Nerve injury elevates functional Cav3.2 channels in superficial spinal dorsal horn.

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial spinal dorsal horn. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2 but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 knockout mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial spinal dorsal horn may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.

1,8- and 1,4-cineole enhance spontaneous excitatory transmission by activating different types of transient receptor potential channels in the rat spinal substantia gelatinosa.

Although transient receptor potential (TRP) channels expressed in the spinal substantia gelatinosa play a role in modulating nociceptive transmission, their properties have not been fully examined yet. In order to address this issue, the effects of 1,8-cineole and its stereoisomer 1,4-cineole on excitatory transmission were examined by applying the whole-cell patch-clamp technique to substantia gelatinosa neurons in adult rat spinal cord slices. Miniature excitatory postsynaptic current frequency was increased by 1,8- and 1,4-cineole. The cineole activities were repeated and resistant to voltage-gated Na+ -channel blocker tetrodotoxin. The 1,8-cineole activity was inhibited by TRP ankyrin-1 (TRPA1) antagonists (HC-030031 and mecamylamine) but not TRP vanilloid-1 (TRPV1) antagonists (capsazepine and SB-366791), whereas the 1,4-cineole activity was depressed by the TRPV1 but not TRPA1 antagonists. Although 1,8- and 1,4-cineole reportedly activate TRP melastatin-8 (TRPM8) channels, their activities were unaffected by TRPM8 antagonist 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide. Monosynaptically evoked C-fiber, but not Aδ-fiber excitatory postsynaptic current amplitude, was reduced by 1,8- and 1,4-cineole. These results indicate that 1,8- and 1,4-cineole increase spontaneous l-glutamate release from nerve terminals by activating TRPA1 and TRPV1 channels, respectively, while inhibiting C-fiber but not Aδ-fiber evoked l-glutamate release. This difference between 1,8- and 1,4-cineole may serve to know the properties of TRP channels located in the central terminals of primary-afferent neurons. The spinal dorsal horn lamina II (substantia gelatinosa; SG) plays a pivotal role in regulating nociceptive transmission from the periphery. We found out in the SG that 1,4- and 1,8-cineole activate TRPV1 and TRPA1 channels, respectively, located in primary-afferent, possibly C-fiber, central terminals. This difference may serve to know the properties of TRP channels expressed in the central terminals.

Spontaneous L-glutamate release enhancement in rat substantia gelatinosa neurons by (-)-carvone and (+)-carvone which activate different types of TRP channel.

Transient receptor potential (TRP) channels in the spinal dorsal horn lamina II (substantia gelatinosa; SG), which are involved in the modulation of nociceptive transmission, have not yet been fully examined in property. Activation of the TRP channels by various plant-derived chemicals results in an increase in the spontaneous release of L-glutamate onto the SG neurons. We examined the effects of a monoterpene ketone (-)-carvone (contained in spearmint) and its stereoisomer (+)-carvone (in caraway) on glutamatergic spontaneous excitatory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. (-)-Carvone and (+)-carvone increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) in a reversible and concentration-dependent manner with a small increase in its amplitude. Half-maximal effective concentrations of (-)-carvone and (+)-carvone in increasing sEPSC frequency were 0.70 mM and 0.72 mM, respectively. The (-)-carvone but not (+)-carvone activity was inhibited by a TRPV1 antagonist capsazepine. On the other hand, the (+)-carvone but not (-)-carvone activity was inhibited by a TRPA1 antagonist HC-030031. These results indicate that (-)-carvone and (+)-carvone activate TRPV1 and TRPA1 channels, respectively, resulting in an increase in spontaneous L-glutamate release onto SG neurons, with almost the same efficacy. Such a difference in TRP activation between the stereoisomers may serve to know the properties of TRP channels in the SG.

Synaptic modulation and inward current produced by oxytocin in substantia gelatinosa neurons of adult rat spinal cord slices.

Cellular mechanisms for antinociception produced by oxytocin in the spinal dorsal horn have not yet been investigated thoroughly. We examined how oxytocin affects synaptic transmission in substantia gelatinosa neurons, which play a pivotal role in regulating nociceptive transmission, by applying the whole-cell patch-clamp technique to the substantia gelatinosa neurons of adult rat spinal cord slices. Bath-applied oxytocin did not affect glutamatergic spontaneous, monosynaptically-evoked primary-afferent Aδ-fiber and C-fiber excitatory transmissions. On the other hand, oxytocin produced an inward current at -70 mV and enhanced GABAergic and glycinergic spontaneous inhibitory transmissions. These activities were repeated with a slow recovery from desensitization, concentration-dependent and mimicked by oxytocin-receptor agonist. The oxytocin current was inhibited by oxytocin-receptor antagonist, intracellular GDPßS, U-73122, 2-aminoethoxydiphenyl borate, but not dantrolene, chelerythrine, dibutyryl cyclic-AMP, CNQX, Ca(2+)-free and tetrodotoxin, while the spontaneous inhibitory transmission enhancements were depressed by tetrodotoxin. Current-voltage relation for the oxytocin current reversed at negative potentials more than the equilibrium potential for K(+), or around 0 mV. The oxytocin current was depressed in high-K(+), low-Na(+) or Ba(2+)-containing solution. Vasopressin V1A-receptor antagonist inhibited the oxytocin current, but there was no correlation in amplitude between a vasopressin-receptor agonist [Arg(8)]vasopressin and oxytocin responses. It is concluded that oxytocin produces a membrane depolarization mediated by oxytocin but not vasopressin-V1A receptors, which increases neuronal activity, resulting in the enhancement of inhibitory transmission, a possible mechanism for antinociception. This depolarization is due to a change in membrane permeabilities to K(+) and/or Na(+), which is possibly mediated by phospholipase C and inositol 1,4,5-triphosphate-induced Ca(2+)-release.

Zingerone enhances glutamatergic spontaneous excitatory transmission by activating TRPA1 but not TRPV1 channels in the adult rat substantia gelatinosa.

Transient receptor potential (TRP) channels are thought to play a role in regulating nociceptive transmission to spinal substantia gelatinosa (SG) neurons. It remains to be unveiled whether the TRP channels in the central nervous system are different in property from those involved in receiving nociceptive stimuli in the peripheral nervous system. We examined the effect of the vanilloid compound zingerone, which activates TRPV1 channels in the cell body of a primary afferent neuron, on glutamatergic excitatory transmission in the SG neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Bath-applied zingerone reversibly and concentration-dependently increased spontaneous excitatory postsynaptic current (EPSC) frequency. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate receptor antagonists. These zingerone effects were repeated and persisted in Na(+)-channel blocker tetrodotoxin-, La(3+)-, or IP3-induced Ca(2+)-release inhibitor 2-aminoethoxydiphenyl borate-containing or Ca(2+)-free Krebs solution. Zingerone activity was resistant to the selective TRPV1 antagonist capsazepine but sensitive to the nonselective TRP antagonist ruthenium red, the TRPA1 antagonist HC-030031, and the Ca(2+)-induced Ca(2+)-release inhibitor dantrolene. TRPA1 agonist allyl isothiocyanate but not capsaicin inhibited the facilitatory effect of zingerone. On the other hand, zingerone reduced monosynaptically evoked EPSC amplitudes, as did TRPA1 agonists. Like allyl isothiocyanate, zingerone enhanced GABAergic spontaneous inhibitory transmission in a manner sensitive to tetrodotoxin. We conclude that zingerone presynaptically facilitates spontaneous excitatory transmission, probably through Ca(2+)-induced Ca(2+)-release mechanisms, and produces a membrane depolarization in SG neurons by activating TRPA1 but not TRPV1 channels.

Enhancement by interleukin-1ß of AMPA and NMDA receptor-mediated currents in adult rat spinal superficial dorsal horn neurons.

BACKGROUND: Proinflammatory cytokine interleukin-1ß (IL-1ß) released from spinal microglia plays an important role in the maintenance of acute and chronic pain states. However, the cellular basis of this action remains poorly understood. Using whole-cell patch-clamp recordings, we examined the action of IL-1ß on AMPA- and NMDA-receptor-mediated currents recorded from substantia gelatinosa (SG) neurons of adult rat spinal cord slices which are key sites for regulating nociceptive transmission from the periphery. RESULTS: AMPA- and NMDA-induced currents were increased in peak amplitude by IL-1ß in a manner different from each other in SG neurons. These facilitatory actions of IL-1ß were abolished by IL-1 receptor (IL-1R) antagonist (IL-1ra), which by itself had no detectable effects on AMPA- and NMDA-induced currents. The AMPA- but not NMDA-induced current facilitated by IL-1ß was recovered to control level 30 min after IL-1ß washout and largely depressed in Na+-channel blocker tetrodotoxin-containing or nominally Ca2+-free Krebs solution. Minocycline, a microglia inhibitor, blocked the facilitatory effect of IL-1ß on AMPA- but not NMDA-induced currents, where minocycline itself depressed NMDA- but had not any effects on AMPA-induced currents. CONCLUSIONS: IL-1ß enhances AMPA and NMDA responses in SG neurons through IL-1R activation; the former but not latter action is reversible and due to an increase in neuronal activity in a manner dependent on extracellular Ca2+ and minocycline. It is suggested that AMPA and NMDA receptors are positively modulated by IL-1ß in a manner different from each other; the former but not latter is mediated by a neurotransmitter released as a result of an increase in neuronal activity. Since IL-1ß contributes to nociceptive behavior induced by peripheral nerve or tissue injury, the present findings also reveal an important cellular link between neuronal and glial cells in the spinal dorsal horn.

Diagnostic Performance of Transient Elastography Versus Two-Dimensional Shear Wave Elastography for Liver Fibrosis in Chronic Viral Hepatitis: Direct Comparison and a Meta-Analysis.

Objective: This systematic review and meta-analysis aimed to compare the diagnostic performance of transient elastography (TE) and two-dimensional shear wave elastography (2D-SWE) for staging liver fibrosis in patients with chronic viral hepatitis (CVH). Methods: Pubmed, Embase, Web of Science, and Cochrane Library were searched (-01/08/2021) for studies comparing TE with 2D-SWE in patients with CVH. Other etiologies of chronic liver disease (CLD) and articles not published in SCI journals were excluded. The bivariate random-effects model was used to pool the performance of the TE and 2D-SWE. Results: Eight articles with a total of 1301 CVH patients were included. The prevalence of significant fibrosis (fibrosis stage ≥ 2), advanced fibrosis (fibrosis stage ≥ 3), and cirrhosis was 50.8%, 44.8%, and 34.7%, respectively. 2D-SWE expressed higher overall accuracy than TE in detecting significant fibrosis (0.93 vs. 0.85, P = 0.04). No significant difference among the overall diagnostic accuracy of TE and 2D-SWE in staging advanced fibrosis and cirrhosis was found. Conclusion: TE and 2D-SWE express good to excellent diagnostic accuracies to stage fibrosis in CVH patients. 2D-SWE compares favorably with TE especially for predicting significant fibrosis.

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons.

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at -70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 µM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.

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.

Enhancement by citral of glutamatergic spontaneous excitatory transmission in adult rat substantia gelatinosa neurons.

Although citral, which is abundantly present in lemongrass, has various actions including antinociception, how citral affects synaptic transmission has not been examined as yet. Citral activates in heterologous cells transient receptor potential vanilloid-1, ankyrin-1, and melastatin-8 (TRPV1, TRPA1, and TRPM8, respectively) channels, the activation of which in the spinal lamina II [substantia gelatinosa (SG)] increases the spontaneous release of L-glutamate from nerve terminals. It remains to be examined what types of transient receptor potential channel in native neurons are activated by citral. With a focus on transient receptor potential activation, we examined the effect of citral on glutamatergic spontaneous excitatory transmission using the whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. Bath-applied citral for 3 min increased the frequency of spontaneous excitatory postsynaptic current in a concentration-dependent manner (half-maximal effective concentration=0.58 mM), with a small increase in its amplitude. The spontaneous excitatory postsynaptic current frequency increase produced by citral was repeated at a time interval of 30 min, albeit this action recovered with a slow time course after washout. The presynaptic effect of citral was inhibited by TRPA1 antagonist HC-030031, but not by voltage-gated Na-channel blocker tetrodotoxin, TRPV1 antagonist capsazepine, and TRPM8 antagonist BCTC. It is concluded that citral increases spontaneous L-glutamate release in SG neurons by activating TRPA1 channels. Considering that the SG plays a pivotal role in modulating nociceptive transmission from the periphery, the citral activity could contribute toward at least a part of the modulation.

Developmental change and sexual difference in synaptic modulation produced by oxytocin in rat substantia gelatinosa neurons.

We have previously reported that oxytocin produces an inward current at a holding potential of -70 mV without a change in glutamatergic excitatory transmission in adult male rat spinal lamina II (substantia gelatinosa; SG) neurons that play a pivotal role in regulating nociceptive transmission. Oxytocin also enhanced GABAergic and glycinergic spontaneous inhibitory transmissions in a manner sensitive to a voltage-gated Na+-channel blocker tetrodotoxin. These actions were mediated by oxytocin-receptor activation. Such a result was different from that obtained by other investigators in young male rat superficial dorsal horn neurons in which an oxytocin-receptor agonist enhanced glutamatergic and GABAergic but not glycinergic spontaneous transmissions. In order to know a developmental change and also sexual difference in the actions of oxytocin, we examined its effect on spontaneous synaptic transmission in adult female and young male rat SG neurons by using the whole-cell patch-clamp technique in spinal cord slices. In adult female rats, oxytocin produced an inward current at -70 mV without a change in excitatory transmission. GABAergic and glycinergic transmissions were enhanced by oxytocin, the duration of which enhancement was much shorter than in adult male rats. In young (11-21 postnatal days) male rats, oxytocin produced not only an inward but also outward current at -70 mV, and presynaptically inhibited or facilitated excitatory transmission, depending on the neurons tested; both GABAergic and glycinergic transmissions were enhanced by oxytocin. The inhibitory transmission enhancements in adult female and young male rats were sensitive to tetrodotoxin. Although the data may not be enough to be estimated, it is suggested that synaptic modulation by oxytocin in SG neurons, i.e., cellular mechanism for its antinociceptive action, exhibits a developmental change and sexual difference.

[Rebound depolarization of substantia gelatinosa neurons and its modulatory mechanisms in rat spinal dorsal horn].

OBJECTIVE: To investigate the rebound depolarization of substantia gelatinosa (SG) neurons in rat spinal dorsal horn and explore its modulatory mechanisms to provide better insights into rebound depolarization-related diseases. METHODS: Parasagittal slices of the spinal cord were prepared from 3- to 5-week-old Sprague-Dawley rats. The electrophysiologic characteristics and responses to hyperpolarization stimulation were recorded using whole-cell patch-clamp technique. The effects of hyperpolarization-activated cyclic nucleotide gated cation (HCN) channel blockers and T-type calcium channel blockers on rebound depolarization of the neurons were studied. RESULTS: A total of 63 SG neurons were recorded. Among them, 23 neurons showed no rebound depolarization, 19 neurons showed rebound depolarization without spikes, and 21 neurons showed rebound depolarization with spikes. The action potential thresholds of the neurons without rebound depolarization were significantly higher than those of the neurons with rebound depolarization and spikes (-28.7∓1.6 mV vs -36.0∓2.0 mV, P<0.05). The two HCN channel blockers CsCl and ZD7288 significantly delayed the latency of rebound depolarization with spike from 45.9∓11.6 ms to 121.6∓51.3 ms (P<0.05) and from 36.2∓10.3 ms to 73.6∓13.6 ms (P<0.05), respectively. ZD7288 also significantly prolonged the latency of rebound depolarization without spike from 71.9∓35.1 ms to 267.0∓68.8 ms (P<0.05). The T-type calcium channel blockers NiCl2 and mibefradil strongly decreased the amplitude of rebound depolarization with spike from 19.9∓6.3 mV to 9.5∓4.5 mV (P<0.05) and from 26.1∓9.4 mV to 15.5∓5.0 mV (P<0.05), respectively. Mibefradil also significantly decreased the amplitude of rebound depolarization without spike from 14.3∓3.0 mV to 7.9∓2.0 mV (P<0.05). CONCLUSION: Nearly two-thirds of the SG neurons have rebound depolarizations modulated by HCN channel and T-type calcium channel.

Action of thymol on spontaneous excitatory transmission in adult rat spinal substantia gelatinosa neurons.

Thymol, which is contained in thyme essential oil, has various actions including antinociception and nerve conduction inhibition. Although thymol activates transient receptor potential (TRP) channels expressed in heterologous cells, it remains to be examined whether this is so in native neurons. It has not yet been examined how thymol affects synaptic transmission. In order to know how thymol modulates excitatory transmission with a focus on TRP activation, we investigated its effect on glutamatergic spontaneous excitatory transmission in lamina II (substantia gelatinosa; SG) neurons with which nerve terminals expressing TRP channels make synaptic contacts. The experiment was performed by using the blind whole-cell patch-clamp technique in adult rat spinal cord slices. Superfusing thymol (1 mM) for 3 min reversibly increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a minimal increase in its amplitude in all neurons examined. Seventy-seven% of the neurons produced an outward current at a holding potential of -70 mV. The sEPSC frequency increase and outward current produced by thymol were concentration-dependent with almost the same half-maximal effective concentration (EC50) values of 0.18 and 0.14 mM, respectively. These activities were repeated at a time interval of 30 min, although the sEPSC frequency increase but not outward current recovered with a slow time course. Voltage-gated Na(+)-channel blocker tetrodotoxin did not affect the thymol activities. The sEPSC frequency increase was inhibited by TRPA1 antagonist HC-030031 but not TRPV1 and TRPM8 antagonist (capsazepine and BCTC, respectively), while these antagonists had no effect on the outward current. This was so, albeit the two thymol activities had similar EC50 values. It is concluded that thymol increases the spontaneous release of L-glutamate onto SG neurons by activating TRPA1 channels while producing an outward current without TRP activation. Considering that the SG plays a pivotal role in modulating nociceptive transmission from the periphery, these actions of thymol could contribute to at least a part of its antinociceptive effect.

TRP Channels Involved in Spontaneous L-Glutamate Release Enhancement in the Adult Rat Spinal Substantia Gelatinosa.

The spinal substantia gelatinosa (SG) plays a pivotal role in modulating nociceptive transmission through dorsal root ganglion (DRG) neurons from the periphery. TRP channels such as TRPV1 and TRPA1 channels expressed in the SG are involved in the regulation of the nociceptive transmission. On the other hand, the TRP channels located in the peripheral terminals of the DRG neurons are activated by nociceptive stimuli given to the periphery and also by plant-derived chemicals, which generates a membrane depolarization. The chemicals also activate the TRP channels in the SG. In this review, we introduce how synaptic transmissions in the SG neurons are affected by various plant-derived chemicals and suggest that the peripheral and central TRP channels may differ in property from each other.

Carvacrol presynaptically enhances spontaneous excitatory transmission and produces outward current in adult rat spinal substantia gelatinosa neurons.

Carvacrol, which is abundantly contained in oregano essential oils, has various pharmacological actions including antinociception. Although the oral administration of carvacrol results in antinociception, cellular mechanisms for this action have not been examined yet. We investigated the action of carvacrol on glutamatergic spontaneous excitatory transmission in substantia gelatinosa neurons which play a pivotal role in regulating nociceptive transmission from the periphery by using the patch-clamp technique in adult rat spinal cord slices. Carvacrol superfused for 2 min produced either spontaneous excitatory postsynaptic current frequency increase or outward current at −70 mV, or both of them in many of the neurons tested. The frequency increase and outward current had the EC(50) values of 0.69 mM and 0.55 mM, respectively. The former action was inhibited by a selective TRPA1 antagonist HC-030031 but not a selective TRPV1 antagonist capsazepine, while the latter action was unaffected by their antagonists. The current­voltage relationship for the outward current indicated an involvement in the current of a change in the membrane permeability of K(+) and its outward rectification. The outward current was inhibited in 10 mM-K((+) 0but not K(+)-channel blockers [tetraethylammonium and Ba(2+)]-containing and 11.0 mM-Cl- Krebs solution. These results indicate that carvacrol increases the spontaneous release of l-glutamate from nerve terminals by activating TRPA1 but not TRPV1 channels and produces membrane hyperpolarization, which is possibly mediated by tetraethylammonium- and Ba(2+)-insensitive K(+) channels, in substantia gelatinosa neurons. It is suggested that the hyperpolarizing effect of carvacrol could contribute to its antinociceptive action.