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: 10006851
• 负责人: Justin O'Hare
• 金额: $ 7.24万
• 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/10006851
• 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; Estrogen receptor positive; 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; conditional knockout; 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+对于确定神经元如何反应传入激发至关重要。而许多研究 已经关注树突Ca2+使用相关性与行为相关的神经元和电路活性的关系 观察结果，目前尚无在体内神经元中精确操纵Ca2+的方法 测试其在电路功能和行为中的作用。在非神经元细胞中，线粒体可以充当Ca2+释放的下沉 通过与这些浓缩细胞内Ca2+存储的直接接触来从内质网（ER）中。最近，Polleux实验室发现蛋白质PDZD8使线粒体可以缓冲ER释放的Ca2+ 在树突中，通过将这些细胞器系在一起；在没有PDZD8的情况下，胞质[Ca2+]明显更高 突触诱发的ER释放后。 使用新开发的PDZD8条件敲除（CKO）鼠标系，多功能重组和标签 策略，以及一种新开发的光遗传学工具，可快速和可逆地诱导新的ER-线粒体 与光线接触，我们现在准备直接操纵树突状的空间和时间动态 Ca2+在清醒和行为小鼠中。通过在头部 - 固定行为，我们将因因果测试树突状CA2+动力学与神经元输出输出之间的关系 转换，电路功能以及学习与内存。使用海马CA1锥体神经元（PNS）作为 一个模型系统，我们将进一步评估有关投入特异性树突隔室的这些关系 被认为接收不同行为与行为相关的信息流。 该提案的长期目标是创建一个用于系统和定量探索的平台 细胞亚细胞CA2+动力学转化为健康和疾病中高阶认知过程。 尽管当前的建议旨在在空间学习的背景下在CA1 PNS中建立这个新颖的平台，但 我们旨在将一般性适用于研究高阶大脑过程中亚细胞CA2+动力学的研究。 假设：我们假设树突状Ca2+以输入特异性方式集成在Ca1 Pn顶端 树突驱动电路动力学基础的空间学习和记忆。我们将在 遵循特定目的： 具体目标1：将ER-Orichons Tewering定为新颖的双向操纵Ca2+的新颖性。 CA1 PNS的输入定义的树突室中的动力学。 特定目标2：因果测试从CA1 PN中的树突状CA2+动力学到电路级神经活动的链接 和体内空间学习。