• International Journal of Oral Biology
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• v.33 no.3
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• pp.83-89
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• 2008

Glutamate-induced cobalt uptake reveals non-NMDA glutamate receptors (GluRs) in rat taste bud cells. Previous studies suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs. Cobaltstained cells were immunoreactive against GluR6 and KA1 subunits of GluRs. However, the functions of those type of receptors are not known yet. It is important question which types of taste cells are cobalt-stained when stimulated by glutamate and whether they express these kinds of GluRs. Circumvallate and foliate papilla of Sprague-Dawley rats (45-60 days old) were used. A cobalt-staining technique combined with immunohistochemistry against specific markers for taste bud cell types, such as blood group H antigen (BGH), $\alpha$-gustducin (Gus), or neural cell adhesion molecule (NCAM) was employed. We also performed double labeling of GluR6 or KA1 subunits of GluR with each specific marker for taste bud cell types. Lots of cobaltstained taste bud cells expressed Gus-like immunoreactivity, and subsets of the cobalt stained cells appeared NCAM- or BGH-like immunoreactivity. Stimulation with 1 mM glutamate significantly increased the number of cobaltstained cells in Gus-like immunoreactive cells, but not in NCAM- or BGH-like immunoreactive cells. In the double labeling experiments, GluR6 and KA1 subunits of GluRs were mainly expressed with Gus. These results suggest that kainate glutamate receptors preferentially expressed in type II taste bud cells in rat.

• The Korean Journal of Pain
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• v.23 no.1
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• pp.1-10
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• 2010

Background: Recent studies indicate that reactive oxygen species (ROS) are involved in persistent pain, including neuropathic and inflammatory pain. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N-methyl-D-aspartate (NMDA) receptors in the dorsal horn after an exogenous supply of three antioxidants in rats with chronic post-ischemia pain (CPIP). This serves as an animal model of complex regional pain syndrome type-I induced by hindpaw ischemia/reperfusion injury. Methods: The application of tight-fitting O-rings for a period of three hours produced CPIP in male Sprague-Dawley rats. Allopurinol 4 mg/kg, allopurinol 40 mg/kg, superoxide dismutase (SOD) 4,000 U/kg, N-nitro-L-arginine methyl ester (L-NAME) 10 mg/kg and SOD 4,000 U/kg plus L-NAME 10 mg/kg were administered intraperitoneally just after O-ring application and on the first and second days after reperfusion. Mechanical allodynia was measured, and activation of the NMDA receptor subunit 1 (pNR1) of the lumbar spinal cord (L4-L6) was analyzed by the Western blot three days after reperfusion. Results: Allopurinol reduced mechanical allodynia and attenuated the enhancement of spinal pNR1 expression in CPIP rats. SOD and L-NAME also blocked spinal pNR1 in accordance with the reduced mechanical allodynia in rats with CPIP. Conclusions: The present data suggest the contribution of superoxide, produced via xanthine oxidase, and the participation of superoxide and nitric oxide as a precursor of peroxynitrite in NMDA mediated central sensitization. Finally, the findings support a therapeutic potential for the manipulation of superoxide and nitric oxide in ischemia/reperfusion related pain conditions.

• Archives of Pharmacal Research
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• v.24 no.1
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• pp.64-68
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• 2001

Endothelin-1 (ET-1 ), a novel and potent vasoconstrictor in blood vessel, is known to have some functions in the rat central nervous system (CNS), In order to investigate the central functions of ET-1 , ET-1 was administered to the periaqueductal gray area (PAC) of anesthetized rats to induce barrel rolling and increase the arterial blood pressure (ABP). ET-1 had a modulatory effect on central cardiovascular and behavioral control. The selective N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (3${u}m/ol/kg$, i.p.) blocked the ET-1 induced responses, and both the nitric oxide synthase (NOS) inhibitor L-NAME (N-nitro-L-arginine mIThyl-ester 1 nmol/rat) and the nitric oxide (NO) scavenger hemoglobin (15 nmol/rat) had similar effects in redtAcing the IT-1 (10 pmol/rat)-induced behavioral changes and ABP elevation. However, NO donor sodium nitroprusside (SNP 10${u}g$, 1${u}g/rat$) decreased the ET-1 induced ABP elevation, and recovered the ET-1 -induced barrel rolling effect that was reduced by MK-801. These results suggest that ET-1 might have neuromodulatory functions such as ABP elevation and barrel rolling induction in the PAG of the rats via the NMDA receptor and NO.

• Applied Microscopy
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• v.40 no.2
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• pp.53-64
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• 2010

Glutamate receptors may play a critical role in the refinement of developing synapses. The lateral superior olivary nucleus (LSO)-medial nucleus of trapezoid body (MNTB) synaptic transmission in the mammalian auditory brain stem mediate many excitatory transmitters such as glutamate, which is a useful model to study excitatory synaptic development. Hearing deficits are often accompanied by changes in the synaptic organization such as excitatory or inhibitory circuits as well as anatomical changes. Owing to this, circling mouse whose cochlea degenerates spontaneously after birth, is an excellent animal model to study deafness pathophysiology. However, little is known about the development regulation of the subunits composing these receptors in circling mouse. Thus, we used immunohistochemical method to compare the N-Methyl-D-aspartate receptor (NMDA receptor) NR1, NR2A, NR2B distribution in the LSO which project glutamergic excitatory input into the auditory brainstem, in circling mouse of postnatal (p) 7 and 16, which have spontaneous mutation in the inner ear, with wild-type mouse. The relative NMDAR1 immunoreactive density of the LSO in circling mouse p7 was $128.67\pm8.87$ in wild-type, $111.06\pm8.04$ in heterozygote, and $108.09\pm5.94$ in homozygote. The density of p16 circling mouse was $43.83\pm10.49$ in wild-type, $40\pm13.88$ in heterozygote, and $55.96\pm17.35$ in homozygote. The relative NMDAR2A immunoreactive density of LSO in circling mouse p7 was $97.97\pm9.71$ in wild-type, $102.87\pm9.30$ in heterozygote, and $106.85\pm5.79$ in homozygote. The density of LSO in p16 circling was $47.4\pm20.6$ in wild-type, $43.9\pm17.5$ in heterozygote, and $49.2\pm20.1$ in homozygote. The relative NMDAR2B immunoreactive density of LSO in circling mouse p7 was $109.04\pm6.77$ in wild-type, $106.43\pm10.24$ in heterozygote, and $105.98\pm4.10$ in homozygote. the density of LSO in p16 circling mouse was $101.47\pm11.5$ in wild-type, $91.47\pm14.81$ in heterozygote, and $93.93\pm15.71$ in homozygote. These results reveal alteration of NMDAR immunoreactivity in LSO of p7 and p16 circling mouse. The results of the present study are likely to be relevant to understand the central change underlying human hereditary deafness.

• Journal of Life Science
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• v.9 no.2
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• pp.34-39
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• 1999

The postsynaptic density (PSD) is a cytoskeletal specialization that is involved in the regulation of synaptic signal transduction. Mainly due to the hydrophobic nature of the PSD proteins, characterization of this intriguing structure at the molecular level has been very intractable until early 1990s. However, recent development in protein microchemistry and molecular cloning techniques allowed identification and characterization of the PSD proteins. As expected, cytoskeletal proteins constitute major components of the PSD. Other major PSD proteins have been identified by protein sequencing, and their genes were used to fish out associating proteins by yeast two-hybrid system expanding our knowledge on the molecular structure of the PSD significantly. In this review, I summarize proteins that are so far identified focusing on the glutamatergic synapses.

• Development and Reproduction
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• v.19 no.2
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• pp.61-67
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• 2015

The immediate early gene c-fos has long been known as a molecular marker of neural activity. The neuron's activity is transformed into intracellular calcium influx through NMDA receptors and L-type voltage sensitive calcium channels. For the transcription of c-fos, neural activity should be strong enough to activate mitogen-activated protein kinase (MAPK) signaling pathway which shows low calcium sensitivity. Upon translation, the auto-inhibition by Fos protein regulates basal Fos expression. The pattern of external stimuli and the valence of the stimulus to the animal change Fos signal, thus the signal reflects learning and memory aspects. Understanding the features of multiple components regulating Fos signaling is necessary for the optimal generation and interpretation of Fos signal.

• The Korean Journal of Pain
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• v.26 no.3
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• pp.255-264
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• 2013

Background: We investigated the effects of pre-emptive administration of ketamine and norBNI on pain behavior and the expression of DREAM, c-Fos, and prodynorphin proteins on the ipsilateral side of the rat spinal cord at 2 and 4 hours after formalin injection. Methods: Eighty-four male Sprague Dawley rats were divided into 4 major groups consisting of control rats (C) (n = 12), rats given only formalin injections (F) (n = 24), and rats treated with pre-emptive administration of either ketamine (K+F) (n = 24) or norBNI (N+F) (n = 24). The non-control groups were further divided into subgroups consisting of rats that were sacrificed at 2 and 4 hours (n = 12 for each group) after formalin injection. Pain behavior was recorded for 1 hour. After 2 and 4 hours, the rats were sacrificed and the spinal cords (L4-L5 sections) were removed for immunohistochemistry and Western blot analysis. Results: The pain behavior response was reduced in the K+F group compared to the other groups during the second phase of the formalin pain response. We detected an increase in the nuclear DREAM protein level in the K+F group at 2 and 4 hours and a transient decrease in the N+F group at 2 hours; however, it increased at 4 hours after injection. Fos-like immunoreactivity (FLI) and Prodynorphin-like immunoreactivity (PLI) neurons decreased in the K+F group but increased in the N+F group at 2 hours after injection. While FLI decreased, PLI increased in all groups at 4 hours after injection. Conclusions: We suggest that NMDA and kappa opioid receptors can modulate DREAM protein expression, which can affect pain behavior and protein transcriptional processes at 2 hours and bring about either harmful or protective effects at 4 hours after formalin injection.

• International Journal of Oral Biology
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• v.33 no.4
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• pp.217-221
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• 2008

Bicuculline is one of the most commonly used $GABA_A$ receptor 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-$GABA_A$ receptor-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 ($20{\mu}M$) using a high $Cl^-$ pipette solution. GABA ($30-100{\mu}M$) also induced membrane depolarization of SG neuron. Although BMI is known to be a $GABA_A$ receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a $GABA_A$ and $GABA_C$ 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 ($Na^+$ channel 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 $GABA_A$ receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.3
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• pp.275-281
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• 2015

Orthostatic hypotension is most common in elderly people, and its prevalence increases with age. Attenuation of the vestibulo-sympathetic reflex (VSR) is commonly associated with orthostatic hypotension. In this study, we investigated the role of glutamate on the vestibulo-solitary projection of the VSR pathway to clarify the pathophysiology of orthostatic hypotension. Blood pressure and expression of both pERK and c-Fos protein were evaluated in the nucleus tractus solitarius (NTS) after microinjection of glutamate into the medial vestibular nucleus (MVN) in conscious rats with sodium nitroprusside (SNP)-induced hypotension that received baroreceptor unloading via sinoaortic denervation (SAD). SNP-induced hypotension increased the expression of both pERK and c-Fos protein in the NTS, which was abolished by pretreatment with glutamate receptor antagonists (MK801 or CNQX) in the MVN. Microinjection of glutamate receptor agonists (NMDA or AMPA) into the MVN increased the expression of both pERK and c-Fos protein in the NTS without causing changes in blood pressure. These results indicate that both NMDA and AMPA receptors play a significant role in the vestibulo-solitary projection of the VSR pathway for maintaining blood pressure, and that glutamatergic transmission in this projection might play a key role in the pathophysiology of orthostatic hypotension.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.259-265
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• 2017

Excessive influx and the subsequent rapid cytosolic elevation of $Ca^{2+}$ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic $Ca^{2+}$ level in normal as well as pathological conditions. Delayed rectifier $K^+$ channels ($I_{DR}$ channels) play a role to suppress membrane excitability by inducing $K^+$ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under $Ca^{2+}$-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of $I_{DR}$ channels to hyperexcitable conditions induced by high $Ca^{2+}$ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high $Ca^{2+}$-treatment significantly increased the amplitude of $I_{DR}$ without changes of gating kinetics. Nimodipine but not APV blocked $Ca^{2+}$-induced $I_{DR}$ enhancement, confirming that the change of $I_{DR}$ might be targeted by $Ca^{2+}$ influx through voltage-dependent $Ca^{2+}$ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated $I_{DR}$ enhancement was not affected by either $Ca^{2+}$-induced $Ca^{2+}$ release (CICR) or small conductance $Ca^{2+}$-activated $K^+$ channels (SK channels). Furthermore, PP2 but not H89 completely abolished $I_{DR}$ enhancement under high $Ca^{2+}$ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for $Ca^{2+}$-mediated $I_{DR}$ enhancement. Thus, SFKs may be sensitive to excessive $Ca^{2+}$ influx through VDCCs and enhance $I_{DR}$ to activate a neuroprotective mechanism against $Ca^{2+}$-mediated hyperexcitability in neurons.