Structure of the Active Zone at Nanometer Scale

描述(由申请人提供)： 电子显微镜层析成像(EMT)技术首次在组织切片上提供纳米级的空间分辨率，为加深我们对突触传递的结构基础的理解提供了强有力的途径。EMT研究已经开始了对活动区的研究，活动区是突触前细胞中突触小泡停靠在质膜并与质膜融合以将其神经递质释放到突触间隙的位置。在一个模型突触，青蛙的神经肌肉接头(NMJ)，定性研究揭示了被称为活动区物质(AZM)的蛋白质聚集体的大分子组成。其成分的组织和关联表明，Azm有助于对接突触小泡，锚定质膜通道和其他调节小泡融合的蛋白质。EMT还暴露了突触小泡管腔内的细丝和细胞骨架与Azm的连接，这也可能在突触传递中发挥作用。这里提出的研究将扩展对青蛙NMJ活动区的EMT分析，通过实施量化方法来表征已经确定的结构和关系，并通过系统地暴露更多的结构和关系。它还将对其他突触的活动区进行EMT分析，这些突触的总体组织与青蛙的NMJ不同。具体目标是：1)在青蛙的NMJ生成Azm的量化建筑地图。2)确定停靠的突触小泡在蛙的NMJ处与AZM和突触前膜的空间关系。3)确定细胞骨架细丝与青蛙NMJ的AZM之间的联系。4)观察蛙NMJ突触小泡内的管腔细丝。5)比较小鼠NMJ和神经元间突触的Azm与青蛙NMJ的Azm的结构。通过EMT获得的活动区成分的定量和系统的结构特征对于产生和检验关于它们在突触传递中的化学性质和功能的假说是必不可少的。在更广泛的水平上，全面了解涉及突触传递的机制是理解调节突触发育和维持、带来疾病和影响再生的因素所必需的。

DESCRIPTION (provided by applicant): The nascent technology of electron microscope tomography (EMT), which for the first time provides nanometer scale spatial resolution in tissue sections, offers a powerful approach to increasing our understanding of the structural basis of synaptic transmission. EMT studies have begun on active zones, the sites in presynaptic cells where synaptic vesicles dock at and fuse with the plasma membrane to release their neurotransmitter into the synaptic cleft. At a model synapse, the frog's neuromuscular junction (NMJ), qualitative studies exposed the macromolecular composition of protein aggregates known as active zone material (AZM). The organization and associations of its components indicate that the AZM helps dock synaptic vesicles and anchor plasma membrane channels and other proteins that regulate vesicle fusion. EMT also exposed filaments in the lumen of synaptic vesicles and connections of cytoskeleton to AZM, which are also likely to play a role in synaptic transmission. The research proposed here will extend EMT analysis of active zones at the frog's NMJ by implementing quantitative methods for characterizing structures and relationships already identified and by systematically exposing additional ones. It will also apply EMT analysis to active zones at other synapses, where the gross organization differs from that at the frog's NMJ. The specific aims are to: 1) Generate a quantitative architectural map of the AZM at the frog's NMJ. 2) Determine the spatial relationship of docked synaptic vesicles to the AZM and presynaptic membrane at the frogs' NMJ. 3) Define the associations of cytoskeletal filaments with AZM at the frog's NMJ. 4) Characterize the lumenal filaments in synaptic vesicles at the frog's NMJ. 5) Compare the structure of the AZM at the mouse's NMJ and at neuron-to-neuron synapses with that of the AZM at the frog's NMJ. The quantitative and systematic structural characterization of constituents of the active zone made accessible by EMT is essential for generating and testing hypotheses as to their chemical nature and function in synaptic transmission. At a broader level, a comprehensive knowledge of the mechanisms involved in synaptic transmission is requisite to understanding factors that regulate synapse development and maintenance, bring about disease and influence regeneration.