Determining the function of TRPC6 channels in a subpopulation of VTA dopamine neurons

项目摘要/摘要： 腹侧被盖区(VTA)的中脑多巴胺(DA)产生神经元在 调节奖赏行为。VTA-DA神经元在功能和遗传上是不同的，并且 神经肽和神经肽受体的遗传标记可用于分离VTA亚群。 尽管已经证明不同的神经肽能通路可以有效地调节DA神经元，但 神经肽受体下游的细胞内信号通路尚不清楚。VTA-DA神经元 表达GQ蛋白偶联受体速激肽受体3(Tacr3)的基因是最低限度的 促进奖励强化行为的DA神经元亚群。Tacr3的激活最近是 表明依赖于瞬时受体电位规范(TRPC)通道信号在 下丘脑。TRPC 6型(TRPC6)通道丰富于DA神经元，并可被刺激激活 GQ偶联受体信号转导。因此，我假设TRPC6可能是中TRPC通道的主要类型 在Tacr3激活下游起作用的VTA-DA神经元。确立TRPC6在调节 VTA-Tacr3神经元的生理和功能，我建议选择性地突变TRPC6基因来产生一个 使用先进的CRISPR/Cas9在成年小鼠的VTA内以细胞类型特异性的方式丧失功能 基因技术。我将进行体外切片电生理和切片钙成像(目标1)，以及 概率贴现范例和累进比率期间钙动力学的活体记录 激励任务(目标2)。确定TRPC6在VTA-Tacr3神经元中的功能将提供重要的 对中枢神经系统中这种未被充分研究的离子通道的治疗潜力的洞察。

Project Summary/Abstract: Midbrain dopamine (DA)-producing neurons of the ventral tegmental area (VTA) play a critical role in modulating reward-seeking behavior. VTA-DA neurons are functionally and genetically heterogeneous, and genetic markers for neuropeptides and neuropeptide receptors can be used to isolate VTA subpopulations. Though distinct neuropeptidergic pathways have been shown to potently modulate DA neurons, the intracellular signaling pathways that act downstream of neuropeptide receptors are unknown. VTA-DA neurons that express the Gq-protein coupled receptor, tachykinin receptor 3 (Tacr3), are a minimally sufficient subpopulation of DA neurons that promote reward reinforcement behavior. Tacr3 activation has recently been shown to be dependent on transient receptor potential canonical (TRPC) channel signaling in the hypothalamus. TRPC type 6 (TRPC6) channels are enriched in DA neurons and are activated by stimulation of Gq-coupled receptor signaling. Therefore, I hypothesize that TRPC6 is likely the main type of TRPC channel in VTA-DA neurons that acts downstream of Tacr3 activation. To establish the role of TRPC6 in regulating the physiology and function of VTA-Tacr3 neurons, I propose to selectively mutate the Trpc6 gene to generate a loss of function in a cell-type specific manner within the VTA of adult mice using an advanced CRISPR/Cas9 genetic technology. I will perform ex vivo slice electrophysiology and slice calcium imaging (Aim 1), as well as in vivo recordings of calcium dynamics during a probabilistic discounting paradigm and progressive ratio motivational task (Aim 2). Determining the function of TRPC6 in VTA-Tacr3 neurons will provide important insights into the therapeutic potential of this understudied ion channel in the central nervous system.