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

在树突中，钙在决定神经元如何对传入的兴奋做出反应方面起着关键作用。虽然众多的研究 重点研究了树突细胞钙是如何与行为相关的神经元和回路活动相关的 据观察，目前还没有方法可以在体内精确地操纵神经元中的钙离子，从而导致 测试其在电路功能和行为中的作用。在非神经细胞中，线粒体可以充当释放钙的汇。 通过与这些浓缩的细胞内钙储存形成直接接触，从内质网(ER)释放。最近，Polleux实验室发现，PDZD8蛋白使线粒体能够缓冲ER释放的钙 在树突中，通过将这些细胞器连接在一起；在没有PDZD8的情况下，胞浆[Ca~(2+)]显著升高 在突触诱发的内质网释放后。 利用新开发的PDZD8条件基因敲除(CKO)小鼠系，多功能重组和标记 策略，以及一种新开发的光遗传工具来快速和可逆地诱导新的ER-线粒体 与光的接触，我们现在准备直接操纵树突的空间和时间动力学 清醒和行为正常小鼠的CA2。通过将这些方法与头部手术过程中的双光子钙离子成像相结合， 固定行为，我们将因果检验树突细胞钙动力学和神经元输入输出之间的关系 转换、电路功能和学习与记忆。以海马CA1区锥体神经元(PNS)为研究对象 作为一个模型系统，我们将进一步评估这些关系与输入特定的树突室 被认为接收与行为相关的不同信息流。 这项建议的长期目标是建立一个平台，系统地和定量地探讨 健康和疾病中亚细胞钙动力学向高级认知过程的转变。 虽然目前的提议寻求在空间学习的背景下在CA1 PNS中建立这一新颖的平台， 我们的目标是普遍适用于高阶大脑过程中亚细胞钙动力学的研究。 假设：我们假设树突状钙离子在CA1PN根尖以一种特定的方式整合 树突驱动空间学习和记忆背后的电路动力学。我们将在 以下是具体目标： 具体目标1：将内质网-线粒体拴系作为双向操纵钙离子的新途径 CA1PNS输入定义的树突室中的动力学。 特定目标2：因果测试CA1PNS中树突状钙动力学与电路水平神经活动之间的联系 以及活体的空间学习。

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.