Vesicle recycling at developing NMJs

描述(由申请人提供)：神经元与其突触伙伴形成的突触在发育过程中是可塑性的，作为经验的结果，在数量、强度和功能特性(如短期和长期可塑性)方面是可塑性的。小鼠神经肌肉突触是发育中活动依赖可塑性的一个研究很好的模型系统，它在发育过程中经历活动依赖可塑性，这是它们在中枢神经系统中较小、较难接近的突触的标志。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，在成年后保持它们的突触，而其他轴突则在竞争中失败，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少数功能的研究，但对其潜在的机制知之甚少，许多重要的问题仍有待解决。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逐步变化、竞争的结果以及活动如何调节这一过程有关。这项拨款提案的目的是了解尚不清楚的突触前竞争方面的动态，包括发育中的哺乳动物神经肌肉突触的突触小泡释放、回收和运输。我们已经建立了Thy1启动子驱动突触素(Thy1-sph)表达的转基因小鼠品系。SPH是一种pH敏感的GFP变体，与囊泡蛋白VAMP2的管腔结构域相连，允许对突触小泡的循环进行光学监测。初步研究表明，SPH突触囊泡团可以很容易地在运动轴突终末和囊泡的释放和运输中显示出来，通过对分离的胸锁乳突神经-肌肉标本以及体内活性诱导的荧光变化的光学测量来评估。我们提出了四个目标，包括：(1)确定发育中和成年神经肌肉接头终末支囊泡释放的空间和时间动力学；(2)确定正在经历突触消除的发育中神经肌肉接头的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行影响发育中和成年神经肌肉接头的突触小泡释放；以及(4)确定突触小泡如何在单个终末内的释放部位之间运输，以及突触前和突触后活动如何调节这种关系。综上所述，下面提出的目标将检验总体假设，即活动调节突触前囊泡的释放和运输，影响突触结构、强度和存活率。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性地改变神经回路。公共卫生相关性：我建议使用神经肌肉突触作为模型系统，研究神经发育过程中突触竞争的潜在机制。利用转基因小鼠，在体内可以监测突触囊泡回收和运输的动态，我将检验总体假设，即活动调节突触前囊泡的释放和运输，影响突触结构、强度和存活率。拟议的实验结果将为活动在正常发育过程中改变突触功能和神经电路的机制提供全新的见解，并有助于理解癫痫、自闭症和智力低下等对公共健康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.