Abnormal Expression Of GLT-1, EAAC1 In Cortex And Hippocampus Of Spontaneously Epileptic Rats

IntroductionEpileptogenic processes involve an imbalance between GABAergic inhibition and glutamatergic excitation.Glutamate transporters as a unique way of clearing extracellular glutamate play an important role in reuptake of glutamate,the normal transmission of synapse and preventing neuron from excitability damage.So far,many resarches worldwide indicate that alternation of the expression and function of glutamate transporter has close relationship with genesis of epilepsy.Spontaneously epileptic rat(SER),is a homozygous double mutant obtained by mating heterozygous tremor rats(tm)(tm/+) and homozygous zitter rats(zi)(zi/zi). SER exhibits spontaneous tonic convulsions and absence-like seizures,characterized by simultaneous appearance of 5-7Hz spike-wave complexes in cortical and hippocampal EEG after the age of 8 weeks.The profiles of conventional antiepileptic drugs in SER are quite similar to the efficacy profile in human epilepsy.Therefore, SER is an ideal model for researching human inheritant epilepsy.Among the five kinds of cloned excitatory amino acid transporters,GLT-1(EAAT2) is a glial transporter,which can absorb over 90%of the total glutamate.It plays a central role in transporting glutmate.EAAC1(EAAT3) is a neuronal transporter.It can not only transport glutamate but also provide precursor for the synthesis ofγ-GABA. The present study investigated the mRNA and protein expressions of GLT-1 and EAAC1 in cortex and hippocampus of SER in order to reveal the probable pathogenesis revolved in epilepsy. Materials and Methods1.Animals and materialsSERs and normal wistar rats at the age of 11-13 weeks 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.Each group includes 5 rats.The GLT-1 and EAAC1 antibodies were purchased from Santa.2.RT-PCR analyzes the expression of GLT-1,EAAC1 mRNATotal RNAs of rats' cortex and hippocampus were prepared with the method of TRIzol,RT-PCR was taken according to instruction.After 1.5%agarose gel electrophoresis,the results were analyzed by FluorChem^TM Imaging System.3.Western blotting analyzes the expression of GLT-1,EAAC1 proteinMembrane proteins of rats' cortex and hippocampus were prepared.Quantitate the protein with Bradford method.After 10%SDS-PAGE gel electrophoresis, antibodies incubated,immunoreactive bands were visualized using ECL.4.Immunohistochemistry detects the localization of GLT-1,EAAC1 proteinAfter perfusion and fixation of rat brain tissue,the paraffin sections were made. Antibodies incubated the sections.At last,immunoreactive bands were visualized with DAB.5.Statistical analysisMean and standard deviation were calculated for all measurements.Student's t-test was performed to determine statistical significance,which was evaluated at p＜0.05.Results1.The EAAC1 mRNA expression in the cortex of SERs was apparently lower than that of Wistar rats(p＜0.01).There were no significant difference of GLT-1 mRNA expression in cortex,EAAC1 and GLT-1 mRNA expressions in hippocampus between SERs and Wistar rats(p＞0.05).2.The GLT-1 protein expression in the cortex of SERs was lower than that of Wistar rats(p＜0.05).The EAAC1 protein expression in the cortex of SERs was apparently lower than that of Wistar rats(p＜0.01).The GLT-1 protein expression in the hippocampus of SERs was higher than that of Wistar rats(p＜0.01).There was no significant difference of EAAC1 protein expressions between SERs and Wistar rats (p＞0.05).3.GLT-1 and EAAC1 protein are expressed in both cortex and hippocampus of SERs and Wistar rats.Moreover,they exist on the cellular membrane.Conclusion1.The EAAC 1 expression increased at both mRNA and protein levels.2.The GLT-1 protein expression reduced in cortex of SERs.The GLT-1 protein expression inhanced in hippocampus of SERs.3.GLT-1 and EAAC1 proteins are expressed on the cellular membrane of both cortex and hippocampus of SERs and Wistar rats.4.The decreased GLT-1 and EAAC1 in cortex of SER may contribute to seizure.5.The increased GLT-1 in hippocampus of SER may be a compensatory neuroprotective mechanism.