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

储存在神经末梢小泡中的神经递质迅速释放，构成了大脑信息传递的基础。这个过程对学习和记忆至关重要，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（SNAREs）偶联到Ca2+信号，从而同步神经传递到神经元放电。囊泡释放机制如何解码Ca2+信号并将其转化为神经递质释放的复杂模式仍然是一个谜。目前，Ca2+诱发的神经递质释放机制主要在活突触中进行研究，采用电生理学、荧光成像和遗传操作相结合的方法。然而，由于内在的可变性，活体突触的实验需要大量的动物。此外，突触前蛋白的基因缺失通常会导致严重甚至致命的表型。活突触实验的解释由于多种蛋白异构体的表达和代偿性体内平衡机制而进一步复杂化。这就要求开发新的技术，以有效地取代活体突触实验，并减少突触生理学领域中使用的动物数量。减少动物数量的一种方法是用生物化学定义的重构囊泡融合试验代替活神经元实验。这种简化的方法，其中变量是有限的，成分可以精确定义，有可能获得Ca2+调节的突触囊泡融合的直接机制见解。然而，低时间分辨率和缺乏精确的Ca2+控制限制了经典体外融合设置的应用。在本项目中，我们建议开发一种新的实验平台，将单囊泡融合测定与Ca2+信号的快速精确控制相结合。该装置将忠实地再现无细胞条件下的突触结构和生理，并将允许以毫秒精度研究Ca2+诱发的囊泡融合。突触生理学是一个庞大而充满活力的研究领域，这项技术的发展将引起学者们的极大兴趣。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准备中进行的许多实验，从而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.