Synaptic transmission at the zebrafish neuromuscular junction

斑马鱼神经肌肉接头处的突触传递

• 批准号: 9069103
• 负责人: PAUL BREHM
• 金额: $ 33.59万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9069103
• 关键词: Antibodies; Attention; Axon; Calcium; Calcium Channel; Calcium Oscillations; Calcium Signaling; Cells; Conotoxin; Data; Elements; Exocytosis; Genetic; Goals; Health; Hot Spot; Human; Image; Immunohistochemistry; L-Type Calcium Channels; Life; Link; Location; Mediating; Membrane; Methods; Molecular; Motor Neurons; Muscle; Mutation; Myasthenic Syndrome; Natural regeneration; Nerve; Neuromuscular Junction; Neurons; Neurosciences; Optics; Pharmacology; Physiological; Physiology; Play; Positioning Attribute; Presynaptic Terminals; Process; Protein Isoforms; Q-Type Calcium Channels; Reporting; Role; Signal Transduction; Signaling Molecule; Source; Synapses; Synaptic Transmission; Synaptic plasticity; Technology; Testing; Zebrafish; base; in vivo; knock-down; mutant; neurotransmitter release; patch clamp; regenerative; research study; sensor; synaptotagmin; transmission process; voltage

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 In this proposal, we present much unpublished data in support of differential locations of these two channel isoforms, with the synchronous channel in the synaptic bouton and the asynchronous isoform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.

描述（由申请人提供）：该项目的重点是我们最近发现的细胞内钙的不同来源，用于斑马鱼神经肌肉接头处突触传递的同步和异步模式。异步释放尤其受到了广泛关注，因为它在突触可塑性中的作用得到了新的认识。然而， 异步模式优先释放的机制是目前争论的热点。我们的实验室是第一个识别出针对异步释放的钙传感器的实验室，而且我们现在发现，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亚型，等待通过 Aim 1 实验进行分子鉴定。在这个提案中，我们提供了许多未发表的数据来支持这两种通道亚型的差异位置，其中同步通道位于突触鲍顿中，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构成了目标 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表明钙波的激活是由异步钙通道亚型激活的，并通过内部钙的主动释放传播到达突触。这是几乎所有研究的突触中异步释放的签名延迟开始和持续的原因。神经肌肉接头和中枢神经元中存在钙波的大量报道，但迄今为止，其相对于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，我们将利用斑马鱼的集体优势来测试每个提议的信号分子在自发、同步和异步释放模式的生理学中的要求。