Role of presynaptic calcium stores

描述（由申请人提供）：细胞内Ca2+信号是神经元回路信息处理的一个组成部分，促进突触前和突触后功能，包括突触传递的长期增强（LTP）和抑制（LTD），长期突触可塑性的电生理相关。由于Ca2+的精细调节变化调节了多种细胞内功能，信号特异性需要对细胞质Ca2+信号进行严格的时空控制。这种需求在快速神经递质释放中最为明显，其中电压依赖性Ca2+信号在亚毫秒尺度上触发突触前末端的突触囊泡融合。内质网（ER）可以作为Ca2+储存/吸收或Ca2+来源，并可能在突触前末端平衡这双重角色，以满足突触对递质释放的特定需求。然而，内质网对突触前Ca2+信号和神经递质释放的贡献尚不清楚。我们假设内质网在果蝇NMJs中作为主要的突触前Ca2+汇。如果是这样，突触前内质网不仅可以通过SERCA泵送暂时缓冲胞质Ca2+，还可以通过将腔内Ca2+“隧穿”到轴突，永久地将Ca从突触末端移除。终端内有限的ER Ca2+释放可能调节神经递质释放，但主要服务于线粒体Ca2+摄取，激活线粒体能量产生。我们将利用基因操纵果蝇和图像去极化诱导的内质网腔、线粒体和幼虫胞浆突触前钙水平的变化来验证这一假设。该提案的核心是开发转基因Ca2+指标，可以忠实地报告内质网和线粒体腔内Ca2+的变化，我们期望这也将广泛用于果蝇任何细胞的线粒体和/或内质网Ca2+动态的遗传分析。具体来说，我们将确定er介导的Ca2+摄取、Ca2+释放和/或Ca2+扩散（隧道）在重复神经刺激下的动态（目的1），并确定er -线粒体相互作用在果蝇NMJs幼虫突触前Ca2+稳态中的作用（目的2）。从这个系统的分析中，一个基本的框架将会出现，以便更好地理解内质网在突触前Ca2+信号/突触前末端稳态中的作用，扩展我们对突触传递的重要调节机制及其与神经系统功能可塑性和人类健康的关系的理解。

DESCRIPTION (provided by applicant): Intracellular Ca2+ signals are an integral part of information processing by neuronal circuits facilitating pre- and postsynaptic functions including long-term potentiation (LTP) and depression (LTD) of synaptic transmission, the electrophysiological correlates of long-term synaptic plasticity. Since finely tuned changes in Ca2+ modulate a variety of intracellular functions, signal specificity requires a tight spatial and temporal control of the cytosolic Ca2+ signal. This need is most apparent for fast neurotransmitter release where voltage-dependent Ca2+ signaling triggers the fusion of synaptic vesicles at presynaptic terminals on a sub-millisecond scale. The endoplasmic reticulum (ER) can act as a Ca2+ store/sink or as a Ca2+ source and is likely to balance these dual roles at presynaptic terminals to meet the specific needs of the synapse for transmitter release. However, the contributions of ER to presynaptic Ca2+ signaling and neurotransmitter release are not well understood. We hypothesize that the ER acts as the primary presynaptic Ca2+ sink at fly NMJs. If so, the presynaptic ER may not only temporarily buffer cytosolic Ca2+ by SERCA pumping but also permanently remove Ca from synaptic terminals by "tunneling" luminal Ca2+ into axons. Limited ER Ca2+ release within terminals may modulate neurotransmitter release but mainly serve mitochondrial Ca2+ uptake activating mitochondrial energy production. We will test this hypothesis by exploiting genetically manipulated Drosophila and image depolarization-induced changes in presynaptic Ca levels of the ER lumen, mitochondria and the cytosol of larval NMJs. At the center of this proposal is the development of transgenic Ca2+ indicators that can faithfully report Ca2+ changes in the lumen of the ER and mitochondria, which we expect will also be of wide use for the genetic analysis of mitochondrial and/or ER Ca2+ dynamics in any cell of Drosophila. Specifically, we will determine the dynamics of ER-mediated Ca2+ uptake, Ca2+ release and/or Ca2+ diffusion (tunneling) upon repetitive nerve stimulation at presynaptic terminals of larval Drosophila NMJs (Aim 1) and determine the role of ER-mitochondria interactions for presynaptic Ca2+ homeostasis at larval Drosophila NMJs (Aim 2). From this systematic analysis a basic framework will emerge for better understanding the role of the ER in presynaptic Ca2+ signaling/homeostasis at presynaptic terminals expanding our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health.