OBJECTIVETo 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.

METHODSA 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.

RESULTSThe 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).

CONCLUSIONSIn 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.

Child ; Child, Preschool ; Epilepsy ; etiology ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Polymorphism, Single Nucleotide ; Receptors, Kainic Acid ; genetics ; Risk Factors

OBJECTIVETo explore the effect of up-regulation of KA1 subunit of the kainate receptor on endoplasmic reticulum stress (ERS)-induced excitotoxic neurodegeneration in mouse hippocampus.

METHODSSeventy 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.

RESULTSAt 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).

CONCLUSIONMicroinjection 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.

Animals ; Apoptosis ; Endoplasmic Reticulum Stress ; Hippocampus ; pathology ; Kainic Acid ; pharmacology ; Male ; Mice ; Neurons ; pathology ; Receptors, Kainic Acid ; metabolism ; Tunicamycin ; pharmacology ; Up-Regulation

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.
Animals ; Apoptosis ; Blood-Brain Barrier ; Brain ; Central Nervous System ; Dentate Gyrus ; Hippocampus* ; In Situ Nick-End Labeling ; Learning ; Lower Urinary Tract Symptoms ; Memory* ; Memory, Short-Term ; N-Methylaspartate ; Neurons ; Patch-Clamp Techniques ; Rats* ; Receptors, Ionotropic Glutamate ; Receptors, Kainic Acid ; Receptors, N-Methyl-D-Aspartate ; Urinary Bladder, Overactive

The subfornical organ (SFO) is one of circumventricular organs characterized by the lack of a normal blood brain barrier. The SFO neurons are exposed to circulating glutamate (60~100 microM), which may cause excitotoxicity in the central nervous system. However, it remains unclear how SFO neurons are protected from excitotoxicity caused by circulating glutamate. In this study, we compared the glutamate-induced whole cell currents in SFO neurons to those in hippocampal CA1 neurons using the patch clamp technique in brain slice. Glutamate (100 microM) induced an inward current in both SFO and hippocampal CA1 neurons. The density of glutamate-induced current in SFO neurons was significantly smaller than that in hippocampal CA1 neurons (0.55 vs. 2.07 pA/pF, p<0.05). To further identify the subtype of the glutamate receptors involved, the whole cell currents induced by selective agonists were then compared. The current densities induced by AMPA (0.45 pA/pF) and kainate (0.83 pA/pF), non-NMDA glutamate receptor agonists in SFO neurons were also smaller than those in hippocampal CA1 neurons (2.44 pA/pF for AMPA, p<0.05; 2.34 pA/pF for kainate, p< 0.05). However, the current density by NMDA in SFO neurons was not significantly different from that of hippocampal CA1 neurons (1.58 vs. 1.47 pA/pF, p>0.05). These results demonstrate that glutamate-mediated action through non-NMDA glutamate receptors in SFO neurons is smaller than that of hippocampal CA1 neurons, suggesting a possible protection mechanism from excitotoxicity by circulating glutamate in SFO neurons.
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Animals ; Blood-Brain Barrier ; Brain ; Central Nervous System ; Glutamic Acid* ; Hippocampus ; Kainic Acid ; N-Methylaspartate ; Neurons* ; Rats* ; Receptors, Glutamate ; Subfornical Organ*

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.
Age Factors ; Animals ; Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Mice ; Receptors, Kainic Acid/*metabolism ; Substantia Gelatinosa/*metabolism

The serine/threonine kinase Akt has been shown to play a role of multiple cellular signaling pathways and act as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI3K). It has been reported that phosphorylated Akt activates eNOS resulting in the production of NO and that NO stimulates soluble guanylate cyclase (sGC), which results in accumulation of cGMP and subsequent activation of the protein kinase G (PKG). It has been also reported that PKG activates PI3K/Akt signaling. Therefore, it is possible that PI3K, Akt, eNOS, sGC, and PKG form a loop to exert enhanced and sustained activation of Akt. However, the existence of this loop in eNOS-expressing cells, such as endothelial cells or astrocytes, has not been reported. Thus, we examined a possibility that Akt phosphorylation might be enhanced via eNOS/sGC/PKG/PI3K pathway in astrocytes in vivo and in vitro. Phosphorylation of Akt was detected in astrocytes after KA treatment and was maintained up to 72 h in mouse hippocampus. 2 weeks after KA treatment, astrocytic Akt phosphorylation was normalized to control. The inhibition of eNOS, sGC, and PKG significantly decreased Akt and eNOS phosphorylation induced by KA in astrocytes. In contrast, the decreased phosphorylation of Akt and eNOS by eNOS inhibition was significantly reversed with PKG activation. The above findings in mouse hippocampus were also observed in primary astrocytes. These data suggest that Akt/eNOS/sGC/PKG/PI3K pathway may constitute a loop, resulting in enhanced and sustained Akt activation in astrocytes.
Animals ; Astrocytes ; Cyclic GMP-Dependent Protein Kinases ; Endothelial Cells ; Guanylate Cyclase ; Hippocampus ; Kainic Acid ; Mice ; Nitric Oxide ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Phosphotransferases ; Receptors, Growth Factor ; Transducers

BACKGROUND: Androsterone is one of the major metabolites from testosterone whose clinical importance remains unclear. This study evaluated the effects of androsterone on seizure susceptibility in mouse models of epilepsy. METHODS: The efficacy of androsterone (10~200 mg/kg, i.p.) against seizures induced by various GABA receptor antagonists and glutamate receptor agonists was evaluated. RESULTS: Androsterone protected mice against seizures induced by PTZ (pentylenetetrazol), PCX (picrotoxin), and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) in a dose-dependent manner. Androsterone did not protect against seizures induced by kainic acid, NMDA (N-methyl-D-aspartic acid), or 4-AP (4-aminopyridine) in mice. CONCLUSIONS: These results suggest that androsterone exhibits anticonvulsant activity that occurs largely via nongenomic mechanisms. Testosterone-derived androsterone might be an endogenous protective neuroactive steroid in the brain.
Androsterone ; Animals ; Carbolines ; Epilepsy ; GABA Antagonists ; gamma-Aminobutyric Acid ; Kainic Acid ; Mice ; N-Methylaspartate ; Receptors, Glutamate ; Seizures ; Testosterone

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.
Animals ; Cobalt ; Epithelium ; Glutamic Acid ; Kainic Acid ; Rats ; Rats, Sprague-Dawley ; Receptors, Glutamate ; Receptors, Kainic Acid ; RNA, Messenger ; Taste Buds ; Tongue

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.
Animals ; Anoxia ; Calcitonin Gene-Related Peptide ; Carisoprodol ; Chronic Pain ; Embryonic Structures ; Fibromyalgia* ; Glutamic Acid ; Humans ; Immunohistochemistry ; N-Methylaspartate ; Rats ; Rats, Sprague-Dawley ; Receptors, Kainic Acid ; Substance P

OBJECTIVEKetogenic 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.

METHODSEpilepsy 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.

RESULTSKD-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.

CONCLUSIONMossy 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.

Animals ; Blotting, Western ; CA1 Region, Hippocampal ; metabolism ; pathology ; CA3 Region, Hippocampal ; metabolism ; pathology ; Chromosome Pairing ; drug effects ; Dentate Gyrus ; metabolism ; pathology ; Diet, Ketogenic ; methods ; Disease Models, Animal ; Epilepsy ; chemically induced ; diet therapy ; genetics ; metabolism ; pathology ; Excitatory Amino Acid Agonists ; Hippocampus ; drug effects ; metabolism ; pathology ; Kainic Acid ; Male ; Mossy Fibers, Hippocampal ; metabolism ; pathology ; Pyramidal Cells ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Rats ; Receptors, Kainic Acid ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction