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

定向诱变阐明中枢神经系统中待研究离子通道的功能

• 批准号: 10045757
• 负责人: Marta E Soden
• 金额: $ 15.55万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2022-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045757
• 关键词: Action Potentials; Address; Adult; Affect; Anxiety; Attention deficit hyperactivity disorder; Automobile Driving; Behavior; Behavioral; Biological Models; CRISPR/Cas technology; Calcium Channel; Cell Separation; Cell membrane; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Cues; Cytomegalovirus; DNA Sequence Alteration; DNA sequencing; Data; Development; Dopamine; Electrophysiology (science); Emotional; Environment; Enzymes; Exhibits; Frequencies; Gene Mutation; Genes; Genome; Goals; Guide RNA; Individual; Injections; Investigation; Ion Channel; Ions; Kinetics; Knock-out; Learning; Light; Link; Measures; Membrane Potentials; Mental disorders; Methods; Modeling; Motivation; Mus; Mutagenesis; Mutation; Neuraxis; Neurons; Neurotransmitters; Nonsense Codon; Nucleus Accumbens; Pattern; Periodicity; Pharmacology; Physiological; Physiology; Play; Polyribosomes; Potassium Channel; Precision therapeutics; Property; Proteins; Research; Rest; Rewards; Role; Scanning; Schizophrenia; Shaker potassium channel; Shapes; Slice; Specificity; Stimulus; Synapses; Synaptic Vesicles; System; Technology; Testing; Therapeutic; Time; Translating; Ventral Tegmental Area; Viral; Viral Vector; Virus; adeno-associated viral vector; autism spectrum disorder; behavior influence; cell type; cost; cost effective; dopamine system; dopaminergic neuron; gene function; in vivo; interest; knockout gene; mature animal; motivated behavior; nervous system disorder; neural circuit; neuropsychiatric disorder; neurotransmitter release; novel; optogenetics; promoter; rapid technique; relating to nervous system; response; therapeutic target; trafficking; vector; voltage

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.

项目摘要/摘要 许多类型的神经元利用复杂且高度特定的动作电势射击来调节 神经递质释放并与下游细胞通信。动作潜力发射主要受到调节 通过神经递质的输入以及通过确定的离子通道基因的表达 单个神经元的基线发射特性。尽管其中一些离子通道很努力，但许多 保持出乎意料或不充实。腹侧盖区（VTA）的多巴胺神经元紧密暴露 受控的活动模式，包括活动中的爆发和停顿，其中编码有关 环境线索和奖励并影响学习和动力。人们认为这些中断 发射模式有助于与精神疾病相关的各种行为扰动。那，VTA 多巴胺神经元提供了一个出色的模型系统，用于研究理解离子的功能 在其本地神经元环境中的渠道。最近的一项研究确定了离子的整个完成 在小鼠多巴胺神经元中表达的通道；我选择了研究其中的三个渠道 出现在照明的可毒基因组（IDG）的蛋白质清单上：cacna2d3，编码 2-3钙通道辅助亚基，KCNA6，编码振动钾通道KV1.6和KCNAB2， 编码KV2钾通道辅助亚基。我将利用一个小说的单矢量adeno相关 利用CRISPR/CAS9基因编辑技术来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）。然后，我将使用切片电生理学 和快速扫描的环状伏安法与光遗传学结合使用，以表征单个离子的影响 通道基因敲除并确定这些理解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病毒 快速，特异性诱导基因敲除技术，这将广泛适用于研究 理解的蛋白质。此外，通过识别多巴胺发射模式的新型调节剂，我们将俩都 提高我们对驱动这些关键神经元的潜在生理学的理解并确定新的 精确治疗的潜在靶标。

项目成果

The potassium channel auxiliary subunit Kvβ2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

钾通道辅助亚基 Kvβ2 (Kcnab2) 调节 Kv1 通道和多巴胺神经元放电。

• DOI: 10.1152/jn.00194.2022
• 发表时间: 2022
• 期刊: Journal of neurophysiology
• 影响因子: 2.5
• 作者: Yee,JoshuaX;Rastani,Ariana;Soden,MartaE
• 通讯作者: Soden,MartaE

Temporal scaling of dopamine neuron firing and dopamine release by distinct ion channels shape behavior.

• DOI: 10.1126/sciadv.adg8869
• 发表时间: 2023-08-11
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Juarez, Barbara;Kong, Mi-Seon;Jo, Yong S.;Elum, Jordan E.;Yee, Joshua X.;Ng-Evans, Scott;Cline, Marcella;Hunker, Avery C.;Quinlan, Meagan A.;Baird, Madison A.;Elerding, Abigail J.;Johnson, Mia;Ban, Derek;Mendez, Adriana;Goodwin, Nastacia L.;Soden, Marta E.;Zweifel, Larry S.
• 通讯作者: Zweifel, Larry S.