Ca2+ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca2+ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in Ca2+ mobilization of muscle, and that Ca2+ 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 Ca2+ 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 Ca2+ imaging system, Ca2+ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular Ca2+ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular Ca2+ increase was completely suppressed by treatment of nifedifine, a L-type Ca2+ 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 Ca2+ increase in myoblasts and then may play an important role in myoblast fusion.
6-Cyano-7-nitroquinoxaline-2,3-dione ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Kainic Acid* ; Muscle Fibers, Skeletal ; Myoblasts ; Receptors, AMPA ; Receptors, Ionotropic Glutamate ; Receptors, Kainic Acid* ; Receptors, N-Methyl-D-Aspartate

metabotropic glutamate receptors on excitatory synaptic transmission. In the presence of strychnine (1 microM) and bicuculline (20 microM), spontaneous inward currents at -70 mV were observed. Spontaneous excitatory postsynaptic currents (sEPSCs) were mediated by non-NMDA and NMDA receptors. The specific AMPA receptor antagonist GYKI 52466 (50 microM) completely blocked the non-NMDA mediated sEPSCs, indicating that they are mediated by an AMPA-preferring receptor. Group I, II metabotropic glutamate receptor (mGluR) agonist ACPD (20 microM) significantly decreased the average sEPSCs frequency. No significant effect on sEPSCs amplitude was observed. Frequency of miniature excitatory postsynaptic currents (mEPSCs) was significantly decreased by 20 microM ACPD, but amplitude of mEPSCs was not changed. The specific mGluR 2, 3 agonist DCG-IV (3 microM) reduced mEPSCs frequency, but did not change mEPSCs amplitude. There were no significant changes in the average sEPSCs frequency and amplitude. The mGluR 1, 5 agonist DHPG (100 microM) decreased sEPSCs and mEPSCs frequency, but had no effects on sEPSCs and mEPSCs amplitude. These data suggest that the activation of metabotropic glutamate receptors, via its presynaptic inhibition, can influence the excitatory synaptic transmission of MDH neurons.]]>
Animals ; Bicuculline ; Brain Stem ; Excitatory Postsynaptic Potentials ; Horns ; Neurons ; Rats ; Receptors, AMPA ; Receptors, Metabotropic Glutamate ; Receptors, N-Methyl-D-Aspartate ; Strychnine ; Synaptic Transmission ; Trigeminal Nucleus, Spinal

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and phosphorylates a large number of synaptic proteins, including major ionotropic glutamate receptors (iGluRs) and group I metabotropic glutamate receptors (mGluRs), to constitutively and/or activity-dependently regulate trafficking, subsynaptic localization, and function of the receptors. Among iGluRs, the N-methyl-D-aspartate receptor (NMDAR) is a direct target of CaMKII. By directly binding to an intracellular C-terminal (CT) region of NMDAR GluN2B subunits, CaMKII phosphorylates a serine residue (S1303) in the GluN2B CT. CaMKII also phosphorylates a serine site (S831) in the CT of α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors. This phosphorylation enhances channel conductance and is critical for synaptic plasticity. In addition to iGluRs, CaMKII binds to the proximal CT region of mGluR1a, which enables the kinase to phosphorylate threonine 871. Agonist stimulation of mGluR1a triggers a CaMKII-mediated negative feedback to facilitate endocytosis and desensitization of the receptor. CaMKII also binds to the mGluR5 CT. This binding seems to anchor and accumulate inactive CaMKII at synaptic sites. Active CaMKII dissociates from mGluR5 and may then bind to adjacent GluN2B to mediate the mGluR5-NMDAR coupling. Together, glutamate receptors serve as direct substrates of CaMKII. By phosphorylating these receptors, CaMKII plays a central role in controlling the number and activity of the modified receptors and determining the strength of excitatory synaptic transmission.
Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Neuronal Plasticity ; Phosphorylation ; Receptor, Metabotropic Glutamate 5 ; metabolism ; Receptors, Metabotropic Glutamate ; metabolism ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Serine ; metabolism ; Synapses ; Synaptic Transmission

BACKGROUND: Excitotoxicity and epileptogenesis have often been associated with glutamate receptor activation. Some evidence indicates that selective down regulation of AMPA receptor may be the mechanism of delayed neuronal cell death in the hippocampus. METHODS: We used in situ hybridization to examine the hybridization density (HD) of NMDA and AMPA receptors on excitotoxicity and epileptogenesis in the hippocampus of the kainic acid (KA)-induced rat seizure model. Some Sprague-Dawley rats were injected with KA, and others with MK-801 prior to KA injection. The rats were killed at 8 hours or 4 weeks after KA or MK-801/KA injection. HD of [3H]MK-801 and [3H]AMPA bind-ing in subfields of the hippocampus was measured by an image analyzer. RESULTS: After 8 hours of KA injection, [3H]MK-801 binding was increased in CA1 and CA3, and decreased in dentate gyrus, and [3H]AMPA binding was decreased in all of CA1, CA3 and fascia dentata, and pretreatment of MK-801 did not affect [3H]AMPA binding in all of CA1, CA3 and dentate gyrus. After 4 weeks, both [3H]MK-801 and [3H]AMPA binding were prominently increased in inner molecular layer of dentate gyrus. CONCLUSIONS: Glutamate receptors, especially NMDA receptor, were associ-ated with excitotoxicity in the hippocampus but the selective down regulation of GluR2 subunit of AMPA receptor without NMDA receptor activation may not be sufficient to cause excitotoxic neuronal cell death in CA1 and CA3. In addition, the synaptic reorganization in inner molecular layer of dentate gyrus was proved to be chronically hyperex-citable in function and may contribute epileptogenesis.
Animals ; Autoradiography* ; Cell Death ; Dentate Gyrus ; Dizocilpine Maleate* ; Down-Regulation ; Glutamic Acid* ; Hippocampus ; In Situ Hybridization ; Kainic Acid ; N-Methylaspartate ; Neurons ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; Receptors, Glutamate* ; Receptors, Ionotropic Glutamate* ; Seizures*

OBJECTIVETo investigate the expression of metabotropic glutamate receptor 5 (mGluR5) and its cellular distribution in the frontal cortex, ventricular zone (VZ) and subventricular zone (SVZ) in human fetuses.

METHODSAccording to the gestational age, the collected fetuses were divided into 4 groups, namely 9-11 weeks, 14-16 weeks, 22-24 weeks and 32-36 weeks. Brain tissue blocks including the frontal lobe or VZ/SVZ were prepared into slices, and the expression pattern and cellular distribution of mGluR5 in the frontal cortex and VZ/SVZ were observed by immunohistochemistry or immunofluorescence.

RESULTSmGluR5 immunoreactivity was present in the cell membrane in the frontal cortex, VZ and SVZ from the 9th to 36th weeks and the immunoreactivity in the marginal zone (MZ) and cortical plate (CP) was markedly stronger than that in VZ and SVZ. The cells expressing mGluR5 included neural stem/progenitor cells in the VZ and SVZ, immature neurons in the VZ and MZ, and numerous mature neurons in the CP.

CONCLUSIONmGluR5 is expressed by a variety of cells such as neural stem cells in the frontal cortex, VZ and SVZ in human fetus, suggesting a role of mGluR5 in the development of human cerebral cortex.

Cerebral Cortex ; cytology ; Cerebral Ventricles ; cytology ; metabolism ; Fetus ; cytology ; metabolism ; Frontal Lobe ; cytology ; metabolism ; Humans ; Neural Stem Cells ; cytology ; metabolism ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; metabolism

OBJECTIVE: To observe the expression of mGluR5 in the medial vestibular nucleus (MVN) following unilateral labyrinthectomy (UL). METHOD: Thirty Wistar rats were randomly divided into two groups. Twenty four animals received unilateral labyrinthectomy while the others maintained labyrinthine well. After setting left labyrinthine, the change of mGluR5 was induced by immunohistochemistry, in situ hybridization. RESULT: mGluR5 was increased in lesioned side MVN after unilateral labyrinthectomy. The 12 h post-UL was highest. Then it was decrease in 36 h post-UL, while 7 d post-UL was same as control group. The change of contralateral was same as that in ipsilateral. CONCLUSION UL can induce increase of mGluR5 in the MVN. The reduced resting discharge in the primary vestibular afferents or in the central vestibular neurons may be responsible for the change of mGluR5. However the significance of the change of mGluR1 in the vestibular compensation is still unknown.
Animals ; Male ; Postoperative Period ; Rats ; Rats, Wistar ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; metabolism ; Vestibular Nuclei ; metabolism ; Vestibule, Labyrinth ; metabolism ; surgery

OBJECTIVE: Studies revealed that cerebellar flocculus-paraflocculus complex plays an important in vestibular compensation. To observe the expression change of Group I mGluRs in flocculus following left unilateral labyrinthectomy (UL). METHOD: After setting left labyrinthectomy, the change of Group I mGluRs was detected by RT-PCR. RESULT: Group I mGluR5 was induced increase in both side flocculus after unilateral labyrinthectomy. By contrast, mGluR1 was induced decrease. However, that of the lesioned side was stronger than that of the unlesioned side. CONCLUSION UL can induce change of Group I mGluRs in the flocculus. The fall in the resting discharge of the primary vestibular afferents and/or in the deafferented central vestibular neurons may cause the change of mGluRs. But the significance of the change of mGluRs in the vestibular compensation is still unknown.
Animals ; Cerebellar Nuclei ; metabolism ; Male ; Rats ; Rats, Wistar ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; metabolism ; Vestibular Nuclei ; metabolism ; Vestibule, Labyrinth ; metabolism

BACKGROUND: Glutamate is the predominant excitatory neurotransmitter in the central and peripheral nervous system and has known to be involved in nociceptive transmission and central sensitization. It acts through ligand gated ionotropic glutamate receptors (iGluRs) and G protein-coupled metabotropic glutamate receptors (mGluRs). And mGlu 1, 5 receptors have been recognized to play a role in nociceptive processing. We want to investigate whether central mGluR1 and mGluR5 antagonists could reverse the behavioral signs of weight bearing and secondary mechanical hyperalgesia induced by chronic knee joint inflammation. METHODS: MGluR1 antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA: 25, 50, 100 microM/10 microliter, n = 7 per group) and selective mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP: 25, 100, 200 nM/10 microliter, n = 7 per group) were injected intrathecally 5 days after Complete Freund's Adjuvant (CFA, 150 microliter) injection into knee joint and behavior signs of weight bearing and secondary mechanical hyperalgesia were observed. RESULTS: CFA significantly reduced the magnitude of right hind paw weight bearing and decreased the withdrawal threshold to mechanical stimulation compared to contralateral side. Higher dose of AIDA (100 microM) significantly reversed the reduction of weight bearing, but MPEP did not. AIDA reversed the decrease of the paw withdrawal threshold to mechanical stimulation at the dosage of 50 and 100 microM respectively. MPEP significantly increased the paw withdrawal threshold to mechanical stimulation in a dose dependent manner. CONCLUSIONS: Group I mGluRs were involved in maintenance of primary and secondary mechanical hyperalgesia.
Animals ; Arthritis ; Central Nervous System Sensitization ; Freund's Adjuvant ; Glutamic Acid ; Hyperalgesia ; Inflammation ; Knee Joint* ; Knee* ; Neurotransmitter Agents ; Peripheral Nervous System ; Rats* ; Receptors, Ionotropic Glutamate ; Receptors, Metabotropic Glutamate ; Weight-Bearing

OBJECTIVETo study the effects on the expression of mGluR5 mRNA and protein levels in primarily cultured hippocampal neurons after lead exposure.

METHODSPrimary embryonic rat hippocampal neuronal culture was prepared. On the 3(rd) day of incubation, lead chloride solution was added into medium to produce four different lead exposure levels: 0, 1 x 10(-8) mol/L, 1 x 10(-6) mol/L, 1 x 10(-4) mol/L Pb(2+). After 10 days of incubation, the neurons were collected to measure the alteration of mGluR5 mRNA expression by real-time fluorescent quantity PCR and the expression of mGluR5 in protein level by Western blot.

RESULTSThe studies revealed that mGluR5 mRNA expression was down-regulated after lead exposure in a dose-dependent manner. The mGluR5 mRNA expression of the lower lead-exposed neurons (Pb(2+) 10(-8) mol/L), the medium lead-exposed neurons(Pb(2+) 10(-6) mol/L), the higher lead-exposed neurons(Pb(2+) 10(-4) mol/L) were 0.724, 0.421, 0.321 times less than that of the controls, respectively. The Western blot demonstrated that mGluR5 expression in protein level should be decreased after lead exposure.

CONCLUSIONThe expression of mGluR5 in mRNA and protein levels should be down-regulated after lead exposure at different lead levels in a dose-dependent manner.

Animals ; Animals, Newborn ; Cells, Cultured ; Female ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Lead Poisoning, Nervous System ; metabolism ; Neurons ; drug effects ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; biosynthesis ; genetics

OBJECTIVETo investigate the expression changes of metabotropic glutamate receptor 5 (mGluR5) in neuropathic pain.

METHODSEighty-four adult male Sprague Dawley rats weighing 180-220 g were randomly divided into 7 groups (n = 12) : control group; S3, S7, and S14 groups: rats received the sham operation, the mechanical pain threshold was measured, and then the rats were decapitated and the ipsilateral lumbar spinal cord dorsal horn and dorsal root ganglion (DRG) samples were obtained on the 3rd, 7th, 14th postoperative day, respectively; C3, C7, and C14 groups: the chronic sciatic nerve constriction (CCI) model was established, the mechanical pain threshold was measured and the samples were obtained on the 3rd, 7th, 14th postoperative day, respectively. The expression level of mGluR5 mRNA and protein in the spinal cord and DRG were measured using the reverse transcriptase polymerase chain reaction and Western blot.

RESULTSIn the CCI group, the mechanical pain threshold in each observation day was significantly lower than in the sham operation group (P < 0.05). In the spinal cord, the expressions of mGluR5 mRNA and protein were significantly elevated in the C3 group than in the S3 and the control group (P < 0.05). On the 7th and the 14th postoperative day, no significant difference was found in the expression of mGluR5 mRNA and protein between CCI groups and the sham operation groups or the control group. No change was detected in DRG mRNA or protein.

CONCLUSIONmGluR5 is differentially expressed in spinal cord in response to neuropathic pain, which suggests that mGluR5 may be involved in the mechanism of neuropathic pain.

Animals ; Disease Models, Animal ; Ganglia, Spinal ; metabolism ; Male ; Neuralgia ; metabolism ; Pain Threshold ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; genetics ; metabolism ; Sciatic Neuropathy ; metabolism ; Spinal Cord ; metabolism