Effects Of Activating GABAc Receptors On Epileptic Seizures And The Expressions Of Glu, EAAT1-mRNA And EAAT1-P In Rats’ Brain

Objective To study the dynamic rule of the content of Glutamate(Glu) and the expressions of EAAT1-mRNA and EAAT1-protein(EAAT1-P) in rats brain in the whole process of epileptic seizures from inducing to the7th day after seizure terminated. To analyze the effects of activating GABAc receptors on epileptic seizures, epileptiform discharge and the content of Glu and the expressions of EAAT1-mRNA and EAAT1-P. To explore the action and elucidate the mechanism of activating GABAc receptors in the seizure process.Methods A rat model of epilepsy was induced by Penicillin(PNC) injecting into motor cortex of healthy adult Sprague-Dawley(SD) rats. EcoG of each rat was recorded simultaneously via RM6240C-type multi-channel biological signal acquisition and processing system. Bicuculline (BIC), the GABAa receptors antagonist, Homotaurine (3APS), the GABAB receptors antagonist and GABAc receptors agonist Muscimol(MUS) were applied as tool remedies. The efficacy index was evaluated according to the seizure duration and Racine’s score. At the peak of seizures, MUS were applied to rats after GABAA,B receptors blocked, the influence of activating GABAc receptors on the duration, frequency, and maximum amplitude of epileptiform discharge was analyzed. The contents of hippocampal Glu and EAAT1-P were determined by immunohisto-chemistry at the following points:the peak and termination of epileptic seizures, the1st day,2nd day,3rd day and7th day after seizure terminated. SYBR-green RT-PCR method was applied to detect the expression of hippocampal EAAT1-mRNA.Results (1) After PNC was injected into rats’ motor cortex (PNC group), the epileptic seizure was induced successfully, the epileptiform EcoG and behaviors were presented in all rats, and manifested the seizures ranged from stage I to stage III. The duration of seizures was extended and the stage of seizures was enhanced after antagonists of GABAA,B receptors were applied, the increased frequency and maximum amplitude of epileptiform discharge were observed, when compared to those of PNC group, the differences were significant(P<0.05). After MUS was applied, the seizure duration was apparently shortened, seizure stage was relieved as well as the frequency and amplitude of spike wave were depressed, there were significant differences as compared to those of groups of PNC and GAB(P<0.05).(2)At the peak of seizures, there was a significantly increased content of the hippocampal Glu in PNC group as compared to those of normal control (CON) group(P<0.05); and the level of Glu was further increased in GAB group(P<0.05). At the termination of seizures, the level of Glu in groups of PNC, GAB and MUS displayed a decreasing tendency, there were significant differences as compared to those of each group at their corresponding peak period(P<0.05). An ongoing decreasing tendency remained in the days of1st-7th day after the seizures terminated, and with no rebound. The decreased range in MUS group was more apparently and swiftly, the level of Glu was significantly lower than those of groups PNC and GAB at the following points: the termination, the1st,2nd and3th day after seizure terminated(P<0.05). On the2nd day, the level of Glu in MUS group approached to normal level(P>0.05); on the3rd day, the level of Glu in PNC group reverted to the normal essentially(P>0.05); while in GAB group, the level of Glu reverted to normal in the7th day(P>0.05).(3)At the peak of seizure, the expression of hippocampal EAAT1-P was significantly decreased in PNC group when compared to those of CON group(P<0.05), while the content of EAAT1-P in GAB group and MUS was further decreased after the antagonists of GABAA,B receptors were applied(P<0.05); the increasing tendency of the expression of EAAT1-P at the termination of seizures was observed in groups of PNC, GAB and MUS, when compared to those of each group at their peaks, there were significant differences(P<0.05). The ongoing increasing tendency manifested in the days of1st-7th day after seizure terminated. Especially, at the termination and the days of lst-3rd day after the termination, the expression of EAAT1-P increased more greatly and swiftly, compared to those of groups of PNC and GAB, there were significant differences(P<0.05). In MUS group, at the termination of seizure, the expression of EAAT1-P approached to normal level(P>0.05); in days of2nd-7th day after seizure terminated, the tendency remained and the content of EAAT1-P exceed normal, although with no significant differences(P>0.05). In PNC group, on the1st day, the content reverted to normal level (P>0.05); in the days of3rf-7th day, the increase remained and exceed normal, but with no significant difference(P>0.05). However, in GAB group the expression was still lower than normal on the7th day, though there were no significant difference (P>0.05).(4)At the peak of seizure, compared to the CON group, the expression of hippocampal EAAT1-mRNA was significantly decreased in PNC group(P<0.05), while the expressions of EAAT1-mRNA in GAB group and MUS group further decreased (P<0.05); at the termination of seizure, when compared to the peak of each group, an increased expression of EAAT1-mRNA was observed in the following groups:PNC, GAB and MUS group, and there were significant differences(P<0.05). The tendency remained in the days of lst-7th day after the seizures terminated. At the termination and in the days of lst-3rd day after seizure terminated, as compared to groups of PNC and GAB, the increased EAAT1-mRNA was greater and to be more swiftly, there were significant differences(P<0.05). The expression of EAAT1-mRNA in MUS group reverted to normal level on the2nd day(P>0.05); in PNC group, the expression reverted to normal level on the3rd day (P>0.05), while, in GAB group the expression reverted to normal on the7th day (P>0.05). Conclusions (1) Epilepsy seizure can be successfully induced by PNC localized injecting into rats’cortex, the blockage GABAA, B receptors was followed by the enhanced seizure degree and prolonged duration of seizures. The activated GABAc receptors could accelerate terminating epilepsy seizure and the epileptiform discharges co-induced by PNC and GABAA,B receptors antagonist.(2)The process of PNC inducing epilepsy involved a significantly increased content of Glu and an apparent reduction in the expressions of EAAT1-P and EAAT1-mRNA. The decreased content of Glu and an apparently increased expressions of EAAT1-P and EAAT1-mRNA were observed in the course of automatic terminating. During the7days after seizure terminated, there were a reducing tendency of Glu and an increasing tendency of the expressions of EAAT1-P and EAAT1-mRNA, and with no epileptic recurrence.(3) Activating GABAc receptors was flowed by a persistent reduction in the content of Glu, as well as further increased expressions of EAAT1-mRNA and EAAT1-P, while the uptake of Glu was enhanced in the synaptic clefts, and the reduced excitability of CNS was the antiepileptic mechanism. Besides, due to the expression of EAAT1-mRNA persistently increased, the content of EAAT1-P was perpetually up-regulated, led to a constantly low level of Glu, it’s the reason for preventing from epileptic recurrence.