Identifying the mechanisms causal to nonequivalent release sites at zebrafish neuromuscular junctions

确定导致斑马鱼神经肌肉接头非等效释放位点的机制

• 批准号: 10452539
• 负责人: PAUL BREHM
• 金额: $ 33.69万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452539
• 关键词: Address; Analysis of Variance; Automobile Driving; Calcium; Collaborations; Data; Development; Drops; Electron Microscopy; Electrons; Electrophysiology (science); Event; Failure; Fatigue; Fluorescence; Frequencies; Heterogeneity; Individual; Kinetics; Learning; Light; Link; Measurement; Mediating; Mental Depression; Methodology; Microscopic; Modeling; Molecular; Monitor; Motor Neurons; Muscle; Myasthenia; Neuromuscular Diseases; Neuromuscular Junction; Nicotinic Receptors; Optics; Permeability; Positioning Attribute; Property; Proteins; Protocols documentation; Readiness; Recovery; Resolution; Role; Series; Site; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; Testing; Transgenic Organisms; Vertebrates; Vesicle; Work; Zebrafish; base; calcium indicator; cholinergic synapse; cytomatrix; detector; experimental study; mutant; neuromuscular; neurotransmission; patch clamp; peripheral membrane protein 43K; postsynaptic; preference; preservation; quantum; reconstruction; recruit; sensor; synaptic depression; tomography; transmission process; vesicular release

The advantages provided by the zebrafish neuromuscular synapse have led to discoveries that have greatly advanced our basic understanding of neurotransmission and neuromuscular diseases. Driving all our studies has been our development of paired motoneuron muscle patch clamp recordings, which has only been possible in zebrafish. We recently used this methodology to explore frequency-dependent depression, one of the most prevalent forms of synaptic plasticity, and obtained evidence for heterogeneity among release sites. The existence of functionally distinct classes of release sites remains an unexplored territory among vertebrates, calling for development of probes adequate to detect their function. Also unique to zebrafish neuromuscular junction is a small number of release sites that are well separated from one another. To probe each of these release sites independently we created a transgenic line wherein the calcium indicator GCaMP6f is fused to postsynaptic rapsyn. Due to the high calcium permeability of the nicotinic receptor, we have been able to optically track release of single quanta from individual motoneuron release sites. With this powerful detector, we now outline a series of experiments that will test for release site heterogeneity and its role in synaptic depression. We also utilize optical quantal analysis to further address an outstanding question asking whether the synchronous, asynchronous and spontaneous modes of synaptic transmission work through the same or different release sites. Finally, to identify the structural counterparts of functional heterogeneity, we are collaborating with Christian Stigloher, an expert in high-resolution tomographic analysis of zebrafish neuromuscular synapses. Individual fast and slow synapses will be identified on the basis of optical quantal analysis and subjected to tomographic reconstruction to test for differences in cytomatrix components of the active release site. Only through the many unique features offered by this vertebrate cholinergic synapse is it possible to perform the proposed experiments to decipher synaptic depression and associated use dependent fatigue, common to almost all myasthenias.

斑马鱼神经肌肉突触提供的优势带来了以下发现： 极大地增进了我们对神经传递和神经肌肉疾病的基本理解。 推动我们所有研究的是配对运动神经元肌肉膜片钳的开发 记录，这只有在斑马鱼中才有可能实现。我们最近使用这种方法来探索 频率依赖性抑制，突触可塑性最普遍的形式之一，以及 获得了释放地点之间异质性的证据。功能上不同的存在 释放位点的类别在脊椎动物中仍然是一个未开发的领域，需要开发 足以检测其功能的探针。斑马鱼神经肌肉接头的另一个独特之处是 少量彼此分开的发布站点。为了探究其中每一个 我们独立地创建了一个转基因系，其中钙指示剂 GCaMP6f 是 与突触后 rapsyn 融合。由于烟碱受体的高钙渗透性，我们有 能够光学跟踪单个运动神经元释放位点的单个量子的释放。和 对于这个强大的探测器，我们现在概述了一系列将测试发布站点的实验 异质性及其在突触抑制中的作用。我们还利用光学量子分析来进一步 解决一个悬而未决的问题，即同步、异步和自发 突触传递的模式通过相同或不同的释放位点起作用。最后，要识别 功能异质性的结构对应物，我们正在与 Christian Stigloher 合作， 斑马鱼神经肌肉突触高分辨率断层扫描分析专家。个人 快突触和慢突触将在光学量子分析的基础上进行识别，并进行 断层扫描重建以测试活性释放的细胞基质成分的差异 地点。只有通过这种脊椎动物胆碱能突触提供的许多独特功能，它才能发挥作用。 可以进行所提出的实验来破译突触抑制和相关用途 依赖性疲劳，几乎是所有肌无力的常见症状。

项目成果

Single-cell RNAseq analysis of spinal locomotor circuitry in larval zebrafish.

• DOI: 10.7554/elife.89338
• 发表时间: 2023-11-17
• 期刊: eLife
• 影响因子: 7.7
• 作者: Kelly JJ;Wen H;Brehm P
• 通讯作者: Brehm P

Primary and secondary motoneurons use different calcium channel types to control escape and swimming behaviors in zebrafish.

初级和次级运动神经元使用不同的钙通道类型来控制斑马鱼的逃逸和游泳行为。

• DOI: 10.1073/pnas.2015866117
• 发表时间: 2020
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Wen,Hua;Eckenstein,Kazumi;Weihrauch,Vivien;Stigloher,Christian;Brehm,Paul
• 通讯作者: Brehm,Paul