• International Journal of Oral Biology
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• v.37 no.1
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• pp.17-23
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• 2012

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 membrane 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 $100{\mu}M$ increased malateinduced 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.

• International Journal of Oral Biology
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• v.39 no.4
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• pp.229-236
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• 2014

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 ($O{_2}^{{\cdot}_-}$) 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 $O{_2}^{{\cdot}_-}$ and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP ($10{\mu}M$) 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-${\beta}$-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.

• Animal cells and systems
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• v.13 no.4
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• pp.357-362
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• 2009

Hesperidin, the most abundant polyphenolic compound found in citrus fruits, has been known to possess neuroprotective, sedative, and anticonvulsive effects on the nervous system. In a recent electrophysiological study, it was reported that hesperidin induced biphasic change in population spike amplitude in hippocampal CA1 neurons in response to both single spike stimuli and theta-burst stimulation depending on its concentration. However, the precise mechanism by which hesperidin acts on neuronal functions has not been fully elucidated. Here, using whole-cell patch-clamp recording, we revealed that hesperidin did not affect excitatory synaptic activities such as basal synaptic transmission and theta-burst LTP. Moreover, in a current injection experiment, spike number, resting membrane potential and action potential threshold also remained unchanged. Taken together, these results indicate that the effects of hesperidin on the neuronal functions such as spiking activity might not be attributable to either modification of excitatory synaptic transmissions or changes in membrane excitability in hippocampal CA1 neuron.

• International Journal of Oral Biology
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• v.42 no.2
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• pp.55-61
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• 2017

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 malate-induced depolarization remained unchanged. In the presence of DNQX, AP5 and AP3 (a group I 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.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.117-123
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• 2008

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 ($IP_3$) receptor antagonist 2-aminoethoxydiphenylbolate (APB), and after depletion of intracellular $Ca^{2+}$ 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 $Ca^{2+}$ store mainly via $PLC-IP_3$ pathway.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.5-12
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• 2013

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 ($[Ca^{2+}]_i$) 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 sulfhydryl-reducing 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 ($H_2DCF$-DA). NaOCl-induced depolarization was not blocked by pretreatment with external $Ca^{2+}$ free solution or by the addition of nifedifine. However, when slices were pretreated with the $Ca^{2+}$ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the $Ca^{2+}$-sensitive fluorescence dye fura-2, the $[Ca^{2+}]_i$ 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.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.4
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• pp.371-376
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• 2017

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion ($O_2{^-}$), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO ($300{\mu}M/30mU$) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near -40 mV. X/XO-induced depolarization reduced in the presence of $La^{3+}$, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of $Na^+$ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

• Annals of Clinical Neurophysiology
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• v.14 no.2
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• pp.64-71
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• 2012

Background: It is generally accepted that upper motor neuron (UMN) lesion can alter lower motor neuron (LMN) function by the plasticity of neural circuit. However there have been only few researches regarding the axonal excitability of LMN after UMN injury especially during the acute stage. The aim of this study was to investigate the nerve excitability properties of the LMNs following an acute to subacute supratentorial corticospinal tract lesion. Methods: An automated nerve excitability test (NET) using the threshold tracking technique was utilized to measure multiple excitability indices in median motor axons of 15 stroke patients and 20 controls. Testing of both paretic and non-paretic side was repeated twice, during the acute stage and subacute stage. The protocols calculated the strength-duration time constant from the duration-charge curve, parameters of threshold electrotonus (TE), the current-threshold relationship from sequential sub-threshold current, and the recovery cycle from sequential supra-threshold stimulation. Results: On the paretic side, compared with the control group, significant decline of superexcitablity and increase in the relative refractory period were observed during the subacute stage of stroke. Additionally, despite the absence of statistical significance, a mildly collapsing in ('fanning in') of the TE was found. Conclusions: Our results suggest that supratentorial brain lesions can affect peripheral axonal excitability even during the early stage. The NET pattern probably suggests background membrane depolarization of LMNs. These features could be associated with trans-synaptic regulation of UMNs to LMNs as one of the "neural plasticity" mechanisms in acute brain injury.

• International Journal of Oral Biology
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• v.32 no.4
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• pp.153-160
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• 2007

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.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.2
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• pp.432-437
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• 2007

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 substantia gelatinosa (SG) neurons in spinal cord slice of neonatal rats to investigate the effects of ROS on neuronal excitability and excitatory synaptic transmission. In current clamp condition, tert-buthyl hydroperoxide (t-BuOOH), an ROS donor, induced a electrical hyperexcitability during t-BuOOH wash-out followed by a brief inhibition of excitability in SG neurons. Application of t-BuOOH depolarized membrane potential of SG neurons and increased the neuronal firing frequencies evoked by depolarizing current pulses. Phenyl-N-tert-buthylnitrone (PBN), an ROS scavenger, antagonized t-BuOOH induced hyperexcitability. IN voltage clamp conditions, t-BuOOH increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). In order to determine the site of action of t-BuOOH, miniature excitatory postsynaptic currents (mEPSCs) were recorded. t-BuOOH increased the frequency and amplitude of mEPSCs, indicating that it may modulate the excitability of the SG neurons via pre- and postsynaptic actions. These data suggest that ROS generated by peripheral nerve injury can induce central sensitization in spinal cord.