3D FINE STRUCTURE OF THE NEUROMUSCULAR JUNCTION

说明(摘自申请人的摘要)以检验以下假设 在神经肌肉连接处组成突触装置的蛋白质有 具体有序的安排，派和他的同事们已经开始 利用电磁层析成像制作高精度三维图像的发展途径 组织切片的重建。从这种重建中获得的数据 提供了关于结构的丰富的新的定量信息来源 小于4 nm的突触结构的组成和组织 所有三个轴上的分辨率。免疫金标记物的位置 突触蛋白也可以被准确地测定。的具体目标 该提案中的实验概述如下： 1.评估不同的组织准备、数据收集、 用于检测细微结构的重建和表示 神经肌肉突触装置的3D图像。 2.对该装置的三维精细结构进行表征。 3.在3D中映射特定突触的免疫金标记物的位置 蛋白质到该装置的特定结构组件。 关于神经肌肉的分子结构的详细知识 连接是理解突触机制的关键 传输和维护。它也是确定如何将 神经肌肉连接在个体发育过程中形成，创伤后再生 并用于表征发病机制。EM断层扫描提供了 在更高的水平上获得此类信息的机会 分辨率高于目前可用的任何其他方法或在 眼前的地平线。此外，由于它也可以用于研究3D 人体内任何组织的精细结构，系统地研究其 在神经肌肉连接处的应用，如这里所建议的，应该提供 洞察力，将加速其他人进入这一新兴领域 研究方法。

DESCRIPTION (from the applicant's abstract) To test the hypothesis that proteins composing the synaptic apparatus at the neuromuscular junction have a specific and orderly arrangements, the PI and his colleagues have begun developing ways of using EM tomography for making accurate 3D reconstructions of tissue sections. Data obtained from such reconstructions furnish a rich new source of quantitative information about the structural composition and organization of the synaptic apparatus to less than 4nm resolution in all three axes. The position of immunogold labels for synaptic proteins can also be accurately determined. The specific aims of the experiments outlines in this proposal are: 1. To assess different methods of tissue preparation, data collection, reconstruction and representation for examining fine structure of the neuromuscular synaptic apparatus in 3D. 2. To characterize the 3D fine structure of the apparatus. 3. To map in 3D the position of immunogold labels for specific synaptic proteins to specific structural components of the apparatus. A detailed knowledge of the molecular architecture of the neuromuscular junction is essential for understanding the mechanisms involved in synaptic transmission and maintenance. It is also necessary for determining how the neuromuscular junction forms during ontogenesis and regenerates after trauma and for characterizing mechanisms of pathogenesis. EM tomography offers the opportunity of obtaining such information at a significantly higher level of resolution than that provided by any other method currently available or on the immediate horizon. Moreover, since it can also be used to study the 3D fine structure of any tissue in the body, a systematic approach to its application at the neuromuscular junction, as proposed here, should provide insights that will expedite the entry of others into this nascent field of research methodology.