AIS结构与功能重塑在癫痫后海马新生神经元自发爆发放电中的作用及机制研究

Prevailing view holds that the increased excitability of dentate newborn granular cells induced by epilepsy and the excitatory-inhibitory imbalance in local circuit contribute to the mechanism of chronic idiopathic epilepsy. Axon initial segment (AIS), as a key area in integrating afferent signal and initiating action potential, its structural and functional plasticity might be vital for excitability regulation of newborn granular cells. To identify our hypothesis, we established Lithium chloride -pilocarpine epileptic rat model, labeled dentate newborn granular cells by transfecting with CAG-EGFP retroviral vector. Both in brain slice and in vivo rat brain, we dynamically monitor the temporal and spatial expression of AIS voltage -gated ion channel(Na+、K+、Ca2+) and the adaptor protein ankyrinG . Also, we perform patch clamp and local field potential recording in single newborn neurons and subgranular zone in dentate gyrus area. We blocked AIS voltage gated ion channel with these specific channel blockers and investigate its effect on the excitability of dentate newborn granular cells in brain slice and in vivo rats. Rat neural stem cells are cultured in high potassium solution to simulate the dentate newborn granular cells with hyperexcitability. High-throughput Gene screening technology is used to investigate differentially expressed miRNA and LncRNA, which might play a great role in AIS structural plasticity and contribute to increase excitability of newborn granular cells in dentate gyrus area after seizure.

癫痫后海马新生神经元异常增生伴兴奋性增高，海马局部神经环路兴奋-抑制稳态失衡，可能是慢性自发性痫性发作的主要原因。轴突起始段（AIS）作为产生并发放动作电位的关键部位，其结构重塑是否参与新生神经元兴奋性增高，其具体调控机制迄今尚不明确。本项目拟建立氯化锂-匹罗卡品颞叶癫痫大鼠模型，CAG-EGFP逆转录病毒特异转染癫痫后海马齿状回新生神经元，离体脑片和在体动物模型上，监测致痫后新生神经元AIS区电压门控离子通道和锚定蛋白的时空改变，膜片钳和双极金属电极记录单个新生神经元及齿状回局部神经场电位变化。应用离子通道阻断剂体内外特异干预，观察靶向调控AIS区电压门控离子通道对新生神经元兴奋性的影响。构建体外高钾溶液下神经干细胞模型，基因芯片高通量筛选可能参与调控AIS区亚细胞结构，介导神经元兴奋性的miRNA和IncRNA，发掘非编码RNA调控癫痫后新生神经元AIS结构与功能重塑的可能信号通路。

癫痫后海马新生神经元异常增生伴兴奋性增高，海马局部神经环路兴奋-抑制稳态失衡，可能是慢性自发性痫性发作的主要原因。轴突起始段（AIS）作为产生并发放动作电位的关键部位，其结构重塑是否参与新生神经元兴奋性增高，其具体调控机制迄今尚不明确。本项目验证了氯化锂-匹罗卡品颞叶癫痫大鼠模型慢性自发痫性发作形成中，海马存在异常增生的新生神经元，且新生神经元轴突起始段（AIS）结构可塑性发生改变；应用高选择性的T型钙离子通道阻断剂Mibefradil特异干预神经元AIS区，可以显著减少致痫后海马新生神经元AIS结构可塑性（长度和距离胞体距离）变化，降低新生神经元兴奋性和颞叶癫痫大鼠自发痫性发作次数及持续时间。进一步应用基因芯片高通量筛选与ankyrinG有靶向关系的miRNA分子，离体细胞和在体动物模型上验证rno-miR485-5P参与海马神经元轴突起始段结构可塑性及慢性致痫网络的调控。项目以“轴突起始段”AIS 为切入点，从“神经元固有可塑性”这个全新的视角，对癫痫后海马新生神经元兴奋性增高的分子机制进行深入探讨；并通过体内、外实验进一步验证其可能的非编码RNA调控路径，以期为慢性自发性癫痫发作发掘新的干预靶点，为难治性颞叶癫痫的治疗提供新的实验基础。