Spinal Substantia Gelatinosa Neurons Research Articles

Suppression of HCN channels in the spinal dorsal horn restores KCC2 expression and attenuates diabetic neuropathic pain

Previous studies have shown that the hyperpolarized cyclic nucleotide gated (HCN) ion channels in the spinal dorsal horn (SDH) might be involved in the development of diabetic neuropathic pain (DNP). Additionally, other studies have shown that the decreased potassium-chloride cotransporter 2 (KCC2) expression in the SDH promotes pain hypersensitivity. Both HCN channels and KCC2 were highly expressed in spinal substantia gelatinosa neurons. However, whether the K+ efflux induced by the activation of HCN channels in DNP modulate KCC2 function and subsequently affect the role of γ-aminobutyric acid (GABA)/GABA-A receptors of neurons in the SDH remains to be clarified. The purpose of this work was to investigate the underlying mechanisms of KCC2 participating in HCN channels to promote DNP. Here, we found that the analgesic role of HCN channels blocker ZD7288 was associated with the up-regulated KCC2 expression and could be prevented by DIOA, a KCC2 blocker. Furthermore, the level of GABA in DNP rats significantly increased, which was decreased by ZD72288. Moreover, DIOA pretreatment could partly block the inhibitory effect of ZD7288 on the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling activation of DNP rats. Finally, inhibition of cAMP-PKA signaling alleviated allodynia and elevated KCC2 expression in DNP rats. Altogether, this study reveals that the role of cAMP-PKA signaling-regulated HCN channels in DNP associated with decreased KCC2 expression in the spinal cord and altered GABA nature.

Engineering 2D Silicene-Based Mesoporous Nanomedicine for In Vivo Near-Infrared-Triggered Analgesia.

The utilization of local anesthetics for postoperative analgesia represents an effective approach, but generally suffers from short half‐lives and brachychronic local neurotoxicity. A desirable anesthetic with controllable and sustainable drug‐releasing performance for adequate analgesia effect is highly required. In this work, the core/shell‐structured two‐dimenional (2D) silicene nanosheets coated with mesoporous silica layer (abbreviated as Silicene@MSNs) have been rationally constructed as localized drug‐delivery system in sciatic nerve block to achieve on‐demand release of loaded ropivacaine (RP) in mesoporous silica layer for local analgesia. Based on the specific photothermal performance of 2D silicene core, this local anesthesia system can be triggered by near‐infrared laser to release the loaded RP, resulting in on‐demand and long‐lasting regional anesthesia. The analgesia effect is assessed by pain behavior tests, which demonstrates that the RP‐loaded Silicene@MSNs core/shell nanosystem behaves almost five times longer analgesia effect than free RP. Furthermore, the activation of pain‐related neurons in nerve conduction pathways is tested to explore the underlying analgesia mechanism, revealing that the designed nanosystem can improve the pain threshold, reduce the activation of neurons in dorsal root ganglion and excitability in spinal substantia gelatinosa neurons. This designed anesthetic nanomedicine provides a facile but effective methodology for long‐lasting regional anesthesia.

Spontaneous excitatory transmission enhancement produced by linalool and its isomer geraniol in rat spinal substantia gelatinosa neurons - involvement of transient receptor potential channels

BackgroundMany of plant-derived compounds inhibiting nerve conduction enhance glutamatergic spontaneous excitatory transmission by activating transient receptor potential (TRP) channels in spinal substantia gelatinosa (SG) neurons that play a crucial role in regulating nociceptive transmission. Although (±)-linalool and its isomer geraniol having antinociceptive effects inhibit nerve conduction, it has not been examined yet how they affect excitatory transmission in SG neurons. We examined the effects of the compounds on action potential-independent spontaneous excitatory transmission with a focus on an involvement of TRP channels. MethodsThe whole-cell patch-clamp technique was applied to SG neurons in adult rat spinal cord slices. Results(±)-Linalool increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a small increase in its amplitude. The (±)-linalool activity was sensitive to TRP vanilloid-1 (TRPV1) antagonist capsazepine and TRP ankyrin-1 (TRPA1) antagonist HC-030,031 while resistant to BCTC that is antagonist for cloned TRPV1 and TRP melastatin-8 (TRPM8) channels and for TRPM8 channels in the SG, indicating TRPV1 and TRPA1 activation. In 73% of the neurons tested, (±)-linalool produced an outward current at -70 mV. In SG neurons sensitive to (±)-linalool, geraniol produced a quantitatively similar effect to (±)-linalool. Geraniol-induced sEPSC frequency increase was sensitive to BCTC but resistant to capsazepine and HC-030,031, indicating TRPM8 activation. Outward currents produced by (±)-linalool and geraniol were unaffected by all of the TRP antagonists. ConclusionIn SG neurons, (±)-linalool and geraniol presynaptically enhanced spontaneous excitatory transmission by activating different types of TRP channel while hyperpolarizing membranes in a manner independent of TRP channels. Such a TRP channel modulation and hyperpolarization could contribute to the antinociceptive effects of (±)-linalool and geraniol.

Electroacupuncture effects on the P2X4R pathway in microglia regulating the excitability of neurons in the substantia gelatinosa region of rats with spinal nerve ligation

Electroacupuncture (EA) has been used to treat neuropathic pain induced by peripheral nerve injury (PNI) by applying an electrical current to acupoints with acupuncture needles. However, the mechanisms by which EA treats pain remain indistinct. High P2X4 receptor (P2X4R) expression levels demonstrate a notable increase in hyperactive microglia in the ipsilateral spinal dorsal horn following PNI. In order to demonstrate the possibility that EA analgesia is mediated in part by P2X4R in hyperactive microglia, the present study performed mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests in male Sprague-Dawley rats that had undergone spinal nerve ligation (SNL). The expression levels of spinal P2X4R were determined using reverse transcription-quantitative PCR, western blotting analysis and immunofluorescence staining. Furthermore, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded using whole-cell patch clamp to demonstrate the effect of EA on synaptic transmission in rat spinal substantia gelatinosa (SG) neurons. The results of the present study demonstrated that EA increased the MWT and TWL and decreased overexpression of P2X4R in hyperactive microglia in SNL rats. Moreover, EA attenuated the frequency of sEPSCs in SG neurons in SNL rats. The results of the present study indicate that EA may mediate P2X4R in hyperactive spinal microglia to inhibit nociceptive transmission of SG neurons, thus relieving pain in SNL rats.

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.

Pre- and Postsynaptic Actions of Reactive Oxygen Species and Nitrogen Species in Spinal Substantia Gelatinosa Neurons

Pre- and Postsynaptic Actions of Reactive Oxygen Species and Nitrogen Species in Spinal Substantia Gelatinosa Neurons

Modulation by orexin A of spontaneous excitatory and inhibitory transmission in adult rat spinal substantia gelatinosa neurons

Hypothalamic neuropeptides, orexins A and B, differently inhibit nociceptive behavior. This difference is possibly due to a distinction between orexins A and B in modulating synaptic transmission in spinal substantia gelatinosa (SG) neurons that play a pivotal role in regulating nociceptive transmission. Although we previously reported a modulatory action of orexin B on synaptic transmission in adult rat SG neurons, it has not been fully examined how the transmission is affected by orexin A. The present study examined the effects of orexin A on spontaneous excitatory and inhibitory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. Like orexin B, orexin A produced an inward current at −70 mV and/or increased the frequency of spontaneous excitatory postsynaptic current without changing its amplitude. Half-maximal effective concentration values for their effects were 0.0045 and 0.030 μM, respectively; the former value was four-fold smaller than that of orexin B while the latter value was comparable to that of orexin B. Orexin A enhanced not only glycinergic but also GABAergic transmission, although only glycinergic transmission was facilitated by orexin B in the majority of neurons tested. Orexin A activities were inhibited by an orexin-1 receptor antagonist (SB334867) but not an orexin-2 receptor antagonist (JNJ10397049), as different from orexin B whose activation was depressed by JNJ10397049 but not SB334867. These results indicate that orexin A has a different action from orexin B in SG neurons in efficacy for inward current production and in GABAergic transmission enhancement, possibly owing to orexin-1 but not orexin-2 receptor activation. This difference could contribute to at least a part of the distinction between orexins A and B in antinociceptive effects.

Presynaptic facilitation by tetracaine of glutamatergic spontaneous excitatory transmission in the rat spinal substantia gelatinosa – Involvement of TRPA1 channels

The amide-type local anesthetic (LA) lidocaine activates transient receptor potential (TRP) ankyrin-1 (TRPA1) channels to facilitate spontaneous l-glutamate release onto spinal substantia gelatinosa (SG) neurons, which play a crucial role in regulating nociceptive transmission. In contrast, the ester-type LA procaine reduces the spontaneous release of l-glutamate in SG neurons. In order to determine whether TRPA1 activation by LAs is specific to amide-types, we examined the actions of tetracaine, another ester-type LA, and other amide-type LAs on glutamatergic spontaneous excitatory transmission in SG neurons by focusing on TRP activation. Whole-cell patch-clamp recordings were performed on SG neurons of adult rat spinal cord slices at a holding potential of −70mV. Bath-applied tetracaine increased spontaneous excitatory postsynaptic current (sEPSC) frequency in a concentration-dependent manner. Tetracaine activity was resistant to the voltage-gated Na+-channel blocker tetrodotoxin, the TRP vanilloid-1 antagonist capsazepine, and the TRP melastatin-8 antagonist BCTC, but was inhibited by the non-selective TRP antagonist ruthenium red and the TRPA1 antagonist HC-030031. With respect to amide-type LAs, prilocaine had a tendency to increase sEPSC frequency, while ropivacaine and levobupivacaine reduced the frequency. In conclusion, tetracaine facilitated spontaneous l-glutamate release from nerve terminals by activating TRPA1 channels in the SG, resulting in an increase in the excitability of SG neurons. TRPA1 activation was not specific to amide-type or ester-type LAs. The facilitatory action of LAs may be involved in pain occurring after recovery from spinal anesthesia.

Effects of Reactive Oxygen Species and Nitrogen Species on the Excitability of Spinal Substantia Gelatinosa Neurons

Effects of Reactive Oxygen Species and Nitrogen Species on the Excitability of Spinal Substantia Gelatinosa Neurons

Reductions in tonic GABAergic current in substantia gelatinosa neurons and GABAA receptor δ subunit expression after chronic constriction injury of the sciatic nerve in mice.

Decreased Gamma-aminobutyric acid (GABA)-ergic phasic inhibitory transmission in the spinal cord is thought to be responsible for the development of neuropathic pain. However, the role of GABAergic tonic current in substantia gelatinosa (SG) neurons in neuropathic pain remains to be fully elucidated. In this study, we assessed GABAergic tonic currents of SG neurons in a sciatic nerve chronic constriction injury (CCI) mouse. Whole-cell patch clamp recordings form lumbar spinal cord slices was performed to evaluate GABAergic currents. We also investigated the expression changes of GABAA receptor subunits which are considered to mediate tonic currents. The percentage of SG neurons receiving GABAergic tonic currents decreased in CCI mice compared with Naïve mice. No significant change was observed in the mean amplitude of GABAergic tonic currents. RT-PCR and Western blot revealed that the expression of GABAA receptor δ subunits decreased following CCI. A reduction in the expression the δ subunit of the GABAA receptor and diminished GABAergic tonic current in SG neurons were observed after CCI in mice. GABAergic tonic current plays a key role in neuropathic pain. The GABAA receptor δ subunit may be a therapeutic target in neuropathic pain. WHAT DOES THIS STUDY ADD?: In spinal SG neurons, GABAergic inhibitory transmission operates through both phasic and tonic currents, but physiological role is largely unknown. In this study, we report dysregulation of GABAA receptor δ subunit-mediated tonic current in SG neurons may result in spinal disinhibition resulting in neuropathic pain in CCI mice.

Intravenous administration of lidocaine directly acts on spinal dorsal horn and produces analgesic effect: An in vivo patch-clamp analysis.

Intravenous lidocaine administration produces an analgesic effect in various pain states, such as neuropathic and acute pain, although the underlying mechanisms remains unclear. Here, we hypothesized that intravenous lidocaine acts on spinal cord neurons and induces analgesia in acute pain. We therefore examined the action of intravenous lidocaine in the spinal cord using the in vivo patch-clamp technique. We first investigated the effects of intravenous lidocaine using behavioural measures in rats. We then performed in vivo patch-clamp recording from spinal substantia gelatinosa (SG) neurons. Intravenous lidocaine had a dose-dependent analgesic effect on the withdrawal response to noxious mechanical stimuli. In the electrophysiological experiments, intravenous lidocaine inhibited the excitatory postsynaptic currents (EPSCs) evoked by noxious pinch stimuli. Intravenous lidocaine also decreased the frequency, but did not change the amplitude, of both spontaneous and miniature EPSCs. However, it did not affect inhibitory postsynaptic currents. Furthermore, intravenous lidocaine induced outward currents in SG neurons. Intravenous lidocaine inhibits glutamate release from presynaptic terminals in spinal SG neurons. Concomitantly, it hyperpolarizes postsynaptic neurons by shifting the membrane potential. This decrease in the excitability of spinal dorsal horn neurons may be a possible mechanism for the analgesic action of intravenous lidocaine in acute pain.

Minocycline enhances inhibitory transmission to substantia gelatinosa neurons of the rat spinal dorsal horn

Minocycline, a second-generation tetracycline, is well known for its antibiotic, anti-inflammatory, and antinociceptive effects. Modulation of synaptic transmission is one of the analgesic mechanisms of minocycline. Although it has been reported that minocycline may suppress excitatory glutamatergic synaptic transmission, it remains unclear whether it could affect inhibitory synaptic transmission, which also plays a key role in modulating pain signaling.To examine the effect of minocycline on synaptic transmission in rat spinal substantia gelatinosa (SG) neurons, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) using whole-cell patch-clamp recording at a holding potential of 0mV. Bath application of minocycline significantly increased the frequency but not the amplitude of sIPSCs in a reversible and concentration-dependent manner with an EC50 of 85.The enhancement of inhibitory synaptic transmission produced by minocycline was not affected by the glutamate receptor antagonists CNQX and D-APV or by the voltage-gated sodium channel blocker tetrodotoxin (TTX). Moreover, the potency of minocycline for facilitating sIPSC frequency was the same in both glycinergic and GABAergic sIPSCs without changing their decay phases. However, the facilitatory effect of minocycline on sIPSCs was eliminated in a Ca2+-free Krebs solution or by co-administration with calcium channel blockers.In summary, our data demonstrate that baseline inhibitory synaptic transmission in SG neurons is markedly enhanced by minocycline. This may function to decrease the excitability of SG neurons, thus leading to a modulation of nociceptive transmission.

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 (1mM) for 3min 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 −70mV. 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 0.14mM, respectively. These activities were repeated at a time interval of 30min, 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.

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

Although 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. Blind 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. Neurotropin (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. This 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.

Isoflurane Suppresses the Excitability and Synaptic Transmission of Spinal Nociceptive Pathway in Rats

Background: Isoflurane (IFL), one of the most widely used volatile anesthetics, has an anti-nociceptive effect at the spinal cord level. However, the reported spinal mechanisms of IFL analgesic action remain controversial. The aim of this study was to examine the effect of IFL on the excitability of spinal substantia gelatinosa (SG, lamina II) neurons and the synaptic transmission from primary nociceptive fibers to SG neurons.Methods: Parasagittal spinal slices with a dorsal root attached were cut from the Sprague-Dawley rats (4-6 weeks old). Whole-cell patch-clamp recordings were made from SG neurons at room temperature. Dorsal root stimulation was used to evoke excitatory postsynaptic currents (eEPSCs). Depolarizing current injection was used to display the firing pattern of action potentials. IFL was given as volume percent (3%) by passing the gas mixture (flow rate, 0.5 L/minute) into equilibrated artificial cerebral spinal fluid (ACSF) for at least 15 minutes at room temperature.Results: IFL significantly suppressed the peak amplitude of monosynaptic eEPSCs mediated by primary Aδ and C fibers. Regardless of neuron types, IFL significantly decreased the frequency of action potentials in all SG neurons tested.Conclusions: IFL may play analgesic role through inhibiting or weakening the excitability and synaptic transmission of spinal nociceptive pathway. Citation: Jie Liu, Gen-Lin Ji, Jian-Min Li, Tun Liu, Qun Wang, Ya-Nan Pu, et al. Isoflurane suppresses the excitability and synaptic transmission of spinal nociceptive pathway in rats. J Anesth Perioper Med 2015; 2: 61-5. doi: 10.24015/JAPM.2015.0010This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reactive Oxygen Species and Nitrogen Species Differentially Regulate Neuronal Excitability in Rat Spinal Substantia Gelatinosa Neurons

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions (O2˙-) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of O2˙- and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP (10μM) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-β-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.

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 2min produced either spontaneous excitatory postsynaptic current frequency increase or outward current at −70mV, or both of them in many of the neurons tested. The frequency increase and outward current had the EC50 values of 0.69mM and 0.55mM, 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 current–voltage 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 10mM-K+ but not K+-channel blockers [tetraethylammonium and Ba2+]-containing and 11.0mM-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 Ba2+-insensitive K+ channels, in substantia gelatinosa neurons. It is suggested that the hyperpolarizing effect of carvacrol could contribute to its antinociceptive action.

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.

Dynamic effects of TNF-α on synaptic transmission in mice over time following sciatic nerve chronic constriction injury

Nerve injury-induced central sensitization can manifest as an increase in excitatory synaptic transmission and/or as a decrease in inhibitory synaptic transmission in spinal dorsal horn neurons. Cytokines such as tumor necrosis factor-α (TNF-α) are induced in the spinal cord under various injury conditions and contribute to neuropathic pain. In this study we examined the effect of TNF-α in modulating excitatory and inhibitory synaptic input to spinal substantia gelatinosa (SG) neurons over time in mice following chronic constriction injury (CCI) of the sciatic nerve. Whole cell patch-clamp studies from SG neurons showed that TNF-α enhanced overall excitability of the spinal cord early in time following nerve injury 3 days after CCI compared with that in sham control mice. In contrast, the effects of TNF were blunted 14 days after CCI in nerve-injured mice compared with sham surgery mice. Immunohistochemical staining showed that the expression of TNF-α receptor 1 (TNFR1) was increased at 3 days but decreased at 14 days following CCI in the ipsilateral vs. the contralateral spinal cord dorsal horn. These results suggest that TNF-α acting at TNFR1 is important in the development of neuropathic pain by facilitating excitatory synaptic signaling in the acute phases after nerve injury but has a reduced effect on spinal neuron signaling in the later phases of nerve injury-induced pain. Failure of the facilatory effects of TNF-α on excitatory synaptic signaling in the dorsal horn to resolve following nerve injury may be an important component in the transition between acute and chronic pain conditions.

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.