钠通道Nav1.6在海马苔藓纤维-CA3通路突触信号传递中的功能研究

Voltage-gated sodium channel Nav1.6 is broadly expressed in nervous system and plays an important role in neuronal excitability. The distinct biophysical properties of Nav1.6 confer neurons the ability to fire repetitively at high frequency. Changes in gating properties or protein expression of Nav1.6 could cause aberrant excitability and lead to neurological disorders. Mutation of Scn8a (encoding Nav1.6) has been recognized as an important genetic cause of epileptic encephalopathy, but the underlying mechanism is still unclear. Hippocampus has been identified as the seizure-initiating zone in many epilepsy patients as well as in animal epileptic models. Here, with the combination of multiple technique approaches, we investigate the function of Nav1.6 in the synaptic transmission at hippocampal mossy fiber (MF)-CA3 pathway. We aim to answer the following questions: 1) the expression and distribution patterns of Nav1.6 in the MF-CA3 pathway, especially at MF boutons; 2) the function and underlying mechanism of Nav1.6 in the synaptic transmission at MF-CA3 pathway; 3) the correlation between the change of Nav1.6 activity and the synaptic transmission at the MF-CA3 pathway in mouse epileptic model. This study will enrich our knowledge on the physiological and pathological roles of Nav1.6 in synaptic transmission, and provide new perspective in exploring the mechanisms underlying Nav1.6-associated neuronal diseases, which will contribute to the development of new therapeutic approaches for sodium channelopathies.

钠通道Nav1.6在神经系统广泛表达，赋予神经元高频放电的能力，Nav1.6功能改变可引起神经系统功能障碍。近年来研究表明Nav1.6基因突变是原发性癫痫的致病因素之一，但其致病机制尚不清楚。海马是脑内最易发生癫痫发作的部位，海马齿状回-CA3-CA1神经环路是研究癫痫及药物成瘾机制的重要神经环路之一。本项目以海马苔藓纤维（MF）-CA3锥体细胞通路为实验对象，研究1）Nav1.6在MF-CA3通路的表达分布特点，特别是在MFB突触/突触邻近结构的分布；2）Nav1.6在MF-CA3通路突触信号传递中的作用和机制；3）癫痫模型动物脑内Nav1.6在MF-CA3通路中的表达和功能变化与癫痫发作的关系。本项目从突触信号传递及突触可塑性角度考察Nav1.6对神经环路中神经元兴奋性的影响，探讨Nav1.6基因突变的可能致病机制，为钠通道疾病的治疗及钠通道阻断剂类药物的临床使用提供新思路和实验依据。

背景：电压门控性钠通道在动作电位（Action potential，AP）产生和传播中起着重要作用。Nav1.6在中枢神经系统广泛表达，同其它中枢神经系统钠通道比较，Nav1.6具有激活电位低，通道失活和复活快速，产生持续电流和再生电流的能力强等特点，是决定神经元能否进行高频放电的重要因素之一。临床报告表明Nav1.6也是癫痫的致病基因之一，但其致病机制尚不明确。.主要研究内容：海马是脑内最易发生癫痫发作的部位，海马齿状回-CA3-CA1神经环路是研究癫痫的重要神经环路。本项目以海马苔藓纤维（mossy fiber， MF）-CA3锥体细胞通路为实验对象，研究：1）Nav1.6在MF-CA3通路中的表达分布特点；2）Nav1.6在MF-CA3通路中的表达对突触信号传递以及神经元兴奋性的影响。从突触信号传递和突触可塑性角度考察Nav1.6对神经元兴奋性的影响，探讨Nav1.6基因改变引发癫痫的可能机制。.重要结果：研究显示1）发育早期，Nav1.6通道在小鼠海马表达分布具有区域差异，Nav1.6首先在新生小鼠CA3区锥体神经元的AIS表达，随后出现在CA1和DG区神经元； 2）在新生小鼠（P5-8和P14-18），Nav1.6表达可降低CA3锥体神经元产生动作电位的阈值，提高AP发放的频率，表明Nav1.6参与CA3神经元AP的发放；3）对临床报道位于DIS3-S4和Loop3上的Nav1.6突变的电生理研究显示功能增强性改变。.关键数据：研究发现Nav1.6在小鼠海马的表达早于以往的报道。Western blot结果表明早在P0小鼠海马即有微弱Nav1.6的表达，到P7时表达水平已同P14相近。免疫荧光实验显示Nav1.6首先出现在CA3锥体神经元，并参与CA3神经元AP的发放。.科学意义：以往研究认为Nav1.6在小鼠出生两周后才出现明显表达，我们的研究结果表明Nav1.6在小鼠脑中的表达要早于过去的认识，这与Nav1.6突变引发癫痫的发病时间早的临床报道也更符合。Nav1.6的电生理特性决定了其在神经元高频放电中的重要性。发育早期Nav1.6首先在海CA3锥体神经元表达并参与神经元AP的发放提示Nav1.6突变有可能通过促进CA3神经元的异常放电导致癫痫发作。开发针对CA3神经元兴奋性的临床药物或干预技术对Nav1.6突变引发癫痫可能具有治疗作用。