Mechanisms of cellular, synaptic and circuit dysfunction in Kcnc1-related epileptic encephalopathy

项目摘要 我申请这个NRSA博士后奖学金的长期职业目标是成为一个 独立研究员，能够在神经科学领域领导一个高产的研究实验室。下 Ethan Goldberg博士和Doug Coulter博士的指导，这个指导奖学金将为我提供 概念和实践培训，以实现我的目标，并在科学企业蓬勃发展。 编码电压门控钾通道亚单位的基因KCNC 1的致病性变体 Kv3.1，导致严重的神经系统疾病，包括癫痫。虽然大多数患者来源的变体是缺失的， 功能，受损的Kv3.1功能改变个体神经元生理学的精确机制， 导致自发性癫痫发作的神经回路功能尚不清楚。Kv3.1主要表达在 小清蛋白阳性的快速尖峰抑制性中间神经元（PV-IN）在各个亚细胞区域，包括 树突、索马、轴突和突触末梢，在这些部位，它对可靠的高频动作起关键作用 潜在的生成和传播。由于PV-IN对网络动态和约束 兴奋性附近的兴奋性锥体神经元在皮层电路，我们假设Kv3.1功能障碍， 损害PV-IN高频动作电位的产生和传播，解除对锥体神经元的抑制， 导致异常的网络兴奋性，以驱动异常行为和癫痫发作。因此， 本研究的目的是确定突变体Kv3.1对PV-IN生理学的影响， KCNC 1相关癫痫的发病机制。在目标1中，我将收集膜片钳电生理学 记录了一种新的PV-IN的体细胞和轴突钾通道功能和神经元兴奋性， KCNC 1癫痫的小鼠模型，其具有癫痫性脑病患者衍生的p.A421V变体。 考虑到在p.A421V变体中观察到的通道活性显著降低，并且Kv 3电流 由于这是快速脉冲生理学所必需的，我预计来自KCNC 1癫痫小鼠模型的PV-IN 脑病将在索马表现出受损的动作电位产生， 轴突在目标2中，我将询问突变Kv3.1引起的抑制性突触传递的缺陷。 使用多个（6-8个）同时记录突触连接的PV-IN和锥体细胞的表达， 皮层微电路中的神经元。最后，在目标3中，我将在体内环境中证实我的体外发现 通过对清醒的、行为正常的老鼠的神经元活动进行钙成像。将神经元活动与 行为，我将检查皮质锥体神经元和PV-IN的兴奋性，以响应感觉 通过晶须偏转刺激，并测试PV-IN中Kv3.1功能改变的假设， 体内网络抑制。总的来说，完成本指导研究金中所述的目标将提供 对KCNC 1相关疾病中癫痫机制的重要见解，以及对 Kv 3通道对PV-IN生理学的贡献。

PROJECT SUMMARY I am applying for this NRSA postdoctoral fellowship with the long-term career goal of becoming an independent investigator capable of leading a highly productive research lab in the field of neuroscience. Under the guidance of Dr. Ethan Goldberg and Dr. Doug Coulter, this mentored fellowship will provide me the conceptual and hands-on training required to achieve my goal and thrive in the scientific enterprise. Pathogenic variants in the gene KCNC1, which encodes the voltage-gated potassium channel subunit Kv3.1, lead to severe neurological disease including epilepsy. While most patient-derived variants are loss-of- function, the precise mechanisms by which impaired Kv3.1 function alters individual neuron physiology and neural circuit function to result in spontaneous seizures are unclear. Kv3.1 is prominently expressed in parvalbumin-positive fast-spiking inhibitory interneurons (PV-IN) in various subcellular regions including the dendrites, soma, axon, and synaptic terminal where it critically contributes to reliable high-frequency action potential generation and propagation. Because PV-INs critically contribute to network dynamics and constrain excitability of nearby excitatory pyramidal neurons in cortical circuits, we hypothesize that Kv3.1 dysfunction impairs PV-IN high-frequency action potential generation and propagation, disinhibits pyramidal neurons, and contributes to aberrant network excitability to drive abnormal behavior and seizures. Therefore, the overarching objective of this study is to determine the effect of mutant Kv3.1 on PV-IN physiology as a potential major contributor to pathogenesis of KCNC1-related epilepsy. In aim 1, I will collect patch-clamp electrophysiology recordings of somatic and axonal potassium channel function and neuronal excitability in PV-INs from a novel mouse model of KCNC1 epilepsy which harbors the epileptic encephalopathy patient-derived p.A421V variant. Given the substantially reduced channel activity observed in the p.A421V variant, and that Kv3 currents are necessary for fast-spiking physiology, I anticipate that PV-INs from the mouse model of KCNC1 epileptic encephalopathy will exhibit impaired action potential generation at the soma, and unreliable propagation through the axon. In aim 2, I will interrogate deficits in inhibitory synaptic transmission in response to mutant Kv3.1 expression using multiple (6-8) simultaneous recordings of synaptically-connected PV-INs and pyramidal neurons in cortical microcircuits. Lastly, in aim 3 I will corroborate my in vitro findings in the in vivo context through calcium-imaging of neuronal activity in awake, behaving mice. To relate neuronal activity to relevant behavior, I will examine the excitability of both cortical pyramidal neurons and PV-INs in response to sensory stimulation by whisker deflection and test the hypothesis that altered Kv3.1 function in PV-INs compromises network inhibition in vivo. Overall, completion of the aims described in this mentored fellowship will provide significant insight into the mechanisms of epilepsy in KCNC1-related disorders and deeper understanding of the contribution of Kv3 channels to PV-IN physiology in general.