Vesicle recycling at developing NMJs

开发 NMJ 中的囊泡回收

• 批准号: 7524495
• 负责人: RITA J. BALICE-GORDON
• 金额: $ 31.5万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7524495
• 关键词: Address; Adult; Affect; Applications Grants; Area; Autistic Disorder; Axon; Biological Models; Botulinum Toxins; Bungarotoxins; CDKN1A gene; Cells; Characteristics; Chromosome Pairing; Couples; Cyclophosphamide/Fluorouracil/Prednisone; Development; Dose; Embryo; Epilepsy; Equilibrium; Facility Construction Funding Category; Fluorescence; Fluorescence Recovery After Photobleaching; Frequencies; Goals; Green Fluorescent Proteins; Image; Individual; Injection of therapeutic agent; Label; Life; Light; Measurement; Measures; Mediating; Mental Retardation; Molecular; Monitor; Motor; Motor Neurons; Mus; Muscle Fibers; Neonatal; Nerve; Nerve-Muscle Preparations; Neuromuscular Junction; Neurons; Neurotransmitters; Numbers; Optics; Outcome; Pattern; Photobleaching; Plastics; Preparation; Presynaptic Terminals; Process; Property; Proteins; Public Health; Rate; Recovery; Recruitment Activity; Recycling; Regulation; Relative (related person); Series; Signal Transduction; Site; Stimulus; Structure; Study models; Synapses; Synaptic Vesicles; Synaptic plasticity; Synaptophysin; Testing; Time; Transgenic Mice; Transgenic Organisms; Variant; Vesicle; Work; base; day; developmental disease; experience; in vivo; insight; neonate; neural circuit; neurodevelopment; neurotransmitter release; neurotrophic factor; oncoprotein p21; postnatal; postsynaptic; presynaptic; prevent; promoter; release factor; research study; size; synaptic function; trafficking

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.

描述（由申请人提供）：神经元与其突触伙伴的突触在开发过程中具有延展性，并且由于经验，在数量，强度和功能特性方面，例如短期和长期可塑性。一个经过良好研究的发展模型系统是小鼠神经肌肉突触，它在发育中经历了活动依赖性的可塑性，这是CNS中较小，易于访问的对应物的标志。在胚胎晚期和早期产后寿命中，神经肌肉突触会消除突触，其中一个轴突的突触与其他轴突的突触相结合，这些轴突支配了相同的靶细胞。根据他们相对于竞争对手的活动模式，将出现一个或少数轴突作为获胜者，将其突触保持在成人生活中，而其他轴突将失去竞争，并从神经电路中永久删除。尽管对神经肌肉突触消除的结构和一些功能研究，但对基本机制和许多重要问题的了解鲜为人知。一组重要的问题包括竞争输入的结构变化与输入强度的逐步变化，竞争结果以及活动如何介导该过程有关。这项赠款提案的目的是了解竞争的突触前方面知之甚少的动态，包括突触囊泡的释放，回收和贩运，以发展哺乳动物神经肌肉突触。我们已经开发了小鼠的转基因线，其中THY1启动子驱动突触氟蛋白（THY1-SPH）的表达。 SPH是GFP的pH敏感变体，该变体将囊泡蛋白Vamp2的腔内域束缚在腔内域，允许突触囊泡回收可以通过光学监测。初步研究表明，SPH+突触小囊泡簇可以在运动轴突末端和囊泡释放和运输中，并使用活性诱导的荧光变化评估的囊泡释放和运输，并在分离的胸骨肌肉神经肌肉制剂中以及体内评估。提出了四个目标，包括（1）确定囊泡释放的空间和时间动力学，跨发育界和成人神经肌肉连接的末端分支； （2）确定囊泡释放，突触强度和竞争输入的突触大小与开发突触消除的神经肌肉连接的关系； （3）确定突触后活性阻滞如何逆行影响突触囊泡在发育和成年神经肌肉连接处的突触囊泡释放； （4）确定突触囊泡如何在单个末端内的释放位点之间运输，以及如何通过突触前和突触后活性调节这一点。综上所述，下面提出的目的将检验一个总体假设，即活动调节突触前囊泡释放和运输，影响突触结构，强度和生存。这将提供一种机制，突触功能的塑性变化可以永久改变神经回路。公共卫生相关性：我建议使用神经肌肉突触作为模型系统来研究神经发育过程中突触竞争的机制。使用转基因小鼠可以在体内监测突触囊泡回收和运输动力学的动力学，我将测试整体假设，即活性调节了突触前囊泡释放和运输，从而影响突触结构，强度和生存。拟议的实验的结果将提供对正常发育过程中活动改变突触功能和神经回路的机制的根本新见解，并有助于理解具有重大公共卫生影响的发育障碍，例如癫痫，自闭症和智力低下。