Optogenetic Analysis of neuropeptidergic regulation of fast synaptic transmission at the zebrafish neuromuscular junction

斑马鱼神经肌肉接头快速突触传递神经肽能调节的光遗传学分析

• 批准号: 459267427
• 负责人: Professor Dr. Alexander Gottschalk
• 金额: --
• 依托单位: Buchmann Institute for Molecular Life Sciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/459267427?language=en
• 关键词: Optogenetic; Analysis; neuropeptidergic; regulation; fast

Regulation of synaptic transmission is central to mechanisms of plasticity and homeostasis, and enables learning and adaptation to new conditions. Maintaining the ability to transmit synaptic signals is based on the availability of neurotransmitter-filled synaptic vesicles (SVs). It is achieved through the “SV cycle”, which involves recycling of SVs. Synaptic function can be modulated in the SV cycle by altering 1) the sensitivity of the presynaptic machinery to the increase in Calcium, 2) the rate at which SVs are recruited from the reserve pool of SVs, and 3) the SV neurotransmitter levels. In recent years, detailed studies have addressed the acute mechanisms of SV release and recycling using optogenetic stimulation and electrophysiological or electron microscopic examinations. My laboratory analyzed how the acute cAMP generation at the neuromuscular synapse of C. elegans modulates its function, and was able to show a previously unknown role of neuropeptides in the regulation of synaptic transmission, by influencing SV mobility and filling status. The synaptic transmission is thus regulated in two ways: 1) Depolarization and Calcium control the acute fusion (rate) of SVs, while 2) cAMP and peptidergic signals determine the SV content. Thus, in addition to the network activity of central pattern generators, motor neurons can also integrate neuromodulatory signals, e.g. in various systemic states. We want to investigate whether this dual control is also preserved in vertebrates, especially in the zebrafish. Danio rerio is unique as a vertebrate model system, as it enables these types of analyses in intact animals, in contrast to neuronal cell cultures, like in similar approaches in mice. We established transgenic lines that enable motor neuron photostimulation (channelrhodopsin, photoactivated adenylate cyclase), and can thus induce (and quantify) light-stimulated behavioral changes, as well as electrophysiological analyses of miniature endplate currents (mEPCs). We want to compare the transgenic lines we generated in wild type background, with knockdowns and knockouts that lack components required for neuropeptide biogenesis, or for their regulated release. Furthermore, we want to detect cAMP-induced neuropeptide release, using fluorescence-based reporters, and analyze a small number of candidate peptides that are expressed in zebrafish motor neurons. This is to show whether these neuropeptides enhance effectivity of the synaptic transmission, e.g. by increasing quantal size. We also established methods to analyze the synaptic ultrastructure of photostimulated synapses. Our work addresses whether the dual-mode control of synaptic transmission is conserved in zebrafish, and thus perhaps in vertebrates in general, and will enable future studies toward an understanding of diseases that are characterized by dysregulation of synaptic transmission.

突触传递的调节是可塑性和稳态机制的核心，并使学习和适应新的条件。保持传递突触信号的能力是基于神经递质填充的突触囊泡（SV）的可用性。这是通过“SV循环”实现的，其中涉及SV的回收利用。通过改变1）突触前机制对钙增加的敏感性，2）SV从SV储备库中募集的速率，以及3）SV神经递质水平，可以在SV周期中调节突触功能。近年来，利用光遗传学刺激和电生理或电子显微镜检查，详细的研究已经解决了SV释放和再循环的急性机制。我的实验室分析了C. elegans调节其功能，并且能够通过影响SV移动性和填充状态来显示神经肽在突触传递调节中的先前未知的作用。因此，突触传递以两种方式调节：1）去极化和钙控制SV的急性融合（速率），而2）cAMP和肽能信号决定SV含量。因此，除了中枢模式发生器的网络活动之外，运动神经元还可以整合神经调节信号，例如在各种系统状态中。我们想研究这种双重控制是否也存在于脊椎动物中，特别是斑马鱼。斑马鱼作为脊椎动物模型系统是独一无二的，因为它能够在完整的动物中进行这些类型的分析，而不是像小鼠中的类似方法那样进行神经元细胞培养。我们建立了转基因系，使运动神经元光刺激（通道视紫红质，光活化腺苷酸环化酶），从而可以诱导（和量化）光刺激的行为变化，以及电生理分析的微型终板电流（mEPCs）。我们想比较我们在野生型背景下产生的转基因株系，其中敲除和敲除缺乏神经肽生物合成或其调节释放所需的组分。此外，我们想检测cAMP诱导的神经肽释放，使用基于荧光的报告，并分析了少量的候选肽，在斑马鱼运动神经元中表达。这是为了显示这些神经肽是否增强突触传递的有效性，例如通过增加量子大小。我们还建立了分析光刺激突触的突触超微结构的方法。我们的工作解决了突触传递的双模式控制是否在斑马鱼中是保守的，因此可能在一般的脊椎动物中，并将使未来的研究朝着了解疾病的特点是突触传递失调。