Vesicle recycling at developing NMJs

描述（由申请人提供）：神经元与其突触伴侣形成的突触在发育过程中具有可塑性，并且由于经验，在数量、强度和功能特性（如短期和长期可塑性）方面具有可塑性。一个充分研究的模型系统发育，活动依赖性的可塑性是小鼠神经肌肉突触，它经历活动依赖性的可塑性在发展中，这是一个标志，其较小的，较难接近的对应在中枢神经系统。在胚胎晚期和出生后早期，神经肌肉突触经历突触消除，其中一个轴突的突触与支配同一靶细胞的其他轴突的突触竞争。根据它们相对于竞争对手的活动模式，一个或少数轴突将成为赢家，将它们的突触维持到成年，而其他轴突则失去竞争，并从神经回路中永久删除。尽管有许多关于神经肌肉突触消除的结构和一些功能研究，但对其潜在机制知之甚少，许多重要问题仍有待解决。一组重要的问题包括竞争性投入的结构变化如何与投入强度的渐进变化相关，如何与竞争的结果相关，以及活动如何介导这一过程。这项拨款提案的目标是了解竞争的突触前方面知之甚少的动态，包括突触囊泡释放，回收和贩运在发展哺乳动物神经肌肉突触。我们已经开发了转基因小鼠品系，其中Thy 1启动子驱动突触荧光蛋白（Thy 1-spH）的表达。SpH是GFP的pH敏感变体，其拴系到囊泡蛋白VAMP 2的管腔结构域，其允许光学监测突触囊泡再循环。初步研究表明，spH+突触囊泡簇可以很容易地可视化内运动轴突末梢和囊泡释放和运输评估使用光学测量活动诱导的荧光变化在分离的胸锁乳突神经肌肉制剂以及在体内。提出了四个目标，包括（1）确定囊泡在发育和成年神经肌肉接头终末分支中释放的时空动力学，（2）确定发育中神经肌肉接头突触消除过程中竞争输入的囊泡释放、突触强度和突触大小之间的关系;（3）确定突触后活动阻断如何逆向影响发育和成年神经肌肉接头处突触囊泡的释放;以及（4）确定突触囊泡如何在单个终末内的释放位点之间运输，以及这如何被突触前和突触后活动调节。总之，下面提出的目标将测试的整体假设，活动调节突触前囊泡的释放和运输，影响突触的结构，强度和生存。这将提供一种机制，通过这种机制，突触功能的可塑性变化可以永久地改变神经回路。公共卫生相关性：我打算以神经肌肉突触为模型系统，研究神经发育过程中突触竞争的机制。使用转基因小鼠的突触囊泡回收和贩运的动态可以在体内监测，我将测试的整体假设，活动调节突触前囊泡的释放和贩运，影响突触的结构，强度和生存。拟议实验的结果将为正常发育过程中活动改变突触功能和神经回路的机制提供全新的见解，并有助于理解癫痫，自闭症和精神发育迟滞等发育障碍，这些障碍对公共卫生具有重大影响。

DESCRIPTION (provided by applicant): Synapses made by a neuron with its synaptic partners are malleable during development, and as a consequence of experience, with respect to number, strength, and functional properties such as short and long term plasticity. A well studied model system for developmental, activity-dependent plasticity is mouse neuromuscular synapses, which undergo activity-dependent plasticity in development that is a hallmark of their smaller, less accessible counterparts in the CNS. During late embryonic and early postnatal life, neuromuscular synapses undergo synapse elimination, in which the synapses of one axon are pitted in competition against the synapses of other axons innervating the same target cell. Based on their activity patterns relative to their competitors, one or a small number of axons will emerge as winners, maintaining their synapses into adult life, while other axons lose the competition and are permanently deleted from neural circuitry. Despite many structural and a few functional studies of neuromuscular synapse elimination, little is known about the underlying mechanisms and many important questions remain to be addressed. One important set of questions includes how the structural changes in competing inputs are related to progressive changes in input strength, to the outcome of competition, and how activity mediates this process. The goal of this grant proposal is to understand the dynamics of the poorly understood presynaptic aspects of competition, including synaptic vesicle release, recycling and trafficking at developing mammalian neuromuscular synapses. We have developed transgenic lines of mice in which the Thy1 promoter drives expression of synaptopHluorin (Thy1-spH). SpH is a pH-sensitive variant of GFP tethered to the luminal domain of the vesicular protein VAMP2 that allows synaptic vesicle recycling to be monitored optically. Preliminary studies suggest that spH+ synaptic vesicle clusters can be readily visualized within motor axon terminals and vesicle release and trafficking assessed using optical measurements of activity- induced fluorescence changes in isolated sternomastoid nerve-muscle preparations as well as in vivo. Four aims are proposed, including (1) to determine the spatial and temporal dynamics of vesicle release across the terminal branches of developing and adult neuromuscular junctions; (2) to determine the relationship between vesicle release, synaptic strength and synaptic size of competing inputs to developing neuromuscular junctions undergoing synapse elimination; (3) to determine how postsynaptic activity blockade retrogradely affects synaptic vesicle release at developing and adult neuromuscular junctions; and (4) to determine how synaptic vesicles are trafficked among release sites within an individual terminal and how this is modulated by pre- and postsynaptic activity. Taken together, the aims proposed below will test the overall hypothesis that activity modulates presynaptic vesicle release and trafficking, affecting synaptic structure, strength and survival. This would provide a mechanism by which plastic changes in synaptic function could permanently alter neural circuitry. PUBLIC HEALTH RELEVANCE: I propose to study the mechanisms underlying synaptic competition during neural development, using neuromuscular synapses as a model system. Using transgenic mice in which the dynamics of synaptic vesicle recycling and trafficking can be monitored in vivo, I will test the overall hypothesis that activity modulates presynaptic vesicle release and trafficking, affecting synaptic structure, strength and survival. The results of the proposed experiments will provide fundamentally new insights into mechanisms by which activity changes synaptic function and neural circuitry during normal development, and contribute to understanding of developmental disorders such as epilepsy, autism and mental retardation, that have a significant public health impact.