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

项目总结 我正在申请NRSA博士后奖学金，我的长期职业目标是成为 能够领导神经科学领域高效率研究实验室的独立调查者。在……下面 在Ethan Goldberg博士和Doug Coulter博士的指导下，这项有指导的奖学金将为我提供 为了实现我的目标并在科学事业中茁壮成长，我需要接受概念性和实践性的培训。 编码电压门控钾通道亚单位的基因KCNC1的致病变异 KV3.1，会导致包括癫痫在内的严重神经疾病。虽然大多数患者衍生的变异体是丢失的- KV3.1功能受损改变个体神经元生理和功能的精确机制 导致自发性癫痫发作的神经回路功能尚不清楚。KV3.1在显著位置表示为 小白蛋白阳性快速放电抑制中间神经元(PV-IN)分布于不同的亚细胞区，包括 树突、胞体、轴突和突触终末对可靠的高频活动起关键作用 潜在的产生和繁殖。因为光伏接入对网络动态和约束具有重要贡献 皮层环路附近兴奋性锥体神经元的兴奋性，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，抑制锥体神经元，以及 有助于异常的网络兴奋性，以驱动异常行为和癫痫发作。因此，最重要的是 本研究的目的是确定突变的KV3.1对PV-IN生理的影响，作为潜在的主要 参与KCNC1相关性癫痫的发病机制。在目标1中，我将收集膜片钳电生理学 新记录的PV-INS的躯体和轴突钾通道功能及神经元兴奋性 KCNC1型癫痫小鼠模型，该模型含有癫痫脑病患者衍生的P.A421V变异。 鉴于在p.A421V变型中观察到的通道活动显著减少，并且Kv3电流是 对于快速放电的生理学来说，我预计Pv-ins来自KCNC1癫痫小鼠模型 脑病将表现为胞体的动作电位产生受损，并通过 轴突。在目标2中，我将询问突变KV3.1对抑制性突触传递的反应缺陷 使用多(6-8)个同时记录突触连接的PV-in和锥体的表达 皮质微回路中的神经元。最后，在目标3中，我将在活体环境中证实我的体外研究结果 通过钙成像对清醒、行为正常的小鼠的神经元活动进行研究。将神经元活动与相关的 行为，我将研究皮层锥体神经元和PV-INS对感官反应的兴奋性 通过晶须偏转刺激并检验改变PV-INS中KV3.1功能的假设 体内网络抑制。总体而言，完成这项指导奖学金中描述的目标将提供 对KCNC1相关疾病的癫痫机制的重要洞察和对KCNC1相关疾病的更深入的理解 Kv3通道在PV-IN生理中的作用

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.