Causally linking dendritic Ca2+ dynamics to CA1 circuit function and spatial learning using novel tools to precisely manipulate an endogenous Ca2+ buffering process

使用新工具将树突 Ca2 动力学与 CA1 电路功能和空间学习因果联系起来，以精确操纵内源 Ca2 缓冲过程

• 批准号: 9788758
• 负责人: Justin O'Hare
• 金额: $ 6.66万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-16 至 2021-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788758
• 关键词: Action Potentials; Acute; Affect; Alzheimer' s Disease; Animals; Apical; Area; Behavior; Behavioral; Behavioral Paradigm; Biological; Biological Models; Brain; Buffers; Calcium; Chronic; Dendrites; Dependence; Disease; Distal; Electrophysiology (science); Endoplasmic Reticulum; Genetic; Genetic Recombination; Glutamates; Goals; Head; Health; Hippocampus (Brain); Image; Injections; Intervention; Knockout Mice; Label; Laboratories; Learning; Ligands; Light; Link; Location; Maps; Memory; Methods; Mitochondria; Monitor; Mus; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neurons; Organelles; Output; Parkinson Disease; Population; Process; Property; Proteins; Research; Resolution; Role; Slice; Stream; Synapses; Synaptic plasticity; Techniques; Testing; autism spectrum disorder; awake; cognitive process; comparative; experimental study; extracellular; hippocampal pyramidal neuron; in vivo; insight; nervous system disorder; neuronal circuitry; novel; optogenetics; relating to nervous system; tool; two-photon; virtual; voltage

In dendrites, Ca2+ is critical in determining how neurons respond to incoming excitation. While numerous studies have focused on how dendritic Ca2+ relates to behaviorally-relevant neuronal and circuit activity using correlative observations, there is currently no method to precisely manipulate Ca2+ in neurons in vivo and thus causally test its role in circuit function and behavior. In non-neuronal cells, mitochondria can act as sinks for Ca2+ released from the endoplasmic reticulum (ER) by forming direct contacts with these concentrated intracellular Ca2+ stores. Recently the Polleux lab discovered that protein PDZD8 enables mitochondria to buffer ER-released Ca2+ in dendrites by tethering these organelles together; in the absence of PDZD8, cytosolic [Ca2+] is markedly higher after synaptically-evoked ER release. Using a newly-developed Pdzd8 conditional knockout (cKO) mouse line, versatile recombination and labeling strategies, and a newly-developed optogenetic tool to rapidly and reversibly induce new ER-mitochondria contacts with light, we are now poised to directly manipulate the spatial and temporal dynamics of dendritic Ca2+ in awake and behaving mice. By combining these approaches with 2-photon Ca2+ imaging during head- fixed behavior, we will causally test the relationship between dendritic Ca2+ dynamics and neuronal input-output transformations, circuit function, and learning & memory. Using hippocampal CA1 pyramidal neurons (PNs) as a model system, we will further assess these relationships with respect to input-specific dendritic compartments thought to receive distinct streams of behaviorally-relevant information. The long-term objective of this proposal is to create a platform for systematically and quantitatively probing the transformation of subcellular Ca2+ dynamics into higher-order cognitive processes in health and disease. While the current proposal seeks to establish this novel platform in CA1 PNs in the context of spatial learning, we aim for general applicability to the study of subcellular Ca2+ dynamics in higher-order brain processes. Hypothesis: We hypothesize that dendritic Ca2+ is integrated in an input-specific manner in CA1 PN apical dendrites to drive circuit dynamics underlying spatial learning and memory. We will test this hypothesis in the following specific aims: Specific Aim 1: Characterize ER-mitochondria tethering as a novel inroad to bidirectionally manipulating Ca2+ dynamics in input-defined dendritic compartments of CA1 PNs. Specific Aim 2: Causally test the link from dendritic Ca2+ dynamics in CA1 PNs to circuit-level neural activity and spatial learning in vivo.

在树突中，Ca2+在决定神经元如何响应传入的兴奋方面至关重要。虽然许多研究 一直专注于树突状细胞Ca2+如何与行为相关的神经元和电路活动相关， 根据观察，目前还没有方法精确地操纵体内神经元中的Ca 2+，因此， 测试其在电路功能和行为中的作用。在非神经元细胞中，线粒体可以作为Ca2+释放的汇 通过与这些浓缩的细胞内Ca2+储存形成直接接触，从内质网（ER）中释放。最近，Polleux实验室发现蛋白PDZD 8能够使线粒体缓冲ER释放的Ca 2 + 在树突中，通过将这些细胞器拴在一起;在没有PDZD 8的情况下，胞质[Ca 2 +]明显高于 在突触诱发的ER释放后。 使用新开发的Pdzd8条件性敲除（cKO）小鼠系， 策略，以及一种新开发的快速可逆诱导新ER线粒体的光遗传学工具 与光接触，我们现在准备直接操纵树突状细胞的空间和时间动力学， Ca2+在清醒和行为小鼠中。通过将这些方法与头部检查期间的双光子Ca2+成像相结合， 固定的行为，我们将因果关系测试树突状细胞钙动力学和神经元的输入输出之间的关系 转换、电路功能以及学习和记忆。使用海马CA1锥体神经元（PNs）作为 一个模型系统，我们将进一步评估这些关系方面的输入特定的树突状隔室 被认为接收不同的行为相关信息流。 这项建议的长期目标是建立一个平台，系统地、定量地探讨 亚细胞Ca2+动力学转化为健康和疾病中的高阶认知过程。 虽然目前的建议试图在空间学习的背景下在CA1 PN中建立这种新的平台， 我们的目标是普遍适用于亚细胞钙离子动力学的研究，在高阶脑过程。 假设：我们假设树突状细胞Ca 2+以输入特异性方式整合在CA1 PN顶端。 树突来驱动空间学习和记忆的电路动力学。我们将在下面的文章中检验这一假设： 具体目标如下： 具体目标1：表征ER-线粒体拴系作为双向操纵Ca2+的新进展 在输入定义的树突状区室的CA1 PN的动力学。 具体目标2：因果检验CA1 PN中树突状Ca2+动力学与回路水平神经活动之间的联系 和空间学习的能力。