Defining the circuit, synaptic, and molecular mechanisms linking intracellular Ca2+ release to learning using subcellularly-targeted manipulations and imaging techniques in dendrites in vivo

项目总结/摘要 候选人目标和使命相关性：申请人的广泛，长期目标是调查有多高- （电路/行为）和低（亚细胞/分子）水平的大脑组织原则合作， 学习拟议的研究活动将为这一长期目标奠定基础， 通过整合新的技术和概念方法，促进BRAIN 2025报告的目标， 细胞内Ca ~（2+）从内质网（ER）释放（ICR）到神经活动动力学和行为。 项目简介：树突状细胞中的钙离子是神经可塑性机制的核心，使动物能够适应环境。 环境ICR长期以来一直被认为塑造了这些机制。申请人最近进行了第一次 研究体内哺乳动物神经元的ICR，以揭示这种亚细胞现象是如何形成的 小鼠锥体神经元树突的经验依赖性特征选择性 海马CA 1区。这项工作提出了关于何时、何地以及如何参与工业和企业代表的重要问题 来支持学习。申请人将在以下目标中解决这些问题： 目标1。表征体内树突中可塑性相关的ER Ca 2+动力学（K99）： 目的是，申请人将对细胞溶质和ER进行同时双色、双平面体内双光子成像。 在新的虚拟环境的头部固定空间导航期间，单个CA 1 PN的树突中的常驻Ca 2+。 目标2.定义将细胞内Ca 2+释放与体内突触可塑性联系在一起的突触逻辑（K99/R 00）： 申请人将首先创建一种新的分子工具来光遗传学诱导ICR（Aim 2.1; K99）。申请人 然后将联合收割机与单细胞成像结合， 释放，以及ICR的光遗传学抑制，以剖析ICR参与可塑性的突触逻辑 诱导行为小鼠。(Aim 2.2; R00）。 目标3：解剖兴奋性回路-体内驱动细胞内Ca 2+释放的分子机制（R 00）： 候选物将光遗传学地激活特定的兴奋性投射到不同的树突隔室， 单个CA 1 PN，同时监测行为小鼠中的ER Ca 2+动力学。局部药理学操作将 剖析两个典型的途径，转换突触前兴奋性输入突触后ICR的贡献。 职业发展计划：申请人将延长一个高度互补的共同导师安排 Franck Polleux博士和Attila Losonczy博士在细胞/分子/遗传学方面拥有深厚的专业知识， in vivo体内/behavioral行为approaches方法，respectively分别.申请人将得到博士的大力咨询支持。 哥伦比亚理论神经科学中心的Stefano Fusi和细胞神经科学中心主任Darcy Peterka博士 哥伦比亚朱克曼研究所的成像。申请人的研究和向独立的过渡将受益 从这个强大的指导团队，国家的最先进的设施，所有必要的设备，以及众多的专业 通过哥伦比亚博士后事务办公室，朱克曼研究所， 和大脑倡议。

Project Summary/Abstract Candidate Goals and Mission Relevance: The applicant’s broad, long-term objective is to investigate how high- (circuit/behavioral) and low- (subcellular/molecular) level organizational principles of the brain cooperate to drive learning. The proposed research activities will build a foundation for this long-term goal and, in so doing, will promote BRAIN 2025 Report goals by integrating new technological and conceptual approaches to causally link intracellular Ca2+ release (ICR) from endoplasmic reticulum (ER) to neural activity dynamics and behavior. Project description: Dendritic Ca2+ is central to neural plasticity mechanisms allowing animals to adapt to the environment. ICR has long been thought to shape these mechanisms. The applicant recently carried out the first investigation of ICR in mammalian neurons in vivo to uncover how this subcellular phenomenon shapes experience-dependent feature selectivity across the dendritic arbor of pyramidal neurons (PNs) in mouse hippocampal area CA1. This work raises important questions regarding when, where, and how ICR is engaged to support learning. The applicant will address these questions in the following Aims: Aim 1. Characterize plasticity-associated ER Ca2+ dynamics in dendrites in vivo (K99): To achieve this Aim, the applicant will perform simultaneous dual-color, dual-plane in vivo 2-photon imaging of cytosolic and ER- resident Ca2+ in dendrites of single CA1 PNs during head-fixed spatial navigation of novel virtual environments. Aim 2. Define the synaptic logic tying intracellular Ca2+ release to in vivo synaptic plasticity (K99/R00): The applicant will first create a novel molecular tool to optogenetically induce ICR (Aim 2.1; K99). The applicant will then combine this precise interventional tool with single-cell imaging, inducible blockade of presynaptic release, and optogenetic dampening of ICR to dissect the synaptic logic by which ICR participates in plasticity induction in behaving mice. (Aim 2.2; R00). Aim 3. Dissect excitatory circuit-molecular mechanisms driving intracellular Ca2+ release in vivo (R00): The candidate will optogenetically activate specific excitatory projections onto distinct dendritic compartments of single CA1PNs while monitoring ER Ca2+ dynamics in behaving mice. Local pharmacological manipulations will dissect contributions of the two canonical pathways that convert presynaptic excitatory input to postsynaptic ICR. Career development plan: The applicant will extend a highly complementary Co-Mentorship arrangement between Drs. Franck Polleux and Attila Losonczy who possess deep expertise in cellular/molecular/genetic and in vivo/behavioral approaches, respectively. The applicant will receive robust consultative support from Dr. Stefano Fusi of Columbia’s Center for Theoretical Neuroscience and Dr. Darcy Peterka, Director of Cellular Imaging at Columbia’s Zuckerman Institute. The applicant’s research and transition to independence will benefit from this strong mentorship team, state-of-the-art facilities, all necessary equipment, and numerous Professional Development resources offered through the Columbia Office of Postdoctoral Affairs, the Zuckerman Institute, and the BRAIN Initiative.