Vesicle recycling at developing NMJs

描述（由申请人提供）：由神经元及其突触伙伴组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。小鼠神经肌肉突触在发育过程中经历了活动依赖的可塑性，这是它们在中枢神经系统中更小、更不易接近的对应体的标志。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目标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，维持它们的突触直到成年，而其他轴突则失去竞争，从神经回路中永久删除。尽管对神经肌肉突触消除的结构和功能进行了许多研究，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和竞争结果相关，以及活动如何调节这一过程。这项拨款提案的目标是了解竞争的突触前方面的动力学，包括发育中的哺乳动物神经肌肉突触的突触囊泡释放、再循环和贩运。我们已经开发了Thy1启动子驱动synaptophorin （Thy1- sph）表达的转基因小鼠系。SpH是GFP的ph敏感变体，连接到囊泡蛋白VAMP2的管腔结构域，使突触囊泡循环能够被光学监测。初步研究表明，spH+突触囊泡簇可以很容易地在运动轴突末梢内可视化，并且通过光学测量活动诱导的荧光变化来评估囊泡释放和运输，在离体胸锁乳突神经肌肉制剂中以及在体内。提出了四个目标，包括：(1)确定发育和成人神经肌肉连接末端分支的囊泡释放的时空动态；(2)确定囊泡释放、突触强度和突触大小之间的关系，以形成经过突触消除的神经肌肉连接；(3)确定突触后活动阻断如何逆行影响发育中和成年神经肌肉连接处的突触囊泡释放；(4)确定突触囊泡是如何在单个末端的释放位点之间运输的，以及这是如何通过突触前和突触后活动调节的。综上所述，以下提出的目标将检验活动调节突触前囊泡释放和运输，影响突触结构、强度和存活的总体假设。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：我建议研究神经发育过程中突触竞争的机制，使用神经肌肉突触作为模型系统。利用体内可监测突触囊泡循环和运输动态的转基因小鼠，我将检验活动调节突触前囊泡释放和运输，影响突触结构、强度和存活的总体假设。这些实验的结果将从根本上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新的见解，并有助于理解癫痫、自闭症和智力迟钝等对公共卫生有重大影响的发育障碍。

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.