Alterations of Glutamate Transporter Gene Expression in Epileptic EL Mice Brains

Abstract

Intmduc%ion: Glutamate is considered to be an important excitatory amino acid and has been implicated in the pathophysiology of cpilcpsy. Glutamate transporters help to maintain the extracellular glutamate concentration and have been identified i n inousc brains, incl tiding GLT‐I and GLAST in astroglia and EAAC‐I in neurons. In an amygdala‐kindling study, alteration of glutamate transporter proteins showed that GLAST protein was down‐regulated in the piriform cortcx/amygclala rcgion of the amygdala‐kindled Iiits. In contrast, kindling induced an increase in EAAC‐ I levels in the pirilorrn cortex/amygdala and hippocampus. No changes i n GLT‐I wcrc obscrvcd in any region. The EL mouse, an inbred mutant strain, is considered to be a model for human complex partial seidures. This study was carried out to reveal the relationship bctwccn cpilcptogcncsis and glutamate transporters in EL mice.Animals: EL micc were tosscd i n thc air approximately 40 times to a height of 10‐15 cm once a week from the age of 4 weeks. They exhihitcd tonic‐clonic scizurcs aftcr 12 wccks of agc and wcrc designated as EL[s] mice. Twenty‐five‐week‐old ELlsl mice developed seixure‐susceptibility and completely cxhibitcd convulsion by the tossing‐up stimulation. On the other hand, the EL mice that did not rcccivc tosscd‐up stimulations and dcmonstratc scizurcs wcrc designated as ELlnsl mice. The progenitor ddY mice were not seizuresusceptible. In this study, all mice wcrc 25 wccks of agc and cach group consisted of 4 mice. Methods: RNrrse Pvowction Asscry: Total RNA was prcparcd from the whole brain without ihe cerebellum by acid guanidium thiocyanate‐ phenol‐chloroform extraction. Ten micrograms of total RNAs was hybridized with antiscnse labeled riboprobcs of glutamate transporters and p‐actin. After RNase digestion, protected probe RNA was dctcctcd by clectrophoresis and autoradiography.In Situ Hyhridiznrion: Transverse frozen sections were cut at I2 pin, fixed in 4% parafomxildchydc, and were stored at ‐8OT bcforc hybridization. Sections were hybridixd ovcrnight at 55°C tising digoxigcnin‐labclcd antisensc riboprobcs. After washing and RNasc digestion, they were incubated at 37°C for I2 hours covered with I 500 dilution of Fab‐fragments from anti‐digoxigenin antibody from sheep conjugated with alkaline phosphatase. After incubation, the color reaction was initiated by the addition of BCIP(S‐bromo‐4‐chloro‐3‐indolyl phosphate) and NBT(nitroh1ue tetrazolium salt). The reaction was terminated by the aiop bufferll0mM Tris(pH7.0), I mM EDTAI. Results: RNase protection assays show that GLT‐ I mRNA expressions of ELls] and ELI nsl wcrc clcvatcd significantly more than that of ddY mice. The mRNA exprcssions of GLAST, EAAC‐ I and p‐actin as a control among the 3 groups were not significantly different. In situ hybridization study showed that GLT‐mRNA in the hippocampus and cerebral cortex of ELIsl and EL[nsl was expressed more than that of ddY mice. Thc expression patterns of GLAST and EAAC‐I mRNA were similar among the 3 groups as shown by in sitti hybridization as well as RNase protection assay. Conclusions: These results demonstrate that the GLT‐ I inRNA expression induced a significant incrcasc in the hippocampus and cerebral cortex of both seizure‐susceptible ELls 1 and seizure‐nonexpericnced ELlnsl mice comparcd with ddY micc. Thcsc results differed from thosc ofthe kindling study. However, GLT‐ I is considered to be a main factor maintaining low glutamate concentration. We proposed that EL micc show hyperactivity of glutamate synthesis and that a compensatory mechanism elcvates GLT‐ I mRNA expression in the hippocampus and cerebral cortex. Thereforc, the findings showed that an increase of glutamate synthesis might be associated with epileptogenesis EL mice.