Presynaptic short- and long-term enhancement of neurotransmitter release: Molecular mechanisms and behavioral relevance

神经元之间的交流依赖于突触接触的快速化学传递，其中突触前神经递质突触囊泡（SVs）的胞吐激活突触后反应。胞吐作用由Ca2+控制，发生在动态病灶，称为活动区（AZs）。随后的内吞作用通过支持囊泡再循环确保突触前的内稳态和持续的活动。显然，外吞作用和内吞作用的同步对于有效的神经传递是必要的。然而，令人惊讶的是，我们对这些反应的分子机制知之甚少。我的研究项目旨在弥补这一差距。AZs被优化用于胞吐；即使在刺激之前，囊泡被靶向释放位点并成熟到接近Ca2+通道的易释放状态，Ca2+通道响应动作电位打开以触发胞吐。细胞基质蛋白决定az结构，这通常被认为是协调胞分泌激活剂（即Ca2+通道）和靶点（SVs）的偶联。然而，这些反应的分子拓扑结构和功能原理在很大程度上仍然不清楚。SV膜的内吞作用发生在靠近AZs的地方。最近的数据表明，从长远来看，内吞作用不仅需要补充SV池，而且还需要快速清除释放位点。此外，内吞作用的模式似乎取决于胞吐作用，但这些过程如何精确地联系起来仍然是难以捉摸的。最后，目前尚不清楚外吞作用和内吞作用的基本调节因子在空间和时间上是如何组织的。我假设AZ的细胞基质蛋白作为反应中心，需要协调有效的胞吐、内吞和部位循环。我的计划将通过结合理论（数学建模）和实验方法（即慢性和急性遗传学，电生理学，活细胞成像，电子显微镜，生物化学）来解决外吞耦合的机制，使用黑腹果蝇神经肌肉连接以及小鼠海马神经元作为模型系统。协同实验工作和理论建模将使我能够测试AZ细胞基质是否以及如何将外吞作用与内吞作用相结合。具体来说，我的目标是阐明(I)通过识别Ca2+通道释放位点拓扑结构，az结构如何优化胞外作用，以及（ii）通过研究单个az的局部蛋白质组成和胞外作用，定义神经元兴奋性的分子机制。此外（iii），我将描述内吞机制适应不同模拟范式的机制。最后（iv），我将通过解剖细胞基质az -蛋白的外吞和内吞功能，直接检验其是否为反应中枢。本提案中提出的工作将阐明突触神经传递的具体机制和一般原理，从而揭示大脑功能。

Communication between neurons relies on fast chemical transmission across synaptic contacts where presynaptic exocytosis of neurotransmitter-laden synaptic vesicles (SVs) activates postsynaptic responses. Exocytosis is controlled by Ca2+ and occurs at dynamic foci, called active zones (AZs). Subsequent endocytosis ensures presynaptic homeostasis and continued activity by supporting vesicle re-cycling. Evidently, synchronization of exo- and endocytosis is warranted for effective neurotransmission. However, surprisingly little is known about molecular mechanisms linking these reactions. My research program is designed to bridge this gap.AZs are optimized for exocytosis; even before stimulation, vesicles are targeted to release sites and mature to a readily releasable state close to Ca2+-channels, which open in response to action potentials to trigger exocytosis. Cytomatrix proteins determine the AZ-architecture and this is generally assumed to orchestrate coupling of exocytosis activators (i.e. Ca2+ channels) and targets (SVs). However, the molecular topologies and functional principles underlying these reactions remain largely unclear. Endocytosis of SV membranes occurs in close proximity to AZs. Recent data suggest that endocytosis not only is required on the long run to replenish SV pools but is also needed for fast clearance of release sites. Furthermore, it appears that the mode of endocytosis depends on exocytosis, but how precisely these processes are connected remains elusive. Finally, it is unknown how essential regulators of exo- and endocytosis are organized in space and time.I hypothesize that cytomatrix proteins of the AZ serve as reaction hubs that are needed to orchestrate effective exocytosis, endocytosis and site recycling. My program will address the mechanism of exo-endocytic coupling by combining theoretical (mathematical modelling) and experimental approaches (i.e. chronic and acute genetics, electrophysiology, live cell imaging, electron microscopy, biochemistry) using Drosophila melanogaster neuromuscular junctions as well as mouse hippocampal neurons as model systems. Synergizing experimental work and theoretical modeling will allow me to test whether and how the AZ cytomatrix integrates exo- with endocytosis. Specifically, I aim to unravel (i) how AZ-architecture optimizes exocytosis by identifying the Ca2+-channel release-site topology and to (ii) define the molecular mechanisms of neuronal excitability by studying local protein composition and exocytosis at single AZs. Moreover (iii), I will characterize the mechanisms by which the endocytosis machinery adapts to diverse simulation paradigms. Finally (iv), I will directly test whether cytomatrix AZ-proteins are reaction hubs by dissecting their exo- and endocytosis functions. The work presented in this proposal will shed light on the specific mechanisms and general principles that underlie synaptic neurotransmission and, thus, brain function.