Synaptic transmission at the zebrafish neuromuscular junction

描述（由申请人提供）：本项目的中心是我们最近发现的斑马鱼神经肌肉接头突触传递同步和异步模式的细胞内钙的不同来源。特别是异步释放，由于其在突触可塑性中的新作用而受到广泛关注。然而，在这方面， 导致通过异步模式优先释放的机制目前是一个激烈争论的主题。我们的实验室是第一个鉴定出特异于异步释放的钙传感器的实验室，我们现在发现，另外，同步与异步释放是由两种不同的电压激活钙通道亚型介导的。同步释放利用P/Q型钙通道，异步释放利用电压依赖性钙通道同种型，其等待通过Aim 1实验进行分子鉴定。在这个建议中，我们提出了许多未发表的数据，在支持这两个通道亚型的差分位置，在突触终扣的同步通道和异步亚型位于突触外的轴突分支点。确定其在细胞中的位置形成了目标2实验的基础。此外，体内钙成像、胞吐指示线和配对记录的组合技术已经指出钙波的激活，所述钙波由异步钙通道同种型激活并且通过内部钙的主动释放传播以到达突触。这是因果关系的签名延迟发作和持续的异步释放在几乎所有的研究突触。神经肌肉接头和中枢神经元都存在钙波的大量报道，但迄今为止，其相对维斯突触传递的生理意义仍然不清楚。在目标2中，我们将通过激活突触外钙电流来确定钙释放的分子基础，建立与异步释放过程的联系。最后，在目标3中，我们将使用斑马鱼的集体优势来测试在自发、同步和异步释放模式的生理学中对每个提议的信号分子的需求。

DESCRIPTION (provided by applicant): This project centers on our recent discovery of separate sources of intracellular calcium for synchronous and asynchronous modes of synaptic transmission at the zebrafish neuromuscular junction. Asynchronous release, in particular, has received much attention, as a result of its newly appreciated role in synaptic plasticity. However, the mechanisms causal to preferential release through the asynchronous mode is presently a hotly contested subject. Our lab was the first to identify a calcium sensor specific to asynchronous release, and we now find that, additionally, synchronous versus asynchronous release is mediated by two distinct voltage- activated calcium channel isoforms. The synchronous release utilizes a P/Q type calcium channel and the asynchronous release utilizes a voltage dependent calcium channel isoform that awaits molecular identification through Aim 1 experiments. In this proposal, we present much unpublished data in support of differential locations of these two channel isoforms, with the synchronous channel in the synaptic bouton and the asynchronous isoform located extrasynaptically at axonal branch points. Establishing its location in the cell forms the basis of aim 2 experiments. Additionally, the combined technologies of in vivo calcium imaging, exocytosis indicator lines, and paired recordings have pointed to activation of a calcium wave that is activated by the asynchronous calcium channel isoform and propagates through active release of internal calcium to reach the synapse. This is causal to the signature delayed onset and persistence of asynchronous release at nearly all studied synapses. Numerous reports of calcium waves exist for both the neuromuscular junction and central neurons but, until now, the physiological significance vis-a-vis synaptic transmission has remained obscure. In aim 2 we will determine the molecular basis of calcium release through activation of the extrasynaptic calcium current establish the links to the asynchronous release process. Finally, in Aim 3 we will use the collective advantages of zebrafish to test the requirement for each proposed signaling molecule in the physiology of spontaneous, synchronous and asynchronous release modes.