory muscles, due to the ingestion of TTX bearing puffers, and found that TTX or toxin extracts preferentially affect motor nerve rather than muscle.

Differential Effects of Alpha 1-Adrenoceptor Antagonists on the Postsynaptic Sensitivity: Using Slice Patch-Clamp Technique for Inhibitory Postsynaptic Current in Substantia Gelatinosa Neurons From Lumbosacral Spinal Cord in Rats.

PURPOSE: Alpha1-adrenoceptors participate in improving storage symptoms of male lower urinary tract symptoms. However, the mechanism of action of these compounds remains unclear. The goal of the present study was to clarify the effect of α1- adrenoceptor antagonists on γ-aminobutyric acid (GABA)/glycine-mediated outward currents of the inhibitory postsynaptic current (IPSC) in substantia gelatinosa (SG) neurons from the lumbosacral spinal cord in rats. METHODS: Male adult Sprague-Dawley rats were used. Blind whole-cell patch-clamp recordings were performed in SG neurons from isolated spinal cord slice preparations. IPSCs were recorded in individual SG neurons to which naftopidil (100µM), tamsulosin (100µM), silodosin (30µM), or prazosin (10µM) were applied sequentially with intervening washout periods. Strychnine (2µM), bicuculline (10µM), or tetrodotoxin (TTX)(1µM) were added before naftopidil. Individual outward currents were analyzed. RESULTS: The bath application of naftopidil, yielded outward IPSCs in 13 of 52 SG neurons. The naftopidil response was unchanged in the presence of TTX. Regression analysis of the outward currents between the 1st and 2nd applications of naftopidil revealed a Pearson correlation coefficient of 0.996 with a line slope of 0.983. The naftopidil-induced outward current was attenuated in the presence of strychnine and/or bicuculline. The GABA/glycine-mediated outward currents induced by tamsulosin, silodosin, and prazosin were smaller than those obtained with naftopidil. CONCLUSION: Naftopidil-induced GABA/glycine-mediated outward currents in a subset of SG neurons prepared from the L6- S1 level of rat spinal cord. The results indicated that α1-adrenoceptor antagonists, particularly naftopidil, induce neural suppression (in part) by mediating hyperpolarization. The response is associated with glycinergic and/or GABAergic neural transmission. Naftopidil may suppress the micturition reflex and improve urinary storage symptoms as a subsidiary effect resulting from hyperpolarization in SG neurons of the spinal cord.

Effect of Alpha 1-Adrnoceptor Antagonists on Postsynaptic Sensitivity in Substantia Gelatinosa Neurons From Lumbosacral Spinal Cord in Rats Using Slice Patch-Clamp Technique for mEPSC.

PURPOSE: Alpha1-adrenoceptors participate in improving storage symptoms of male lower urinary tract symptoms (LUTS). However, the mechanism of action of these compounds remains unclear. To clarify the mechanism of the α1-adrenoceptor antagonists, the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was analyzed in the lumbosacral spinal cord in rats. METHODS: Male adult Sprague-Dawley rats were used. Blind whole-cell patch-clamp recordings were performed on substantia gelatinosa (SG) neurons in spinal cord slice preparations. The amplitude of mEPSCs was recorded in individual SG neurons to which α1-adrenoceptors (100µM naftopidil, 100µM tamsulosin, and 30µM silodosin) were applied sequentially with intervening washout periods. Individual amplitudes were analyzed. RESULTS: Pearson correlation coefficients (r) for the amplitudes of mEPSCs between the baseline and postadministration of α1- adrenoceptor antagonists indicated changes of the amplitude ranked in the order of naftopidil (r =0.393), tamsulosin (r=0.738), and silodosin (r=0.944). Together, the α1-adrenoceptor antagonists yielded significant increases in the amplitude of mEPSCs in SG neurons (n=108, P=0.012). However, the effects of each α1-adrenoceptor antagonist on the amplitude were as follows (relative to the baseline; n=36 each): naftopidil, P=0.129; tamsulosin, P=0.201; and silodosin, P=0.005. The rate of response to naftopidil for the outward current was relatively high among the α1-adrenoceptor blockers. An inward current was observed only with the naftopidil application. CONCLUSION: Alpha1-adrenoceptor antagonists changed the amplitudes of mEPSCs in a subset of SG neurons in slices prepared from the L6-S1 levels of rat spine. Although the α1-adrenoceptor antagonists generated inward or outward currents in the SG neurons, different rates of response were observed with each antagonist. These results are important for understanding the mechanisms of action (at the spinal level) of α1-adrenoceptor antagonists for the storage symptoms of male LUTS.

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model.

BACKGROUND: Fibromyalgia patients experience cardiovascular complications in addition to musculoskeletal pain. This study aimed to investigate the cardiac effects of a prolonged shallow water gait in a fibromyalgia-induced young mouse model. METHODS: To produce a fibromyalgia mouse model, wild-type mice were administered an intraperitoneal injection of reserpine once a day for three days, and two primary experiments were performed. First, three types of gait tests were performed before and after the reserpine injections as follows: (i) 5 minutes of free gait outside the water, (ii) 1 minute of free gait in shallow warm water, and (iii) 5 minutes of free gait in shallow warm water. Second, electrocardiogram recordings were taken before and after the three gait tests. The average heart rate and heart rate irregularity scores were analyzed. RESULTS: Exercise-induced cardiac arrhythmia was observed at 1-minute gait in shallow water during the acute stage of induced FM in young mice. Further, both cardiac arrhythmia and a decrease in HR have occurred at 5-minute gait in shallow water at the same mice. However, this phenomenon was not observed in the wild-type mice under any test conditions. CONCLUSION: Although a short-term free gait in shallow warm water may be advantageous for increasing the motor activity of FM-model mice, we should be aware of the risk of prolonged and excessive exercise-induced cardiac arrhythmia. For gait exercises in shallow water as a treatment in FM patients. We suggest a gradual increase in exercise duration may be warranted.

Free gait in a shallow pool accelerates recovery after exercise in model mice with fibromyalgia.

This study aimed to determine the effect of pool gait exercise using fibromyalgia-induced model mice. The sensory threshold, locomotive behavior, electrocardiogram, and onset time after the gait test in shallow water using male C57BL/6J mice (weight, 30-35 g; n=21) were investigated. To induce fibromyalgia in model mice, reserpine was injected intraperitoneally into wild-type mice once a day for 3 days. Subsequently, the fibromyalgia-induced model mice were randomly classified into two groups as follows: the control group (n=11) and the pool gait group (n=10). The mice in the pool gait group walked in the same cage containing shallow warm water 5 times per week. Both groups underwent sensory thresholds and video recordings to determine locomotive behaviors weekly. Further, both heart rate and video recordings for observation of a recovery after the gait test in shallow water were undertaken (control group; n=5, pool gait group; n=5). The pool gait did not affect sensory thresholds and locomotive behavior; however, in the pool gait group, both the recovery after the test, such as onset time and gait distance, were considerably better than those of the control group. Furthermore, changes in heart rate and heart rate irregularity after the test were more apparent in the control group than in the pool gait group. The free gait in a shallow pool accelerated recovery after exercise, unlike the sensory threshold.

Actions of propofol on substantia gelatinosa neurones in rat spinal cord revealed by in vitro and in vivo patch-clamp recordings.

Propofol, an intravenous general anaesthetic, exerts anaesthetic actions through interaction with gamma-aminobutyric acid type A (GABA(A)) receptors in the supraspinal nervous system. However, whether propofol has any significant effects on synaptic transmission at the spinal level and whether it exhibits antinociceptive action is still not fully clarified. Spontaneous activity and stimulus-evoked responses of substantia gelatinosa (SG) neurones to noxious pinch stimuli were recorded using spontaneously breathing rats under propofol anaesthesia using in vivo whole-cell patch-clamp techniques. Precise actions of propofol on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) as well as excitatory postsynaptic currents (EPSCs) in SG neurones were also analyzed in spinal cord slice preparations. At clinical doses (5 mg/kg), propofol reversibly depressed action potentials elicited by noxious mechanical stimuli applied to the skin in the majority (6/8) of SG neurons recorded under in vivo conditions. This depression may have been caused by interactions of propofol with GABA(A) receptors, as decay time of GABAergic sIPSCs was prolonged after propofol injection (128 +/- 11% of control, n = 5) with minimal effect on EPSCs. Although prolongation of IPSCs in vivo was reversible, IPSCs were progressively prolonged even after washout of propofol when the effect was tested using spinal cord slices. Propofol had a mild depressant effect on Adelta- and C-afferent-mediated EPSCs. We conclude that systemic bolus injection of propofol reversibly depressed nociceptive transmission, at least in part, by enhancing postsynaptic GABA(A) receptor-mediated responses in the SG.