癫痫慢性期BDNF-TrkB-PLCγ通路调控突触外GABAA受体对自发性癫痫发作的作用及机制研究

It is controversial about role of BDNF in epileptogenesis and development of seizure. Our previous work discovered that BDNF and its receptor TrkB are decreased in the chronic recurrent phase of the epileptic rats; and the tonic inhibition mediated by the extrasynaptic GABAA receptors is closely related to the epileptogenesis. BDNF has been reported to regulate the transportation of GABAA receptors to the synapse. We hypothesis that increasing BDNF levels by continuous intrahippocampal infusion will up regulate the extrasynaptic GABAA receptor expression and function, and in turn to inhibit the epileptogenesis in the chronic phase of the epilepsy. We plan, by using behavioral, electrophysiological, Western blot and immunohistochemistry techniques in the chronic CTZ and KA seizure animal model，to study: 1) whether any change of the BDNF, TrkB and the extrasynaptic GABAA receptor expression occurred in the chronic phase of the epilepsy, 2) what the effect on the recurrent seizure by artificial increasing of the BDNF level in the hippocampus, and 3) how the BDNF-TrkB-PLCγ and its down stream signaling pathway regulate the expression and the function of the δ and α5 subunit of the extrasynatic GABAA receptors. The completion of this project will demonstrate that the mechanism of BDNF-TrkB-PLCγ and its down stream signaling pathway regulating extrasynaptic GABAA receptor in chronic recurrent phase of epilepsy and the role of BDNF-TrkB-PLCγ signaling pathway inhibit recurrent seizure.

癫痫发生发展不同时期BDNF在脑内的作用存在很大的争议。我们前期研究发现慢性发作期癫痫鼠海马内BDNF和TrkB的表达减少，而突触外GABAA受体介导的紧张性抑制与癫痫的发生发展有密切的关系。BDNF可调节GABAA受体亚单位的运输，我们推测在癫痫慢性期持续性注射BDNF恢复海马内BDNF水平可通过调节突触外GABAA受体表达与功能抑制癫痫发作。本项目将在CTZ/KA癫痫动物模型上应用行为学、电生理、Western blot以及免疫组织化学等方法研究阐明在癫痫慢性发作期：1）BDNF-TrkB以及突触外GABAA受体表达分布变化；2）增加海马内BDNF含量对癫痫发作的影响作用；3）BDNF-TrkB-PLCγ调控突触外GABAA受体δ和α5亚单位表达与功能的机制。本项目的完成将阐明癫痫慢性期BDNF-TrkB-PLCγ通路调控突触外GABAA受体中的作用机制及其抑制自发癫痫发作的作用

我们前期研究中发现BDNF-TrkB通路在癫痫发生发展中有重要作用，不过在癫痫发作的急性期和慢性期的作用完全相反。抑制BDNF受体TrkB后明显降低了Cyclothiazide （CTZ）诱发的急性癫痫发作，慢性癫痫发作期癫痫鼠海马内BDNF和其受体TrkB的表达却明显减少（Kong SZ et al, Neural Plasticity, 2014），可能慢性期BDNF减少是造成癫痫自发性发作及神经元损伤的原因之一。我们在该项目通过侧脑室注射BDNF、PLCγ抑制剂U73122来研究和探索BDNF-TrkB-PLCγ通路在CTZ诱发癫痫发生、发展中的作用，并进一步探索了突触外GABAA受体在癫痫发作中的作用及机制。实验结果显示：（1）低剂量注射BDN不仅F可以抑制CTZ诱发癫痫的发生，而且在癫痫慢性发作期连续注射低剂量BDNF降低了自发性癫痫的发作，对CTZ诱发癫痫大鼠海马神经元有一定的保护作用；（2）侧脑室注射PLC-γ抑制剂U73122抑制PLC-γ通路后，CTZ诱发癫痫行为明显受到抑制，而且海马内神经元生存数目增加；（3）特异性激动剂THIP能够通过提高突触外GABAA受体功能来抑制海马神经元癫痫样放电的发生；（4）GIRK激动剂ML297通过调节GIRK1通道抑制癫痫样放电。因此，BDNF-TrkB-PLCγ通路、GABAARs和GIRK通道在癫痫发生发展中具有非常重要的作用，该项目的完成对筛选、评价新的抗癫痫药物都具有重要的意义。