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导致了对 极大地提高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我们对运动神经元肌肉膜片钳的开发 录音，这只在斑马鱼身上才有可能。我们最近使用了这种方法来探索 频率依赖性抑郁，突触可塑性最普遍的形式之一，以及 获得了释放部位之间异质性的证据。在功能上不同的存在 在脊椎动物中，释放地点的类别仍然是一个未开发的领域，需要开发 足够检测其功能的探头。斑马鱼神经肌肉接头也是独一无二的 少量彼此分开的投放点。为了探测其中的每一个 我们独立地创建了一个转基因品系，其中钙指示剂GCaMP6f是 融合到突触后的突触突触。由于尼古丁受体的高钙渗透性，我们有 能够光学跟踪单个运动神经元释放部位的单量子释放。使用 这个强大的探测器，我们现在概述一系列的实验，将测试释放地点 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 回答了一个悬而未决的问题，即同步、异步和自发 突触传递的方式通过相同或不同的释放部位起作用。最后，要确定 功能异构性的结构对应者，我们正在与克里斯蒂安·斯蒂格勒合作， 斑马鱼神经肌肉突触的高分辨率断层分析专家。个体 快突触和慢突触将在光学量子分析的基础上被识别并接受 断层重建以检测活性释放物中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，才能 有可能进行拟议的实验来破译突触抑制和相关用途 依赖性疲劳，几乎所有的肌无力患者都有。

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