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

突触传递的调节是可塑性和内稳态机制的核心，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小泡(SVS)的可用性。它是通过“SV循环”实现的，该循环涉及到SVS的回收。在SV周期中，突触功能可以通过改变1)突触前结构对钙离子增加的敏感性，2)SVS从SVS储备池中招募的速率，以及3)SV神经递质水平来调节。近年来，通过光遗传刺激和电生理或电子显微镜检查，详细研究了SV释放和再循环的急性机制。我的实验室分析了线虫神经肌肉突触上的急性cAMP生成是如何调节其功能的，并能够通过影响SV的流动性和充盈状态来展示神经肽在突触传递调节中先前未知的作用。因此，突触传递受到两种方式的调节：1)去极化和钙离子控制SVS的急性融合速度；2)cAMP和肽能信号决定SV的含量。因此，除了中枢模式生成器的网络活动外，运动神经元还可以整合神经调制信号，例如在各种全身状态下。我们想要调查这种双重控制是否也在脊椎动物中保存下来，特别是在斑马鱼中。Danio Rerio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，与神经细胞培养形成对比，比如在小鼠身上进行类似的方法。我们建立了能够使运动神经元光刺激(通道视紫红质，光激活的腺苷环化酶)的转基因系，从而可以诱导(和量化)光刺激的行为变化，以及对微型终板电流(MEPCS)的电生理分析。我们希望将我们在野生型背景下产生的转基因株系与缺乏神经肽生物发生所需成分或其调节释放所需成分的敲除和敲除进行比较。此外，我们希望使用基于荧光的记者检测cAMP诱导的神经肽释放，并分析在斑马鱼运动神经元中表达的少量候选肽。这是为了表明这些神经肽是否通过增加量子大小来增强突触传递的有效性。我们还建立了分析光刺激突触超微结构的方法。我们的工作针对的是，突触传递的双模式控制是否在斑马鱼中保守，因此可能在整个脊椎动物中也是如此，并将使未来的研究能够理解以突触传递失调为特征的疾病。

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.