Molecular and cellular mechanisms governing interneuron development and connectivity

项目概要/摘要 新皮层中神经元回路的正确组装和功能，这是一种对所有高级神经元至关重要的大脑结构。 顺序功能，依赖于兴奋性锥体神经元之间特定突触连接的形成 （PyNs）和不同类型的抑制性GABA能中间神经元。中间神经元的一个子集， 控制PyN尖峰的是枝形吊灯细胞（ChC），它专门在作用部位形成连接 PyN中的潜在起始，称为轴突起始段（AIS）。由于形成的独特连接 在ChC轴突乔木的末端和大量PyN的AIS之间，ChC是理想的适合 来控制兴奋性皮层网络的输出。因此，这并不奇怪，ChC连接缺陷是 与精神分裂症和自闭症谱系障碍等脑部疾病有关。尽管ChC很重要， PyNAIS的亚细胞神经支配的分子机制尚不清楚。为了解决这个问题， 我们启动了一项基于子宫内电穿孔（IUE）的体内RNAi筛选， 轴突细胞粘附分子（CAM）和选择的Eph/ephrin。引人注目的是，在所有测试的分子中，我们发现 CAM L1 CAM是ChCs对PyNAIS神经支配所需的唯一蛋白质。此外，基于单- ChC RNAseq数据，我们研究了ChC表达的netrin受体Unc 5 b在ChC/PyN AIS神经支配中的作用 并发现它在ChC轴突终末发育和连接中起关键作用。我们的发现提供了一个 ChC/PyN连接的分子基础研究的独特切入点。本申请旨在阐明 PyN L1 CAM如何支配选择性AIS神经支配，以鉴定ChC上L1 CAM的突触前结合伴侣， 探讨Unc 5 b在ChC轴突终末发育和连接中的作用机制。为此，瞄准 1将使用分子工具破坏L1 CAM和AIS细胞骨架之间的相互作用，以评估是否 在AIS处的细胞色素介导的L1 CAM聚集是适当的ChC/PyN AIS神经支配所需要的。高 将进行分辨率成像以研究AIS和远端轴突上表面L1 CAM的分布。 此外，将使用由IUE递送以暂时调节体内PyN L1 CAM水平的诱导型RNAi构建体 以确定PyN L1 CAM是否是建立和/或维持ChC/PyN神经支配所必需的。 目的2将鉴定ChC末端上L1 CAM的突触前结合伴侣。我们的初步数据显示 neuropilin-1（Nrp 1）是ChC/PyN神经支配所需的ChC上的L1 CAM伴侣。这将由以下人员进行测试： 使用RNAi技术和条件性Nrp 1敲除小鼠，消除ChCs中的Nrp 1。Nrp 1结构域 L1 CAM绑定所需的参数也将定义。目标3将确定Unc 5 b是否支配ChC测试卡片 通过调节末端轴突分支来发育。我们还将测试LARG/RhoA/ROCK通路是否 使用分子/细胞工具介导Unc 5 b对ChC轴突末端发育和连接的影响， ChC靶向IUE。总之，我们的研究将首次深入了解ChC/PyN的调控机制 连通性，并揭示了常见大脑疾病的连通性缺陷。 !

PROJECT SUMMARY/ABSTRACT Proper assembly and functioning of neuronal circuits in the neocortex, a brain structure critical for all higher- order functions, relies on the formation of specific synaptic connections between excitatory pyramidal neurons (PyNs) and different types of inhibitory GABAergic interneurons. One subset of interneurons that exerts powerful control over PyN spiking is the chandelier cell (ChC), which forms connections specifically at the site of action potential initiation in PyNs, referred to as the axon initial segment (AIS). Due to the unique connections formed between the terminals of ChC axonal arbors and the AISs of large populations of PyNs, ChCs are ideally suited to control the output of excitatory cortical networks. Hence, it is not surprising that ChC connectivity defects are linked to brain disorders, such as schizophrenia and autism spectrum disorder. Despite the importance of ChCs, the molecular mechanisms underlying their subcellular innervation of PyN AISs are unknown. To approach this, we initiated an in utero electroporation (IUE)-based in vivo RNAi screen of known neocortical PyN-expressed axonal cell adhesion molecules (CAMs) and select Ephs/ephrins. Strikingly, of all the molecules tested, we found the CAM L1CAM to be the only protein required for PyN AIS innervation by ChCs. In addition, based on single- ChC RNAseq data, we investigated the role of ChC-expressed netrin receptor Unc5b in ChC/PyN AIS innervation and found that it plays a key role in ChC axon terminal development and connectivity. Our findings provide a unique entry point for studies on the molecular basis of ChC/PyN connectivity. This application aims to elucidate how PyN L1CAM governs selective AIS innervation, to identify L1CAM’s presynaptic binding partner(s) on ChCs, and to scrutinize the mechanism of Unc5b in ChC axon terminal development and connectivity. To this end, Aim 1 will use molecular tools to disrupt interactions between L1CAM and the AIS cytoskeleton to assess whether cytoskeleton-mediated L1CAM clustering at the AIS is required for proper ChC/PyN AIS innervation. High resolution imaging will be performed to investigate the distribution of surface L1CAM on the AIS and distal axon. Also, inducible RNAi constructs delivered by IUE to temporally regulate PyN L1CAM levels in vivo will be used to determine whether PyN L1CAM is required for the establishment and/or maintenance of ChC/PyN innervation. Aim 2 will identify the presynaptic binding partner(s) of L1CAM on ChC terminals. Our preliminary data suggest that neuropilin-1 (Nrp1) is the L1CAM partner on ChCs required for ChC/PyN innervation. This will be tested by depleting Nrp1 in ChCs, using RNAi technology and conditional Nrp1 knockout mice. The Nrp1 domain(s) required for L1CAM binding will also be defined. Aim 3 will determine whether Unc5b governs ChC cartridge development by regulating terminal axon branching. We will also test whether the LARG/RhoA/ROCK pathway mediates Unc5b’s effect on ChC axon terminal development and connectivity using molecular/cellular tools and ChC-targeting IUE. Together, our studies will provide first insight into the mechanisms governing ChC/PyN connectivity and shed new light on the connectivity defects underlying common brain disorders. !