Glial cells, including astrocytes and microglia, interact closely with neurons and modulate pain transmission, particularly under pathological conditions. In this study, we examined the excitability of substantia gelatinosa (SG) neurons of the spinal dorsal horn using a patch clamp recording to investigate the roles of microglial activation in the nociceptive processes of rats. We used xanthine/xanthine oxidase (X/XO), a generator of superoxide anion (O2∙–), to induce a pathological pain condition. X/XO treatment induced an inward current and membrane depolarization. The inward current was significantly inhibited by minocycline, a microglial inhibitor, and fluorocitrate, an astrocyte inhibitor. To examine whether toll-like receptor 4 (TLR4) in microglia was involved in the inward current, we used lipopolysaccharide (LPS), a highly specific TLR4 agonist. The LPS induced inward current, which was decreased by pretreatment with Tak-242, a TLR4-specific inhibitor, and phenyl N-t-butylnitrone, a reactive oxygen species scavenger. The X/XO-induced inward current was also inhibited by pretreatment with Tak-242. These results indicate that the X/XO-induced inward current of SG neurons occurs through activation of TLR4 in microglial cells, suggesting that neuroglial cells modulate the nociceptive process through central sensitization.

Reactive oxygen species (ROS) and nitrogen species (RNS) are involved in cellular signaling processes as a cause of oxidative stress. According to recent studies, ROS and RNS are important signaling molecules involved in pain transmission through spinal mechanisms. In this study, a patch clamp recording was used in spinal slices of rats to investigate the action mechanisms of O2 ⦁- and NO on the excitability of substantia gelatinosa (SG) neuron. The application of xanthine and xanthine oxidase (X/XO) compound, a ROS donor, induced inward currents and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSC) in slice preparation. The application of S-nitroso-N-acetyl-DLpenicillamine (SNAP), a RNS donor, also induced inward currents and increased the frequency of sEPSC. In a single cell preparation, X/XO and SNAP had no effect on the inward currents, revealing the involvement of presynaptic action. X/XO and SNAP induced a membrane depolarization in current clamp conditions which was significantly decreased by the addition of thapsigargin to an external calcium free solution for blocking synaptic transmission. Furthermore, X/XO and SNAP increased the frequency of action potentials evoked by depolarizing current pulses, suggesting the involvement of postsynaptic action. According to these results, it was estblished that elevated ROS and RNS in the spinal cord can sensitize the dorsal horn neurons via pre- and postsynaptic mechanisms. Therefore, ROS and RNS play similar roles in the regulation of the membrane excitability of SG neurons.

Recent studies indicate that mitochondria are an important source of reactive oxygen species (ROS) in the spinal dorsal horn. In our previous study, application of malate, a mitochondrial electron transport complex I substrate, induced a membrane depolarization, which was inhibited by pretreatment with ROS scavengers. In the present study, we used patch clamp recording in the substantia geletinosa (SG) neurons of spinal slices, to investigate the cellular mechanism of mitochondrial ROS on neuronal excitability. DNQX (an AMPA receptor antagonist) and AP5 (an NMDA receptor antagonist) decreased the malate-induced depolarization. In an external calcium free solution and addition of tetrodotoxin (TTX) for blockade of synaptic transmission, the malateinduced depolarization remained unchanged. In the presence of DNQX, AP5 and AP3 (a groupⅠ metabotropic glutamate receptor (mGluR) antagonist), glutamate depolarized the membrane potential, which was suppressed by PBN. However, oligomycin (a mitochondrial ATP synthase inhibitor) or PPADS (a P2 receptor inhibitor) did not affect the substrates-induced depolarization. These results suggest that mitochondrial substrate-induced ROS in SG neuron directly acts on the postsynaptic neuron, therefore increasing the ion influx via glutamate receptors.

Reactive oxygen species (ROS) and nitrogen species (RNS) are both important signaling molecules involved in pain transmission in the dorsal horn of the spinal cord. Xanthine oxidase (XO) is a well-known enzyme for the generation of superoxide anions (O2 ⦁-), while S-nitroso-N-acetyl-DLpenicillamine (SNAP) is a representative nitric oxide (NO) donor. In this study, we used patch clamp recording in spinal slices of rats to investigate the effects of O2 ⦁- and NO on the excitability of substantia gelatinosa (SG) neurons. We also used confocal scanning laser microscopy to measure XO- and SNAP-induced ROS and RNS production in live slices. We observed that the ROS level increased during the perfusion of xanthine and xanthine oxidase (X/XO) compound and SNAP after the loading of 2′,7′-dichlorofluorescin diacetate (H2DCF-DA), which is an indicator of intracellular ROS and RNS. Application of ROS donors such as X/XO, β -nicotinamide adenine dinucleotide phosphate (NADPH), and 3-morpholinosydnomimine (SIN-1) induced a membrane depolarization and inward currents. SNAP, an RNS donor, also induced membrane depolarization and inward currents. X/XO-induced inward currents were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger) and manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP; superoxide dismutase mimetics). Nitro-L-arginine methyl ester (NAME; NO scavenger) also slightly decreased X/XO-induced inward currents, suggesting that X/XO-induced responses can be involved in the generation of peroxynitrite (ONOO-). Our data suggest that elevated ROS, especially O2 ⦁-, NO and ONOO-, in the spinal cord can increase the excitability of the SG neurons related to pain transmission.

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium (BKca) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of Ca²+ that induces Ca²+-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective BKCa channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove Ca²+ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through BKca channels, which are activated by intracellular Ca²+ increase via activation of RyR of Ca²+ stores.

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.

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ([Ca²⁺]_i) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydrylreducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2′,7′-dichlorofluorescin diacetate (H₂DCF-DA). NaOCl-induced depolarization was not blocked by pretreatment with external Ca²⁺ free solution or by the addition of nifedifine. However, when slices were pretreated with the Ca²⁺ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the Ca²⁺-sensitive fluorescence dye fura-2, the [Ca²⁺]_i was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, and that mitochondria are the main source of ROS in the spinal dorsal horn. To investigate whether mitochondrial ROS can induce changes in mem¬brane excitability on spinal substantia gelatonosa (SG) neurons, we examined the effects of mitochondrial electron transport complex (ETC) substrates and inhibitors on the membrane potential of SG neurons in spinal slices. Application of ETC inhibitors, rotenone or antimycin A, resulted in a slowly developing and slight membrane depolarization in SG neurons. Also, application of both malate, a complex I substrate, and succinate, a complex II substrate, caused reversible membrane depolarization and enhanced firing activity. Changes in membrane potential after malate exposure were more prominent than succinate exposure. When slices were pretreated with ROS scavengers such as phenyl-N-tert-buthylnitrone (PBN), catalase and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), malate-induced depolarization was significantly decreased. Intracellular calcium above 100 µM increased malate-induced depolarization, witch was suppressed by cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor. These results suggest that enhanced production of spinal mitochondrial ROS can induce nociception through central sensitization.

Substantia gelatinosa (SG) neurons receive synaptic inputs from primary afferent Aδ- and C-fibers, where nociceptive information is integrated and modulated by numerous neurotransmitters or neuromodulators. A number of studies were dedicated to the molecular mechanism underlying the modulation of excitability or synaptic plasticity in SG neurons and revealed that second messengers, such as cAMP and cGMP, play an important role. Recently, cAMP and cGMP were shown to downregulate each other in heart muscle cells. However, involvement of the crosstalk between cAMP and cGMP in neurons is yet to be addressed. Therefore, we investigated whether interaction between cAMP and cGMP modulates synaptic plasticity in SG neurons using slice patchclamp recording from rats. Synaptic activity was measured by excitatory post-synaptic currents (EPSCs) elicited by stimulation onto dorsal root entry zone. Application of 1 mM of 8- bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) for 15 minutes increased EPSCs, which were maintained for 30 minutes. However, simultaneous application of 8-BrcAMP and 8-Br-cGMP failed to increase EPSCs, which suggested antagonistic cross-talk between two second messengers. Application of 3-isobutyl-1-methylxanthine (IBMX) that prevents degradation of cAMP and cGMP by blocking phosphodiesterase (PDE) increased EPSCs. Co-application of cAMP/cGMP along with IBMX induced additional increase in EPSCs. These results suggest that second messengers, cAMP and cGMP, might contribute to development of chronic pain through the mutual regulation of the signal transduction.

Somatostatin (SST) is a known neuromodulator of the central nervous system. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives many thinmyelinated Að-fiber and unmyelinated C primary afferent fibers and is involved in nociceptive processing. Many studies have demonstrated that SST plays a pivotal role in pain modulation in the spinal cord. However, little is yet known about the direct effects of SST on the SG neurons of the Vc in adult mice. In our present study, we investigated the direct membrane effects of SST and a type 2 SST receptor agonist, seglitide (SEG), on the SG neurons of the Vc using a gramicidin-perforated current clamp in adult mice. The majority (53%, n = 27/51) of the adult SG neurons were hyperpolarized by SST (300 nM) but no differences were found in the hyperpolarization response rate between males and females. When SST was applied successively, the second response was smaller (76±9.5%, n=19), suggesting that SST receptors are desensitized by repeated application. SST-induced hyperpolarization was also maintained under conditions where presynaptic events were blocked (75±1.0%, n=5), suggesting that this neuromodulator exerts direct effects upon postsynaptic SG neurons. SEG was further found to induce membrane hyperpolarization of the SG neurons of the Vc. These results collectively demonstrate that SST inhibits the SG neuronal activities of the Vc in adult mice with no gender bias, and that these effects are mediated via a type 2 SST receptor, suggesting that this is a potential target for orofacial pain modulation.

Bicuculline is one of the most commonly used GABAд eceptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide are preferred. However, a number of studies have shown that BMI has non-GABAд eceptor-mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. In this study, we investigated whether BMI has non-GABA receptor-mediated activity in Vc SG neurons using a whole cell patch clamp technique. SG neurons were depolarized by application of BMI (20M) using a high Cℓ⁻ipette solution. GABA ( 30-100μM) also induced membrane depolarization of SG neuron. Although BMI is known to be a GABAд receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a GABAд and GABAс receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PTX or an antagonist cocktail consisting of tetrodotoxin (Nα+ nnel blocker),AP-5 (NMDA receptor antagonist), CNQX (non-NMDA receptor antagonist), and strychnine (glycine receptor antagonist). Thus BMI induces membrane depolarization by directly acting on postsynaptic Vc SG neurons in a manner which is independent of GABAд receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.

Reactive oxygen species (ROS) are toxic agents that may be involved in various neurodegenerative diseases. Recent studies indicate that ROS can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In the present study, whole cell patch clamp recordings were carried out to investigate the effects of tert-buthyl hydroperoxide (t-BuOOH), an ROS, on neuronal excitability and the mechanisms underlying changes of membrane excitability. In current clamp condition, application of t-BuOOH caused a reversible membrane depolarization and firing activity in substantia gelatinosa (SG) neurons. When slices were pretreated with phenyl-N-tert-buthylnitrone (PBN) and ascorbate, ROS scavengers, t-BuOOH failed to induce membrane depolarization. However, isoascorbate did not prevent t-BuOOH-induced depolarization, suggesting that the site of ROS action is intracellular. The t-BuOOH-induced depolarization was not blocked by pretreatment with dithiothreitol (DTT), a sulfhydryl-reducing agent. The membrane-impermeant thiol oxidant 5,5-dithiobis 2-nitrobenzoic acid (DTNB) failed to induce membrane depolarization, suggesting that the changes of neuronal excitability by t-BuOOH are not caused by the modification of extrathiol group. The t-BuOOH-induced depolarization was suppressed by the phospholipase C (PLC) blocker U-73122 and inositol triphosphate (IP₃)receptor antagonist 2-aminoethoxydiphenylbolate (APB), and after depletion of intracellular Cα²+ pool by thapsigargin. These data suggest that ROS generated by peripheral nerve injury can induce central sensitization in spinal cord, and t-BuOOH-induced depolarization may be regulated by intracellular Cα²+ store mainly via PLC-IP₃pathway.

The superficial dorsal horn, particularly substantia gelatinosa (SG) in the spinal cord, receives inputs from small-diameter primary afferents that predominantly convey noxious sensation. Reactive oxygen species (ROS) are toxic agents that may be involved in various neurodegenerative diseases. Recent studies indicate that ROS are also involved in persistent pain through a spinal mechanism. In the present study, whole cell patch clamp recordings were carried out on SG neurons in spinal cord slice of young rats to investigate the effects of hydrogen peroxide on neuronal excitability and excitatory synaptic transmission. In current clamp condition, tert-buthyl hydroperoxide (t-BuOOH), an ROS donor, depolarized membrane potential of SG neurons and increased the neuronal firing frequencies evoked by depolarizing current pulses. When slices were pretreated with phenyl-N-tert-buthylnitrone (PBN) or ascorbate, ROS scavengers, t-BuOOH did not induce hyperexcitability. In voltage clamp condition, t-BuOOH increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), and monosynaptically evoked excitatory postsynaptic currents (eEPSCs) by electrical stimulation of the ipsilateral dorsal root. These data suggest that ROS generated by peripheral nerve injury can modulate the excitability of the SG neurons via pre- and postsynaptic actions.