Targeted mutagenesis to elucidate the function of understudied ion channels in the central nervous system

项目总结/摘要 许多类型的神经元利用复杂和高度特异性的动作电位放电模式来调节 神经递质释放并与下游细胞沟通。动作电位放电主要是由 通过神经递质的输入以及通过离子通道基因的表达， 单个神经元的基线放电特性。虽然这些离子通道中的一些已经得到了很好的研究，但许多离子通道仍然存在。 仍然未被探索或探索不足。腹侧被盖区（VTA）的多巴胺神经元紧密地表现出 受控的活动模式，包括活动中的突发和暂停， 环境线索和奖励并影响学习和动机。据信，这些中断 放电模式导致了与精神疾病相关的各种行为紊乱。因此，VTA 多巴胺神经元提供了一个很好的模型系统，用于研究未充分研究的离子的功能。 在它们的天然神经元环境中的通道。最近的一项研究发现， 在小鼠多巴胺神经元中表达的通道;我选择了其中三个通道进行研究， 出现在未充分研究的蛋白质的照亮可药用基因组（IDG）列表中：Cacna 2d 3，编码 β 2 β-3钙通道辅助亚基，Kcna 6，编码Shaker钾通道KV 1.6，和Kcnab 2， 编码KV-12钾通道辅助亚基。我将利用一种新的，单一载体腺相关病毒 利用CRISPR/Cas9基因编辑技术快速诱导基因突变的病毒系统， 成年小鼠神经元中的细胞类型特异性（Cre依赖性）方式。然后我会用切片电生理学 和快速扫描循环伏安法结合光遗传学来表征单个离子的影响 通道基因敲除，并确定这些未充分研究的离子通道如何调节多巴胺神经元 生理学和多巴胺释放动力学。这项研究的完成将建立一个简单的，单一的病毒 一种快速、特异地诱导基因敲除技术，将广泛应用于研究 研究不足的蛋白质。此外，通过识别多巴胺放电模式的新型调节器，我们将 增加我们对驱动这些关键神经元的潜在生理学的理解， 精确治疗的潜在目标。

Project Summary/Abstract Many types of neurons utilize complex and highly specific patterns of action potential firing to regulate neurotransmitter release and communicate with downstream cells. Action potential firing is primarily regulated through neurotransmitter input as well as through the expression of ion channel genes that determine the baseline firing properties of an individual neuron. While some of these ion channels are well studied, many remain unexplored or underexplored. Dopamine neurons in the ventral tegmental area (VTA) exhibit tightly controlled activity patterns, including bursts and pauses in activity, which encode information about environmental cues and rewards and influence learning and motivation. It is believed that disruptions in these firing patterns contribute to a variety of behavioral perturbations associated with mental illness. Thus, VTA dopamine neurons provide an excellent model system for investigating the function of understudied ion channels within their native neuronal environment. A recent study identified the entire complement of ion channels expressed in dopamine neurons in mice; I have selected for study three of these channels that also appear on the Illuminating the Druggable Genome (IDG) list of understudied proteins: Cacna2d3, encoding the 2-3 calcium channel auxiliary subunit, Kcna6, encoding the Shaker potassium channel KV1.6, and Kcnab2, encoding the KV2 potassium channel auxiliary subunit. I will utilize a novel, single vector adeno associated viral system that takes advantage of CRISPR/Cas9 gene editing technology to rapidly induce gene mutation in a cell-type specific (Cre-dependent) manner in neurons of adult mice. I will then use slice electrophysiology and fast-scan cyclic voltammetry in combination with optogenetics to characterize the effects of individual ion channel gene knockout and determine how these understudied ion channels regulate dopamine neuron physiology and dopamine release dynamics. Completion of this research will establish a simple, single virus technique for rapidly and specifically inducing gene knockout, which will be widely applicable to investigations of understudied proteins. Additionally, by identifying novel regulators of dopamine firing patterns we will both increase our understanding of the underlying physiology driving these critical neurons and identify new potential targets for precision therapeutics.