Abnormal Expression Of MGluR1 In Hippocampus Of Spontaneously Epileptic Rats

IntroductionThe spontaneously epileptic rats(SERs),a kind of hereditary epileptic animal model,was introduced from Kyoto University in Japan by our college in 2000.It is a homozygous double mutant obtained by mating heterozygous tremor rats(tm)(tm/+) and homozygous zitter rats(zi)(zi/zi).Both of the two mutant genes are autosomal recessive inheritance.SERs exhibit spontaneous tonic convulsions and absence-like seizures,characterized by simultaneous appearance of 5-7 Hz spike-wave complexes in cortical and hippocampal EEG after the age of 8 weeks.The profiles of conventional antiepileptic drugs in SERs are quite similar to the efficacy profile in human epilepsy. The absence-like seizures are inhibited by Ethosuximide.Phenytoin can inhibit the tonic seizures without affecting the absence-like seizures.In contrast,Phenobarbital and Valproate inhibit both seizures to a similar degree.Therefore,the SER is a useful model for researching human epilepsy and evaluation of antiepileptic drugs(AEDS). MGluR1 is a member of metabotropic glutamate recepors,which belongs to G-protein coupled receptors.It has seven transmembrane motifs.The extracellular domain of mGluR1,transmembrane region and peptide chain links in or outside the cells are highly conserved.At present,it is presumed that mGluR1 is mainly coupled to phosphatidase C(PLC)and intracellular Ca²⁺signal.MGluR1 can accommodate neuronal excitability and synaptic transmission by regulating many transmembrane ion channels and intracellular second messenger system. The etiology of epilepsy is complicated,the imbalance of excitatory amino acid and inhibitory amino acids is considered to play a critical role in pathopoiesis.The present study therefore investigated the expression of mGluR1 in the hippocampus of SERs by several experimental methods,in order to reveal the possible pathogenesy in epilepsy.Materials and Methods1.RT-PCR analysisExperimental animals:normal Wistar rats and SERs at the age of 2-3 months were housed in individual cages under a controlled environment(12:12 h light/dark cycle, 50～70%humidity,24℃).Food and water were available ad libitum.Eight SERs were in experimental group and eight Wistar rats were in control group.Specimen preparation:the brains of SERs and Wistar rats were dissected out under anesthesia.The samples of the whole hippocampus were stripped and freezed in -80℃. Total RNA was prepared using Trizol according to manufacturer's instructions and measured purity.If OD₂₆₀/OD₂₈₀is between 1.8～2.0,indicating that the specimen RNA is pure.RT-PCR reaction:Primer pairs used as follows:mGluR1 452bp(forward prime: 5'-CATGCCCATTTTGTCCTACC-3',reverse primer:5'-CTTCTTTCTCCGGAA AATGTTG-3');β-actin 300bp(forward primer 5'-AGAGCTACGAGCTGCCTGAC -3',reverse primer:5'-AGTACTTGCGCTCAGGAGGA-3'),RT reaction conditions: 42℃30min,99℃5min,5℃5min.PCR reaction conditions:initial denaturation at 94℃for 4min,followed by 33cycles(94℃1min,52℃1min,72℃90s),After1.5% agarose gel electrophoresis,the results are analysised by FluorChem^TMImaging System.2.Immunohistochemistry analysisExperimental animals:normal Wistar rats and SERs at the age of 2-3 moths were housed in individual cages under a controlled environment(12:12h light/dark cycle, 50～70%humidity,24℃).Food and water were available ad libitum.Five SERs were in experimental group and five Wistar rats were in control group.Steps:Perfusion fixation of rat brain tissue—paraffin imbedding,Paraffin sections,antigen Retrieval,an anti-incubated,second-incubated,DAB coloration, image acquisition and analysis.3.Western blotting analysisExperimental animals:normal Wistar rats and SERs at the age of 2-3 months were housed in individual cages under a controlled environment(12:12h light/darkcycle, 50-70%humidity,24℃).Food and water were available ad libitum.Five SERs were in experimental group and five Wistar rats were in control group.Steps:membrane protein extraction,Protein quantitation,SDS-PAGE gel electrophoresis,PVDF trarsmembrane,an anti-incubated,second-incubated,DAB coloration,gray scale scanning and analysis.4.Statistical analysesStudent's t-test was performed to determine statistical significance,which was evaluated at p＜0.05.Results1.The mRNA expressions of mGluR1 by RT-PCRThe mRNA expression of mGluR1 was lower than that of Wistar groups in hippocampus(0.76±0.15 in SERs versus 1.02±0.26 in Wistar rats;p＜0.05).2.The protein expressions of mGluR1 by immunohistochemistryThese proteins were easily detected in SERs and control rats hippocampus.The protein density of mGluR1 positive cells in the hippocampus of SERs was higher than those in Wistar rats.The population of mGluR1 Positive cells in the hippocampus DG region of SERs were significant lower than those in Wistar rats,p＜0.01.The population of mGluR1 positive cells in the hippocampus CA3 region of SERs were lower than those in Wistar rats,p＜0.05.The population of mGluR1 positive cells in the hippocampus CA1 region of SERs were a little lower than those in Wistar rats,p＞0.05.3.The protein expressions of mGluR1 by Western blottingThe results are consistent with immunohistochemistry.The expression of mGluR1 in the hippocampus of SERs were lower than those in Wistar rats,p＜0.05.Conclusion1.The mRNA expressions of mGluR1 were down-regulated in SERs hippocampus.2.The protein expressions of mGluR1 were down-regulated in SERs hippocampus.3.MGluR1 Positive cells in the hippocampus of SERs were higher than those in Wistar rats,and the population of positive cells was down-regulated.