Structure of the Active Zone at Nanometer Scale

描述（由申请人提供）： 新生的电子显微镜断层扫描（EMT）技术，首次提供了纳米级的空间分辨率在组织切片，提供了一个强大的方法来增加我们的理解突触传递的结构基础。EMT的研究已经开始于活动区，即突触前细胞中突触囊泡停靠并与质膜融合以将其神经递质释放到突触间隙中的部位。在一个模型突触，青蛙的神经肌肉接头（NMJ），定性研究暴露了蛋白质聚集体的大分子组成，称为活性区材料（AZM）。其组成部分的组织和协会表明，AZM有助于对接突触囊泡和锚质膜通道和其他蛋白质，调节囊泡融合。EMT还暴露了突触囊泡内腔中的细丝和细胞骨架与AZM的连接，这些也可能在突触传递中起作用。这里提出的研究将扩展EMT分析的活动区在青蛙的NMJ通过实施定量方法表征结构和关系已经确定，并通过系统地暴露额外的。它还将EMT分析应用于其他突触的活跃区域，这些区域的总体组织与青蛙的NMJ不同。具体目标是：1）生成青蛙NMJ处AZM的定量结构图。2)确定对接的突触囊泡的空间关系的AZM和突触前膜在青蛙的NMJ。3)定义青蛙NMJ处细胞骨架丝与AZM的关系。4)描述青蛙NMJ突触囊泡中的管腔细丝。5)比较小鼠NMJ和神经元-神经元突触的AZM结构与青蛙NMJ的AZM结构。通过EMT获得的活性区成分的定量和系统的结构表征对于产生和测试关于其化学性质和在突触传递中的功能的假设是必不可少的。在更广泛的层面上，对突触传递机制的全面了解是理解调节突触发育和维持、引起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.