ClC-2对慢性颞叶癫痫大鼠海马CA1区α5-GABAA受体介导紧张性抑制的影响

ClC-2 is a member of the supergene family of voltage-gated chloride channels. It is proved to be inwardly rectifying, and plays an important role in setting the intracellular chloride concentration in neurons expressing inhibitory GABAA receptors (GABAARs). Considering the conductance of ClC-2 is large and does not display time-dependent inactivation, it is well suited for stabilize ECl. However, whether ClC-2 contributes to epilepsy or not is controversial. Our previous studies showed that there is an increased ClC-2 IR in CA1 region in pilocarpine-treated rats, especially in the apical dendrites (strata radiatum). Immunoblot was performed to quantify this change that ClC-2 protein level was significantly higher than that in control group. Moreover, ClC-2 currents is increased in CA1 PCs in pilocarpine-treated rats, and blockage the tonic inhibition mediated by α5-GABAARs reversed the increased ClC-2 currents in vitro, indicating that ClC-2 modification is probably to assist the chloride extrusion and maintain the inwardly directed driving force for chloride ions, that is a prerequisite for hyperpolarizing inhibition of extrasynaptic GABAARs. Therefore, we will further investigate whether the changes of ClC-2 and tonic inhibition in pilocarpine-treated rats are contributed to the generation and propagation of spontaneous recurrent seizures (SRSs) or cognivive defict. Using the pilocarpine-induced experimental TLE model, we will perform behavioral tests to identify the distinction of chronic SRSs and spatial memory from wildtype and ClC-2 KO mice. Then we will record the currents of ClC-2 in vitro and the hippocampal LTP in vivo to reveal the difference in the excitability and synaptic plasticity between control and pilocarpine-treated rats. Finally, using biochemical methods, we will explore the mechanism of α5-GABAARs in modulating the excitability and synaptic transmission efficacy.

ClC-2是一种电压门控性氯通道，在稳定细胞膜电位、调节细胞兴奋性方面有重要意义，但其在癫痫发病中所起的作用还不清楚。我们研究发现：致痫鼠海马锥体细胞的顶树突区ClC-2 的表达量显著上调、全细胞模式下ClC-2电流明显增大；且ClC-2与α5-GABAAR介导的紧张性抑制有关。本项目将进一步探讨ClC-2和紧张性抑制的改变对于海马神经元兴奋性、癫痫发作及学习记忆功能的影响及机制。应用匹罗卡品致痫模型，首先行为学研究ClC-2 KO小鼠和野生型小鼠癫痫发作的频率、持续时间及空间记忆的差异；然后膜片钳记录各组小鼠致痫前、后海马CA1区锥体细胞的兴奋性、ClC-2电流及α5-GABAAR介导的紧张性抑制电流，在体场电位记录各组小鼠致痫前、后海马LTP/LTD等；最后应用分子生物学技术阐明ClC-2和紧张性抑制在调节海马神经元电活动及突触可塑性方面的作用机制。为揭示癫痫发病机制提供新的实验依据。

ClC-2是一种电压门控性氯通道，在维持神经元内氯离子浓度、调节细胞兴奋性方面起重要作用，但其在癫痫发病中所起的作用还不清楚。我们研究发现：在癫痫病理状态下，海马神经元ClC-2功能性上调和α5-GABAAR 介导紧张性抑制的代偿性增强，对于维持细胞膜电位、避免神经元过度兴奋有着积极的保护作用；但同时，对于学习记忆功能存在一定程度的抑制。从而解释了颞叶癫痫患者合并认知功能障碍的普遍现象。离体海马脑片给予特异性α5-GABAAR阻断剂，ClC-2电流明显减小。由此证明，ClC-2和α5-GABAAR介导的紧张性抑制有关。此外，我们课题组发现，侧脑室注射特异性α5-GABAAR阻断剂，可以改善癫痫动物的空间记忆功能。说明在病理状态下，分布于突触外的GABAAR受体介导的紧张性抑制参与调节海马突触可塑性。本项目进一步探讨了紧张性抑制调节海马突触可塑性的机制，观察到阻断海马CA1区GABAergic的紧张性抑制作用以后，再诱导海马LTP，PI3K/Akt-mTOR信号通路的表达出现显著性改变，通过对该信号通路限速酶的磷酸化水平定量分析，明确了该信号通路参与调节致痫鼠海马的突触可塑性。为揭示癫痫合并认知功能损伤的机制提供新的实验依据。