The Effects Of GABA_C Receptor Activation On The Epileptic Seizures And The Expressions Of GABA,GAT1 And ρ2mRNA In Rat's Brain

Objective To explore the dynamic rules of the GABA content and the expressions of GAT1, GABAC receptorρ2mRNA in rats'brain in the process of the epileptic seizures and the natural termination and during 7days after terminating seizures; to investigate the influence of agitating GABAC receptor on the antiepileptic effects, the epileptiform discharge and the GABA content and the expression of GAT1, GABAC receptorρ2mRNA in order to find out the effects of GABAC receptor activation on the endogenous antiepileptic mechanism.Methods A rat model of penicillin-induced epilepsy was established after the PNC localized injected in cortex; GABAA receptor antagonist (bicuculline, BIC), GABAB receptor antagonist (homotaurine,3APS) and GABAC receptor agonist (muscimol,MUS) were used as pharmacological tools. The seizure duration and Racine's behavior study graduation were used to evaluate the efficacy index. RM6240C-type multi-channel biological signal acquisition and the processing system simultaneously recorded EcoG of epileptic rats. After blocked the GABA_A,B receptors at the peak period of seizure, the rats were administrated by intraperitoneally injection(ip) with MUS (1.0 mg·kg^-1) to analyze the influence following the activation of GABAC receptors on the epileptiform discharge latency, the frequency, and the maximum amplitude. The immunohistochemistry technology was performed to determine the content of GABA and GAT1 at the peak period of seizures, the termination period of seizures and the 1st day ,3rd day and the 7th day after terminating seizures. The SYBR-greenⅠRT-PCR was applied to detect the expression of hippocampalρ2mRNA at the same time.Results (1) The seizure ranging fromⅠtoⅢgrade, the spike wave and the spike slow wave were observed after the PNC localized injected in cortex. Compared with the PNC model group, the duration of seizures were prolonged and the grade of seizures was enhanced in the rats treated with GABAA and GABAB receptor antagonists at the peak period of seizures. There were statistical differences between the two groups (P<0.05). After agitating GABAC receptor, the duration of seizures was apparently shortened, the frequency of spike wave was diminished and the amplitude of vibration was depressed. There were significant differences (P<0.05) compared with the PNC and the GAB groups. (2) The level of GABA was decreased at the peak period of seizures (P<0.05). On the contrary, the GAT1 was raised (P<0.05).There was syntropic change in the alteration of GAT1 and GABA levels. The level of GAT1 kept pace with that of GABA from the natural termination period of seizures to 1-7 days after terminating seizures, until GABA approached the normal level. They were rising in the natural termination period of seizures (P<0.05), then decreasing in 24h after terminating seizures. There was a consistent decrease during 1-7 days, but compared with the control rats still an increasing trend (P<0.05), and then it returned to the normal level on the 7th day. The changes of GAT1 and GABA levels were consistent with the PNC groups when GABA_A,B antagonists was administered. However both increased more significantly. There was a significant difference compared with the penicillin group (P<0.05). Meanwhile the content curve of GAT1 and GABA rose compared with the PNC group. After GABAC agonist was administered, the GAT1 and GABA levels were consistent with those of the PNC groups but returned to normal on the 7th day. The levels of GABA and GAT1 increased in the termination period of seizures (P<0.05). But the increased extent of GABA and GAT1 was lower than PNC and GAB groups and the dynamic curve moved downward by compared with the PNC groups. (3) In the PNC groups, the expression of hippocumpalρ2mRNA decreased at the peak period of seizures, there was a significant difference compared with the control groups(P<0.05), while it was continuing decrease in natural termination period of seizures and 1-3 days after terminating seizures (P<0.05), and reached the normal level on the 7th day. In GAB groups,the expression of hippocumpalρ2mRNA decreased at the peak period of seizure, and there were no significant differences compared with the PNC groups (P>0.05). Theρ2mRNA expression was increase in natural termination period of seizures (P<0.05), while it was decrease during 1-3 days after terminating seizures, and reached the normal level on the 7th day. After agitating GABAC receptor,the tendency ofρ2mRNA expression was the same as that in GAB groups. In the termination period of seizures, the expression ofρ2mRNA increased,there was a significant difference compared with the PNC groups (P<0.05), while it was decrease during 1-3 days after terminating seizures, and reached the normal level on the 7th day,there were no significant differences compared with the control groups (P>0.05).Conclusions (1) A rat model of epileptic seizures and the epileptic discharge was induced after the PNC localized injected in cortex.In GAB groups the grade of epileptic seizures and epileptic discharge could be enhanced. This effect was related to cancel the inhibition of CNS rapid response which was mediated by the GABAA, B receptors. After agitating GABAC receptor, it could inhibit the epileptic discharge, and terminate the epileptic seizures. It proved that a "double insurance" mechanism existed in the body's endogenous antiepileptic system.(2) The level of GABA decreased and the GAT1 increased at the peak period of seizure. GAT1 as the GABA regulator in brain was in concordance with the dynamic change rules of the GABA level in natural termination period of seizures and 1-7 days. Both of them increased in natural termination period of seizures and then decreased in 24h after terminating seizures,but the level of GABA and GAT1 showed a continuing rising trend during 1-7 days compared with normal rats, which was the main reason for avoiding recurrence.(3) The content of GABA and GAT1 was significantly higher than that in the PNC groups when the GABAA, B receptors was blocked. The dynamic change curve was upward moved in the period of 1-7 days after terminating seizures. The result showed that the expression of GABA and GAT1 was increased by the body self-regulatory mechanism. After agitating GABAC receptor, endogenous GABA was antiported to the synaptic cleft by GAT1, resulting in a slow and long-lasting inhibitory effect, and thus terminated the epileptic seizures.(4) After GABAC agonist was administered under the condition of blocked GABAA, B receptors, the increased levels of GABA and GAT1 were significantly lower than that of the PNC and GAB groups. The dynamic change curve downward moved compared with the PNC groups. It was shown that after agitating GABAC receptor, the inhibition of brain was enhanced and the body's self-regulatory mechanism was weakened and the content of GABA and GAT1 were reduced relatively.Thus the inhibition and excitement in the brain maintained balance.(5)The expression ofρ2mRNA in the rat's hippocampus could be continuing decreased by epileptic seizures in the model of penicillin-induced epilepsy.After agitating GABAC receptor, the expression ofρ2mRNA increased, and then decreased in 24h after terminating seizures. There was a continuing down regulation in 1-3 days. It was suggested that a short term up-regulation in the expression ofρ2mRNA might be one of the mechanisms for the termination of epileptic seizures.