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

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.

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

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-DL-penicillamine (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.

Ryanodine Receptor-mediated Calcium Release Regulates Neuronal Excitability in Rat Spinal Substantia Gelatinosa Neurons

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.

Mechanism of Glutamate-inducedCa2+i Increase in Substantia Gelatinosa Neurons of Juvenile Rats

The glutamate receptors (GluRs) are key receptors for modulatory synaptic events in the central nervous system. It has been reported that glutamate increases the intracellularCa (2+) concentration ([Ca2+]i) and induces cytotoxicity. In the present study, we investigated whether the glutamate-induced[Ca2+]i increase was associated with the activation of ionotropic (iGluR) and metabotropic GluRs (mGluR) in substantia gelatinosa neurons, using spinal cord slice of juvenile rats (10~21 day) .[Ca2+]i was measured using conventional imaging techniques, which was combined with whole-cell patch clamp recording by incorporating fura-2 in the patch pipette. At physiological concentration of extracellularCa (2+), the inward current and[Ca2+]i increase were induced by membrane depolarization and application of glutamate. Dose-response relationship with glutamate was observed in bothCa (2+) signal and inward current. The glutamate-induced[Ca2+]i increase at holding potential of 70 mV was blocked by CNQX, an AMPA receptor blocker, but not by AP-5, a NMDA receptor blocker. The glutamate-induced[Ca2+]i increase inCa (2+) free condition was not affected by iGluR blockers. A selective mGluR (group I) agonist, RS-3, 5-dihydroxyphenylglycine (DHPG), induced[Ca2+]i increase at holding potential of 70 mV in SG neurons. These findings suggest that the glutamate-induced[Ca2+]i increase is associated with AMPA-sensitive iGluR and group I mGluR in SG neurons of rats.