Association between glutamate receptor 2 polymorphisms and epilepsy in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the association between two single nucleotide polymorphisms (SNPs), rs9390754 and rs4840200, in the glutamate receptor 2 (GRIK2) gene and the genetic susceptibility to epilepsy (EP) in the Han population in Central China.</p><p><b>METHODS</b>A case-control study was performed in 284 EP children (including 132 children with refractory epilepsy) and 315 normal children from Central China. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotypes of the two SNPs rs9390754 and rs4840200. The genotype frequency was compared between groups.</p><p><b>RESULTS</b>The frequencies of GG, GA, and AA genotypes of SNP rs9390754 showed a significant difference between the EP and normal control groups (P=0.016). The allele frequency also showed a significant difference between the two groups (P=0.002). The frequencies of CC, CT, and TT genotypes of SNP rs4840200 and allele frequency showed no significant differences between the two groups. The C allele frequency of SNP rs4840200 in the refractory EP subgroup was significantly higher than in the non-refractory EP subgroup (OR=1.435, 95% CI: 1.021-2.016, P=0.037).</p><p><b>CONCLUSIONS</b>In the Han population in Central China, the polymorphisms of SNP rs9390754 in the GRIK2 gene may be associated with EP susceptibility, and the C allele of SNP rs4840200 may be a genetic risk factor for the development of drug resistance in children with EP.</p>

Alpha1-Adrenoceptor Antagonists Improve Memory by Activating N-methyl-D-Aspartate-Induced Ion Currents in the Rat Hippocampus / 대한배뇨장애요실금학회지

PURPOSE: Alpha1 (alpha1)-adrenoceptor antagonists are widely used to treat lower urinary tract symptoms. These drugs not only act on peripheral tissues, but also cross the blood-brain barrier and affect the central nervous system. Therefore, alpha1-adrenoceptor antagonists may enhance brain functions. In the present study, we investigated the effects of tamsulosin, an alpha1-adrenoceptor antagonist, on short-term memory, as well as spatial learning and memory, in rats. METHODS: The step-down avoidance test was used to evaluate short-term memory, and an eight-arm radial maze test was used to evaluate spatial learning and memory. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) staining was performed in order to evaluate the effect of tamsulosin on apoptosis in the hippocampal dentate gyrus. Patch clamp recordings were used to evaluate the effect of tamsulosin on ionotropic glutamate receptors, such as N-methyl-D-aspartate (NMDA), amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate receptors, in hippocampal CA1 neurons. RESULTS: Tamsulosin treatment improved short-term memory, as well as spatial learning and memory, without altering apoptosis. The amplitudes of NMDA-induced ion currents were dose-dependently increased by tamsulosin. However, the amplitudes of AMPA- and kainate-induced ion currents were not affected by tamsulosin. CONCLUSIONS: Tamsulosin enhanced memory function by activating NMDA receptor-mediated ion currents in the hippocampus without initiating apoptosis. The present study suggests the possibility of using tamsulosin to enhance memory under normal conditions, in addition to its use in treating overactive bladder.

Role of KA1 receptor in excitotoxic neurodegeneration in mouse hippocampus triggered by kainic acid- or tunicamycin-induced endoplasmic reticulum stress / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of up-regulation of KA1 subunit of the kainate receptor on endoplasmic reticulum stress (ERS)-induced excitotoxic neurodegeneration in mouse hippocampus.</p><p><b>METHODS</b>Seventy adult male KM mice were subjected to microinjections into the hippocampus of kainic acid (KA) or 500, 1000, or 2000 µg/ml tunicamycin (TM). At 1, 2, 3, 4, 5, 8, and 12 h after the injections, the mice were assessed for Bederson scores and sacrificed for FJB staining and immunofluorescence observation of the brain slices.</p><p><b>RESULTS</b>At 3, 4, 5, and 8 h after KA injection and at 4 and 5 h after of 2000 µg/ml TM injection, the mice showed severe central nervous system dysfunction, and FJB staining revealed increased cell death in the hippocampus, where up-regulated expressions of KA1 receptor and ERS marker P-eIF2α were found by immunofluorescence staining (P<0.05).</p><p><b>CONCLUSION</b>Microinjection of KA or TM into the hippocampus causes neuronal death and ERS with up-regulated expression of KA1. In this process of neuronal apoptosis, the membrane receptor KA1 receives the apoptosis signal and transfers it to the inside of the cells to cause cell endoplasmic reticulum dysfunction and ERS response, which ultimately leads to neuronal death.</p>

Expression of KA1 kainate receptor subunit in the substantia gelatinosa of the trigeminal subnucleus caudalis in mice

The KA1 kainate receptor (KAR) subunit in the substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. This study compared the expression of the KA1 KAR subunit in the SG of the Vc in juvenile, prepubescent and adult mice. RT-PCR, Western blot and immunohistochemistry analyses were used to examine the expression level in SG area. The expression levels of the KA1 KAR subunit mRNA and protein were higher in juvenile mice than in prepubescent or adult mice. Quantitative data revealed that the KA1 KAR subunit mRNA and protein were expressed at levels approximately two and three times higher, respectively, in juvenile mice than in adult mice. A similar expression pattern of the KA1 KAR subunit was observed in an immunohistochemical study that showed higher expression in the juvenile (59%) than those of adult (35%) mice. These results show that the KA1 KAR subunits are expressed in the SG of the Vc in mice and that the expression level of the KA1 KAR subunit decreases gradually with postnatal development. These findings suggest that age-dependent KA1 KAR subunit expression can be a potential mechanism of age-dependent pain perception.

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Effect of ketogenic diet on hippocampus synaptic reorganization and GluR5 expression in kainic acid induced rat model of epilepsy / 中华儿科杂志

<p><b>OBJECTIVE</b>Ketogenic diet (KD) is a high fat, low protein, low carbohydrate diet. Its antiepileptic effect is certain but the underlying mechanism is unknown. The aim of the study was to reveal the possible mechanism from the view points of synaptic reorganization and GluR(5) expression in hippocampus.</p><p><b>METHODS</b>Epilepsy was induced in Sprague-Dawley rats by kainic acid at postnatal day 28, all control animals were fed with normal rodent chow, whereas experimental rats were fed with ketogenic feed for 8 weeks. Spontaneous recurrent seizures were recorded. Mossy fiber sprouting and neuron damage in hippocampus were investigated by Timm staining and Nissl staining. Western blot and RT-PCR methods were applied to detect the expression of GluR(5) and GluR(5) mRNA in hippocampus.</p><p><b>RESULTS</b>KD-fed rats (1.40 +/- 1.03) had significantly fewer spontaneous recurrent seizures than control diet-fed rats (7.36 +/- 3.75). The mean A of mossy fiber sprouting in the inner molecular layer of dentate gyrus was markedly higher in KA induced animals than that in saline control animals but it was similar in different diet fed groups. No significant differences were found in the mean A of Timm staining in CA(3) area and Nissl staining of neuron in hilus, CA(3) and CA(1) area. After KA kindling, KD-fed animals [(189.38 +/- 40.03)/mg pro] had significantly higher GluR(5) expression in hippocampus than control diet-fed animals [(128.79 +/- 46.51)/mg pro] although their GluR(5) mRNA was the same.</p><p><b>CONCLUSION</b>Mossy fiber sprouting may be responsible for epileptogenesis in KA induced model and KD can suppress seizures in these animals. KD may upregulate young rat GluR(5) in inhibitory interneurons of CA(1) thus lead to an increased inhibition to prevent the propagation of seizure.</p>

Hypoxia-A Possibility in Fibromyalgia Syndrome Pathogenesis / 대한류마티스학회지

OBJECTIVE: We studied the expression of pain-related molecules such as substance P involved in chronic pain of fibromyalgia syndrome (FMS) patients using rat cortical cells in hypoxia. METHODS: We sacrificed pregnant Sprague-Dawley rat and got embryo. We cultured the cortical cells and compared the expression of pain-related molecules in 1st, 3rd, 5th day cortical cells exposed to hypoxia (37 degrees C, 5% CO2, 98% N2) to control by immunohistochemistry. We measured the density at soma using softwear 'Scion image'. RESULTS: The expression of substance P was increased in hypoxic cortical cell group than control (control mean: 49.9 vs. hypoxia 1st day: 75.4 (p<0.001), 3rd day: 65.6 (p<0.001), 5th day: 79.9 (p<0.001)). The expression of kainate receptor was increased in hypoxic cortical cell group than control (control mean: 58.4 vs. hypoxia 1st day: 64.9 (p<0.001), 3rd day: 63.3 (p<0.001), 5th day: 62.9 (p<0.001)). The expression of N-methyl-D-aspartate receptor 2B was increased in hypoxic cortical cell group than control (control mean : 59.4 vs. hypoxia 1st day: 60.8 (p<0.001), 3rd day: 62.6 (p<0.001), 5th day: 67.1 (p<0.001)). But, the expression of calcitonin gene related peptide was decreased in hypoxic cortical cell group than control (control mean: 76.8 vs. hypoxia 1st day: 76.4 (p<0.001), 3rd day: 71.5 (p<0.001), 5th day: 61.3 (p<0.001)). CONCLUSION: Hypoxia during night could increase the expression of some pain-related molecules, which might be the cause of chronic pain in FMS patients.

Glutamate and GABA levels in the frontal cortex of rats with chronic epilepsy

Recently published data (Baran et al., Neurosignals 2004; 13: 290-7) have shown significantly increased activity of glutamic acid decarboxylase, the neuronal marker for gamma-aminobutyrate (GABA)-neurons, in the frontal cortex of rat brains, 6 months after kainic acid (KA) injection. In present study glutamate and GABA levels in the frontal cortex of rats in the KA (10 mg/kg, subcutaneously)-induced spontaneous recurrent seizure model of epilepsy, 6 months after the initial KA-induced seizures, were investigated. Six months after KA injection there was found a slightly reduced glutamate level in the frontal cortex (89.7 % of control), whereas the GABA level was moderately increased (119.6 % of control). The ratio GABA:glutamate level was significantly increased in the frontal cortex (134.5 % of control; P<0.001). Obtained data would indicate an enhancement of GABAergic activities in the frontal cortex in the chronic KA epileptic model. Interaction within GABAergic parameters, thus the GABAA receptors, the GABAB receptors, glutamate and GABA transporters may play a role in the modulation but also in the exertion of epileptic events in chronic KA epileptic model, which needs to be clarified.

O sistema glutamatérgico e desordens neuropsiquiátricas: aspectos básicos / The glutamatergic system and neuropsychiatric disorders: basic aspects

O sistema glutamatérgico, um dos mais extensos do sistema nervoso central, é formado por neurônios, vias e receptores específicos. O agonista natural corresponde a um aminoácido excitador de constituição relativamente simples, o glutamato. Este atua em uma ampla gama de subtipos de receptores, ionotrópicos e metabotrópicos, que oferecem a possibilidade de uma variada atividade sináptica, desde a neurotransmissão rápida até às respostas lentas que induzem modificações sinápticas de longa duração. Essa ampla distribuição dos receptores influencia neurônios que transmitem com o mesmo ou com outros neurotransmissores. Os receptores ionotrópicos, os mais bem estudados, apresentam funções diferenciadas, sendo que o do tipo AMPA é responsável pela maior parte das transmissões sinápticas excitadoras rápidas, a do tipo KA contribui nas respostas pós-sinápticas nas sinapses excitadoras, podendo ainda modular a liberação pré-sináptica do transmissor em determinadas sinapses, enquanto o receptor NMDA é fundamental na indução de formas específicas de plasticidade sináptica. Os variados receptores metabotrópicos também contribuem de modo importante em diversas dessas atividades. Dessa maneira, o sistema glutamatérgico encontra-se relacionado a diversas funções normais, como a plasticidade neural, subjacente a processos cognitivos, assim como a situações patológicas, responsáveis por diversas desordens neuropsiquiátricas agudas e crônicas. O conhecimento detalhado do sistema glutamatérgico, portanto, é essencial para a compreensão de variados processos normais e patológicos, assim como das possibilidades de intervenção farmacológica com objetivos terapêuticas

A Possible Role of Kainate Receptors in C2C12 Skeletal Myogenic Cells

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.

Distribution and Expression of Kainate(KA) Receptor Subunits in Moderate Hypoxic Newborn Piglet Brain

PURPOSE: The mechanism of hypoxic damage is mainly intracellular influx of calcium ions through the glutamate ionotropic receptor. This study was performed to determine alterations in distribution and expression of kainate receptor subunits after 1 hour of moderate hypoxia in the newborn piglet brain, as in a condition of mild to moderate perinatal hypoxic-ischemic encephalopathy. METHODS: Ten newborn piglets were ventilated at PaO2 over 80mmHg for 30min. Thereafter, the control group(n=5) was ventilated with 21% oxygen, and hypoxic group(n=5) with 6% oxygen at PaO2 below 25mmHg for 1 hour. Concentrations of protein, ATP and phosphocreatine were determined. The proteins were immunostained with anti-rat GluR6/7 and anti-rat KA2 antibody. RESULTS: Hypoxia(PaO2 20+/-1mmHg) and acidosis(pH 7.06+/-0.09) developed significantly in the hypoxic group compared to the control group(PaO2 104+/-4mmHg, pH 7.44+/-0.03, respectively, Phippocampus, thalamus, hypothalamus>basal ganglia, cerebellum>white matter, and KA2 subunits were ordered : hippocampus, basal ganglia>cerebral cortex>thalamus, cerebellum>hypothalamus, white matter. The distribution of the subunits between the hypoxic group and control group were similar. CONCLUSION: Cerebral cortex, hippocampus and basal ganglia may be the most vulnerable to excitotoxic injury. Kainate receptor subunits did not change after 1 hour of moderate hypoxia.

Distribution and Expression of Kainate(KA) Receptor Subunits in Moderate Hypoxic Newborn Piglet Brain

PURPOSE: The mechanism of hypoxic damage is mainly intracellular influx of calcium ions through the glutamate ionotropic receptor. This study was performed to determine alterations in distribution and expression of kainate receptor subunits after 1 hour of moderate hypoxia in the newborn piglet brain, as in a condition of mild to moderate perinatal hypoxic-ischemic encephalopathy. METHODS: Ten newborn piglets were ventilated at PaO2 over 80mmHg for 30min. Thereafter, the control group(n=5) was ventilated with 21% oxygen, and hypoxic group(n=5) with 6% oxygen at PaO2 below 25mmHg for 1 hour. Concentrations of protein, ATP and phosphocreatine were determined. The proteins were immunostained with anti-rat GluR6/7 and anti-rat KA2 antibody. RESULTS: Hypoxia(PaO2 20+/-1mmHg) and acidosis(pH 7.06+/-0.09) developed significantly in the hypoxic group compared to the control group(PaO2 104+/-4mmHg, pH 7.44+/-0.03, respectively, Phippocampus, thalamus, hypothalamus>basal ganglia, cerebellum>white matter, and KA2 subunits were ordered : hippocampus, basal ganglia>cerebral cortex>thalamus, cerebellum>hypothalamus, white matter. The distribution of the subunits between the hypoxic group and control group were similar. CONCLUSION: Cerebral cortex, hippocampus and basal ganglia may be the most vulnerable to excitotoxic injury. Kainate receptor subunits did not change after 1 hour of moderate hypoxia.

Excitotoxic Cell Death in Cultured Retinal Neurons

We examined excitotoxicity, putatively a major mechanism of ischemic neuronal death, in primary rat retinal cultures. Retinal cultures were prepared from newborn rats (day 1 or 2). Exposure of these cultures (DIV8-10)to NMDA or kainate induced neuronal death. Furthermore, MK-801 or CNQX each partially attenuated glutamateinduced neuronal death, suggesting that both NMDA and kainate receptors mediate it. Thy-1(+) retinal ganglion neurons, like neurons as a whole, were equally injured by NMDA and by kainate. However, GABA(+) or calbindin (+) neurons of the inner nuclear layer were resistant to NMDA, but highly vulnerable to kainate. These neurons may have AMPA/kainate receptors that are highly permeable to Ca2+, as they take up cobalt with kainate stimulation. These results suggest that the AMPA/kainate receptor, rater than the NMDA receptor, may mediate this pattern of selective neurnonal death.

Domoic acid induces neurotoxicity and ip3 mobilization in cultured cells of embryonic chick retina

Domoic acid (DOM), 1 to 50 µM, a glutamate agonist responsible for several neurological effects such as loss of memory and confusion, induced the death of cultured neurons of chick embryonic retina, in a concentration- and Ca2+ -dependent manner. This effect was blocked by 100 µM CNQX, a competitive antagonist of the non-NMDA receptor, but not by 10 µM MK-801, a non-competitive antagonist of the NMDA receptor. DOM also induced inositol triphosphate (ip3) accumulation 4 to 7 times above basal levels. This effect was also dependent on external Ca2+ and was entirely blocked by 100 µM CNQX, but not by 10 µM MK-801. These results suggest that DOM interaction with non-NMDA glutamate receptors mediates signal transduction with ip3 accumulation and cell death

Excitotoxic change of Hippocampal Neuron by Kainic Acid in Rat Brain

The excitotoxic effect of kainic acid on dendrites and neuronal cell bodies of hippocampus and dentate gyrus was studied with time (1, 4, 8, 16 hours, 2, 7, 14 days) light and electron microscopically by intraperitonial injection into rat. The results obtained were as follows. 1) The acute dendrotoxic effect was observed as laminar pattern of swelling along pyramidal cell body layer and dendritic fields and was most prominently at 2-4 hours after kainic acid injection. In ultractructural study, the acute change occurred in dendrites of pyramidal cells in hipocampus because the synapses between nerve terminals and swollen components were not destroyed and remained intact and, identified the swollen structures as dendrites. So, it was obvious from the results that the acute change by kainic acid was osmolysis and was continued till initial 4 hours but was finally faded out. 2) The distribution of kainic acid receptor within hippocampus was different because the prominent dendritic swelling occurred in proximal basilar dendritic field of CA 3 and 4 and the proximal and distal basilar dendritic fields of CA 1 and 2, and no change was observable in dentate granule cell. The sensitivity of hippocampal dendritic fields to kainic acid could be put in decreasing order as CA3, CA4, CA1, CA2 and dentate granule cell 3) With the elapse of time, the acute change disappeared and pyramidal cells began to degenerate by the chronic reaction about 7 days after kainic acid injury, and the pyramidal cell density in CA regions greatly decreased. Almost all pyramidal cells degenerated the dentate granule cells were not affected to kainic acid throughout the time. In conclusions, hippocampal neurons were postulated to be very sensitive to kainic acid, and in contrast to the gradual disappearance of acute change within several hours, the degeneration of pyramidal neurons by chronic change was developed within several days regardless of acute change.