Novel in vitro platform to study molecular mechanisms of neurotransmitter release and synaptic plasticity

储存在神经末梢的小泡中的神经递质的快速释放形成了大脑中信息传递的基础。这个过程对于学习和记忆至关重要，并且在许多神经系统疾病中被破坏。在神经末梢，Ca2+感应蛋白（synaptotagmins）将囊泡释放机制（SNARE）与Ca2+信号偶联，从而使神经传递与神经元放电同步。囊泡释放机制如何解码Ca 2+信号并将其转化为复杂的神经递质释放模式仍然是一个谜，目前，Ca 2+诱发的神经递质释放机制主要是在活突触中，使用电生理学，荧光成像和遗传操作相结合的方法进行研究。然而，由于内在的变异性，活突触的实验需要大量的动物。此外，突触前蛋白的遗传缺失通常导致严重的甚至致命的表型。活突触实验的解释由于多种蛋白质异构体的表达和补偿性稳态机制而进一步复杂化。这就需要开发新的技术来有效地取代活突触实验，减少突触生理学领域使用的动物数量。减少动物数量的一种方法是用生化定义的重组囊泡融合试验取代活神经元实验。这种简化的方法，其中的变量是有限的，组件可以精确定义，有可能获得直接的机械见解钙调节突触囊泡融合。然而，低的时间分辨率和缺乏精确的Ca2+控制限制了经典的体外融合setups.In本项目中，我们建议开发一种新的实验平台，结合了一个单一的囊泡融合检测的快速和精确的控制的Ca2+信号。这种设置将忠实地重演突触结构和生理无细胞条件下，将允许研究钙离子诱发的囊泡融合毫秒精度。开发的技术将对参与突触生理学研究的学者产生重大兴趣，这是一个庞大而动态的研究领域。这一新的创新平台的实施具有很大的潜力，可以取代目前只能在动物制剂中进行的许多实验，因此可以取代每年需要生产数万只转基因动物的实验。

Rapid release of neurotransmitters stored in small vesicles at the nerve terminals forms the basis of information transfer in the brain. This process is essential for learning and memory and is disrupted in many neurological disorders. At nerve terminals, Ca2+-sensing proteins (synaptotagmins) couple vesicular release machinery (SNAREs) to Ca2+ signals, thus synchronising neurotransmission to neuronal firing. How the vesicular release machinery decodes Ca2+ signals and translates them into the complex patterns of neurotransmitter release remains enigmatic.At present, the mechanisms of Ca2+-evoked neurotransmitter release are predominantly studied in live synapses, using a combination of electrophysiology, fluorescence imaging and genetic manipulations. However, due to intrinsic variability, experiments in live synapses require large numbers of animals. Moreover, genetic deletion of presynaptic proteins often results in severe or even lethal phenotypes. The interpretation of experiments in live synapses is further complicated by the expression of multiple protein isoforms and compensatory homeostatic mechanisms. This calls for developing new technologies that can effectively replace experiments in live synapses and reduce the number of animals used in the field of synaptic physiology.One way to reduce the number of animals is to replace experiments in live neurons with biochemically defined reconstituted vesicle fusion assays. This reductionist approach, where the variables are limited, and the components can be precisely defined, has the potential to gain direct mechanistic insights into Ca2+-regulated synaptic vesicle fusion. However, the low temporal resolution and lack of precise Ca2+ control limit the application of the classical in vitro fusion setups.In this project, we propose to develop a novel experimental platform that combines a single vesicle fusion assay with the fast and precise control of the Ca2+ signal. This setup will faithfully recapitulate synaptic architecture and physiology under cell-free conditions and will allow studying Ca2+-evoked vesicle fusion with millisecond precision. The developed technology will be of significant interest to the academics involved in the research of synaptic physiology, which is a large and dynamic research field. The implementation of this new innovative platform has a large potential to replace many experiments that are now only possible in animal preparations and therefore to allow for the replacement of experiments that require the production of over several tens of thousands of transgenic animals per year.