The role of acetylcholine signaling in the axonal wiring of cortical interneurons

项目总结/摘要 胆碱能系统，使用乙酰胆碱（ACh）作为神经递质，塑造可塑性和认知功能。 通过调节皮质活动在成人皮质中起作用，并且与脑疾病如癫痫， 注意力缺陷多动障碍抑郁症和精神分裂症。然而，ACh信号转导在 在正常和患病条件下的皮层回路的发育仍然知之甚少。特别是， 关于ACh信号传导是否以及如何调节抑制性中间神经元（IN）的布线， 皮质计算的关键部件。皮质IN通常发育高度分支的轴突以建立 局部密集电路模块。尽管代表了IN电路布线过程中的关键事件，但蜂窝和 IN轴突树枝化的分子机制仍然是难以捉摸的。 我们的建议的目的是建立ACh信号在塑造IN轴突乔木中的作用 in vivo.我们还将提供证据表明，ACh所必需的基因中的疾病相关突变 信号传导可以影响IN轴突分支，并且轴突表型可以通过 操纵ACh信号传导中的下游成分。为了实现这一目标，我们将执行一系列 实验中使用枝形吊灯细胞（ChC），它专门支配锥体神经元的轴突起始段 神经元（PN），从而有力地控制PN中的尖峰生成。 ChC由于其轴突结构的定型性，是研究IN轴突的理想模型 形态发生我们的初步资料表明：（1）由静脉曲张引起的轴突丝状伪足， （2）ChC轴突静脉曲张中丝状伪足的起始以及基础Ca ~（2+）水平 通过烟碱型AChR（nAChR）向T型电压依赖性钙通道（T-Ca 2+）的信号传导来调节 （3）CRISPR/Cas9介导的T-VDCC缺失， 单个ChC中的LOF功能（LOF）显著降低其轴突分支点，以及（4）全身性尼古丁 对发育中的出生后小鼠给药增加ChC轴突主干。基于这些结果，我们建议 为了检验nAChR-T-VDCC信号通路形成ChC轴突分支和疾病的假设， ACh信号分子中的相关突变导致ChC中的布线缺陷。在目标1中，我们将阐明 nAChR-T-VDCC信号通路在体内ChC轴突分支中的作用。在目标2中，我们将确定 nAChRs中癫痫相关的功能获得性（GOF）突变对ChC轴突分支的影响， 阐明操纵T-VDCC是否可以是逆转nAChR GOF表型的一种方式。 我们的研究将为ACh信号在神经元连接中的发育作用提供第一个证据。 正常和患病的大脑皮层，以及一个新的提示，发展战略，以防止和改善大脑 与ACh信号相关的疾病。

Project Summary/Abstract The cholinergic system, using acetylcholine (ACh) as a neurotransmitter, shapes plasticity and cognitive functions in the adult cortex by tuning cortical activity, and has been implicated in brain disorders such as epilepsy, attention-deficit hyperactivity disorder, depression, and schizophrenia. However, the role of ACh signaling in development of cortical circuits in normal and diseased conditions remains poorly understood. In particular, little is known about whether and how ACh signaling regulates the wiring of inhibitory interneurons (INs), cellular components critical for cortical computations. Cortical INs generally develop highly branched axons to establish local, dense circuit modules. Despite representing a crucial event during the wiring of IN circuits, the cellular and molecular mechanisms underlying IN axonal arborization remain elusive. The objective of our proposal is to establish the role of ACh signaling in shaping IN axonal arbors in vivo. We will also provide evidence that disease-relevant mutations in genes that are essential for ACh signaling could impact IN axonal branching, and that the axonal phenotype could be ameliorated by manipulating downstream components in ACh signaling. To achieve this goal, we will perform a series of experiments using the chandelier cell (ChC), which exclusively innervates axon initial segments of pyramidal neurons (PNs) and thus powerfully controls spike generation in PNs. Because of its stereotypy of the axonal organization, the ChC serves as an ideal model to study IN axonal morphogenesis. Our preliminary data has shown that (1) Axonal filopodia arising from varicosities serve as precursors of branches in vivo, (2) Filopodia initiation as well as the basal Ca2+ levels in ChC axonal varicosities are regulated by signaling from nicotinic AChRs (nAChRs) to T-type voltage dependent calcium channels (T- VDCCs), independently of action potentials/network activity, (3) CRISPR/Cas9-mediated T-VDCC loss-of- function (LOF) in single ChCs significantly reduces their axonal branching points, and (4) Systemic nicotine administration to developing postnatal mice increases ChC axonal arbors. Based on these results, we propose to test the hypothesis that the nAChR-T-VDCC signaling pathway shapes ChC axonal arborization, and disease- relevant mutations in ACh signaling molecules cause wiring defects in ChCs. In Aim 1, we will elucidate the role of the nAChR-T-VDCC signaling pathway in ChC axonal arborization in vivo. In Aim 2, we will determine the effect of the epilepsy-related gain-of-function (GOF) mutation in nAChRs on ChC axonal arborization, and elucidate whether manipulating T-VDCCs can be a way to revert the nAChR GOF phenotype. Our study will provide first evidence for the developmental role of ACh signaling in the wiring of INs in the normal and diseased cortices as well as a novel hint for developing strategies to prevent and ameliorate brain disorders associated with ACh signaling.