• The Korean Journal of Pain
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• 제9권2호
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• pp.311-317
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• 1996

Background: Following peripheral nerve injury, rats will show a tactile allodynia and hyperalgesia. But the mechanism of allodynia is still obscure. Previous studies have shown this allodynia was reversed by intrathecal alpha-2 agonists and NMDA antagonists, but not by morphine. In formalin test, either the pretreatment of NMDA antagonist or morphine prevents the hyperalgesia. The present studies, using rats rendered allodynic by ligation of the left L5 and L6 nerves, aimed to investigate the effects of pretreatment of MK-801 and morphine on the development of tactile allodynia. Methods and Material: Male Sprague-Dawley rats (100~150g) were anesthetized with halothane, the left L5 and L6 spinal nerves were ligated tightly by 6-0 black silk. For sham operation muscle dissection was performed but the spinal nerve was not ligated. For pretreatment of drugs, MK-801 (NMDA antagonist; 0.3 mg/kg). CNQX (non-NMDA) antagonist; 0.3 mg/kg), morphine (1 mg/kg) or saline (placebo) was administered subcutaneously 30 minutes before operation. A second dose was administered subcutaneously 24 hours after operation and further doses were given daily for 2 days further. The volume of injection was 5 ml/kg. To assess the mechanical allodynia, paw withdrawal thresholds of ipsilateral limb were determined using 8 von Frey hairs. Results: Within 2 days saline, CNQX or morphine injected rats developed tactile allodynia (paw withdrawal threshold was about 2g), and persisted for over 2 weeks. Pretreatment of MK-801 delayed the development of tactile allodynia for 3 days comparing to that of saline injected rat. Conclusion: NMDA receptor in the central nerve system plays an important role in the development of tactile allodynia induced by peripheral nerve injury. But the mechanism may be different from hyperalgesia developed in formalin test.

• Journal of Korean Neurosurgical Society
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• 제29권9호
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• pp.1161-1170
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• 2000

Objective : The transverse hippocampal slice is one of the most commonly studied in vitro models of mammalian brain physiology. However, despite its broad usage, there has been no standardization of slice preparation techniques or recording condition. It is well known that variations in recording conditions can result in profound different effects to neuronal responses. Evoked field potentials, recorded extracellularly, were used to investigate the effects of variations in hippocampal slice preparation protocol on hypoxia responses of CA1 neurones. Material & Methods : Before hypoxic injury, hippocampal slices were incubated for 4 hours. During incubation period, the slices were placed in a incubation chamber($21^{\circ}C$) for recovery from preparation injury and then transferred to recording chamber($34^{\circ}C$) for more recovery and baseline electric recording with current stimulation(0.1Hz). Various time periods in incubation chamber and recording chamber were applied to each experimental group(group 1=60min : 180min, group 2=90min : 150min, group 3=180min : 60min, time in incubation chamber : time in recording chamber) before 10 min hypoxia produced by replacing 95% $O_2$+5% $CO_2$ mixed gas to 95% $N_2$+5% $CO_2$ gas. Calcium, Magnesium ions and several drugs effecting on glutamate receptor also were studied. Recoveries from hypoxic injury of hippocampal slices were estimated by percent recovery of population spike(PS). Statistic analysis of study were performed using paired t-test. Results : The percent recovery of PS after 10min hypoxia was considerably enhanced by increasing the period of current stimulation during incubation period before hypoxic injury. Temperature effect on the result of this experiment was also studied(group 4) but the result from this showed no statistic significance. Low magnesium ion concentration of artificial CSF(Mg-free aCSF) during incubation period enhanced the recovery of PS but low calcium (calcium-free) and high magnesium ion concentration(2mM) reduced it after hypoxic injury. L-glutamate($100{\mu}M$) and AP-5($50{\mu}M$) had no effect on the recovery of PS but CNQX($10{\mu}M$) in artificial CSF during incubation period markedly enhanced the recovery of PS. Co-treatment of AP-5($50{\mu}M$), CNQX($10{\mu}M$) and high magnesium concentration(2mM) enhanced recovery of PS in immediate following period of hypoxic injury but the effect of cotreatment after then decayed rapidly and lost statistic significance. Conclusions : Judging from above results, the condition of baseline recording is important in observing the recovery of population spike after hypoxia, and the time and the condition should be controled more strictly to obtain reliable results.

• The Korean Journal of Physiology and Pharmacology
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• 제5권6호
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• pp.457-466
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• 2001

Glutamate is the most common excitatory amino acid in the brain. Responsiveness of dendrites to the glutamate greatly varies depending on the application sites. Especially, a point of the maximal response to the glutamate of the dendrite is called as 'hot spot'. In our experiment, the responsiveness of the hot spot to the glutamate was investigated in the CA1 pyramidal neuron of the rat hippocampal slice. CNQX, the antagonist of AMPA receptor, blocked 95% of membrane current to the glutamate focal application $(I_{gl}).$ Train ejection of glutamate on one point of the dendrite increased or decreased the amplitude of $I_{gl}$ with the pattern of train, and the changes were maintained at least for 30 min. In some cases, glutamate train ejection also induced calcium dependent action potentials. To evoke long-term change of synaptic plasticity, we adopted ${\theta}-burst$ in the glutamate train ejection. The ${\theta}-burst$ decreased the amplitude of glutamate response by 60%. However, after ${\theta}-burst$ glutamate train ejection, the calcium dependent action potential appeared. These results indicated that the focal application of glutamate on the neuronal dendrite induced response similar to the synaptic transmission and the trains of glutamate ejection modulated the change of AMPA receptor.

• The Korean Journal of Physiology and Pharmacology
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• 제11권3호
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• pp.79-84
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• 2007

Because synaptic refinement of medial nucleus of trapezoid body (MNTB) - lateral superior olive (LSO) synapses is most active during the first postnatal week and the long term depression (LTD) has been suggested as one of its mechanisms, LTD of MNTB-LSO synapses was investigated in neonatal rat brain stem slices with the whole cell voltage clamp technique. In $Mg^{2+}$ free condition, tetanus (10 stimuli at 10 Hz for 2 min) in the current clamp mode induced a robust LTD of isolated D, L-APV-sensitive postsynaptic currents (PSCs) for more than 30 min ($n=6,\;2.4{\pm}0.4%$ of the control), while isolated CNQX-sensitive PSCs were not suppressed ($n=6,\;95.3{\pm}1.6%$). Tetanus also elicited similar LTD in the isolated GABAergic/glycinergic PSCs ($n=6,\;3.6{\pm}0.5%$) and mixed PSCs (GABAergic/glycinergic/glutamatergic) ($n=4,\;2.2{\pm}0.7%$). However, such a strong LTD was not observed in the mixed PSCs when 10 mM EGTA was added in the internal solution (n=10), indicating that postsynaptic $Ca^{2+}$ rise is needed for the strong LTD. This robust LTD might contribute to the active synaptic refinement occurring during the first postnatal week.

• 동의생리병리학회지
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• 제17권4호
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• pp.996-1001
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• 2003

It is well known that oxidative stress of reactive oxygen species(ROS) may be a causative factor in the pathogenesis of bone disorder. The purpose of this study was to evaluate the cytotoxicity of oxidative stress. Cell viability by MTS assay or INT assay, activity of glutathione peroxidase(GPx), lipid peroxidation(LPO) activity and cell viablity. And also protctive effect of glutamate receptors against ROS-induced osteotoxicity was examined by protein synthesis, alkaline phosphatase (ALP) activity and lactate dehydrogenase (LDH) activity in cultured rat osteoblasts and osteoclasts. XO/HX decreased cell viability and GPx activity, protein synthesis and ALP activity, but increased LPO activity and LDH activity. In the protective effect, N-methyl-D-aspartate (NMDA) receptor antagonists or AMPA/kainate receptor antagonists such as D-2-amino-5-phosphonovaleric acid (APV), 7-chlorokynurenic acid (CKA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), NMDA receptor antagonists but AMPA/kainate receptor antagonists showed protective effect on xanthine oxidase (XO) and hypoxanthine (HX) in these cultures by the increse of protein synthesis, ALP activity.

• Journal of Acupuncture Research
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• 제17권4호
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• pp.5-17
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• 2000

To research the characteristics of ion currents induced by Bujaijung-tang and Bojungikgi-tang, nystatin-perforated patch clamp technique under voltage-c(amp condition was used. Periaqueductal gray neuron was dissociated from Sprauge-Dawley rat, 10-15 days old. Cytotoxicity of Bujaijung-tang and Bojungikgi-tang showed incubation time and concentration dependent manner. Ion current activated by Bujaijung-tang and Bojungikgi-tang were inhibited by bicuculline and strychnine and CNQX. It can be suggested that Bujaijung-tang and Bojungikgi-tang modulate inhibitory and excitatory neurotransmitters-, GABA, glycine and non-NMDA, acticvated ion channels. Modulatory effect of Bujaijung-tang and Bojungikgi-tang was more greater in inhibitory neurotransmitters. Low concentration of Bujaijung-tang which dose not elicit ion current itself, activated GABA and glycine induced chloride currents. In this study, we can found that the activation of Bujaijung-tang and Bojungikgi-tang on non-NMDA subtypes of glutamate receptor is its major action mechanism and can be used as very effective Herb treatment on Myasthenia gravis patient.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.209-214
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• 2009

The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by ${\alpha}$-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at - 70 and + 40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between + 50 and - 50 mV. NMDA/AMPA ratio was 0.20${\pm}$0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26${\pm}$0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32${\pm}$0.03. The rectification index (control 2.39${\pm}$0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02${\pm}$0.11 and 0.93${\pm}$0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.

• The Korean Journal of Physiology and Pharmacology
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• 제7권6호
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• pp.375-379
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• 2003

$Ca^{2+}$ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how $Ca^{2+}$ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in $Ca^{2+}$ mobilization of muscle, and that $Ca^{2+}$ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in $Ca^{2+}$ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a $Ca^{2+}$ imaging system, $Ca^{2+}$ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular $Ca^{2+}$ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular $Ca^{2+}$ increase was completely suppressed by treatment of nifedifine, a L-type $Ca^{2+}$ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular $Ca^{2+}$ increase in myoblasts and then may play an important role in myoblast fusion.

• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.467-475
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• 1997

Although one of the major physiological functions of taurine(2-aminoethanesulfonic acid) is the inhibitory action on the central nervous system(CNS), the mechanism of taurine in controlling the neuronal excitation in the CNS has been in controversy. Electrically evoked pEPSP and spontaneous activity induced by the perfusion of low $Mg^{++}-ACSF$ were recorded in the CA1 pyramidal cell layer of the hippocampal slice. To test the inhibitory effect of taurine on spontaneous responses, taurine was treated for 2 min at various concentrations(1 mM-10 mM). Taurine reduced the spontaneous activity by 22.2% at 1 mM, and 100% at 2 mM in low $Mg^{++}-ACSF$. Evoked response was induced by electrical stimulation of Schaffer collateral-commissural fibers. Taurine reduced the evoked response by 11.68% at 3 mM, and 24.25% at 5 mM. Even 20 mM of taurine reduced the evoked response only by 24 % after 5 min treatment. That is, the inhibitory efficacy was much higher in spontaneous activity than in evoked response. The $GABA_A$ receptor antagonist, 100 uM bicuculline, blocked the inhibitory action of taurine, while $GABA_B$ receptor antagonist, 700 uM phaclofen, did not. Taurine blocked the spontaneous activity in the presence of CNQX, and did not block the electrically evoked responce in the presence of APV. The results suggest that taurine causes hyperpolarization in the cell by binding to $GABA_A$ receptor and preferentially attenuates NMDA receptor-mediated hyperexcitation, leaving synaptic transmission unmodified.

• The Korean Journal of Physiology and Pharmacology
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• 제7권2호
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• pp.53-57
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• 2003

The glutamate receptors (GluRs) are key receptors for modulatory synaptic events in the central nervous system. It has been reported that glutamate increases the intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) and induces cytotoxicity. In the present study, we investigated whether the glutamate-induced $[Ca^{2+}]_i$ 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${\sim}21 day). $[Ca^{2+}]_i$ 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 extracellular $Ca^{2+}$, the inward current and $[Ca^{2+}]_i$ increase were induced by membrane depolarization and application of glutamate. Dose-response relationship with glutamate was observed in both $Ca^{2+}$ signal and inward current. The glutamate-induced $[Ca^{2+}]_i$ 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 $[Ca^{2+}]_i$ increase in $Ca^{2+}$ free condition was not affected by iGluR blockers. A selective mGluR (group I) agonist, RS-3,5-dihydroxyphenylglycine (DHPG), induced $[Ca^{2+}]_i$ increase at holding potential of -70 mV in SG neurons. These findings suggest that the glutamate-induced $[Ca^{2+}]_i$ increase is associated with AMPA-sensitive iGluR and group I mGluR in SG neurons of rats.