SUPRAMOLECULAR ORGANIZATION OF POSTSYNAPTIC DENSITY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。脑内兴奋性神经传递主要通过突触后膜上的嗜离子性谷氨酸受体介导，并通过突触后密度（PSD）集中的细胞质蛋白介导。这些PSD蛋白有助于形成对谷氨酸的突触后反应，但即使在体外记录了这些分子的全部可能的生化和电生理作用之后，它们在体内的作用将取决于它们在突触中的物理排列和组织方式。现代生物化学技术允许在PSD的大分子复合体内构建蛋白质-蛋白质相互作用的拓扑图，但不能提供这些分子的超分子结构的直接信息。我目前的n111资助的研究将在皮层和海马体中进行定量免疫金EM，以绘制出与组织受体和相关信号分子有关的几种蛋白质在PSD内的相对位置。使用常规技术，我们可以将表位定位到-20 nm的精度；通过平均技术，我们可以得到精度为5纳米的平均位置估计。这项研究的总体目的是促进我们对突触化学结构的理解。我希望得到比标准方法更准确的抗原定位估计。我目前的方法的一个主要问题来自切片厚度：我检测到的金颗粒仅与100 nm切片厚度的“涂抹”投影相关。电子断层扫描可以为这个问题提供一个优雅的解决方案。通过允许我只考虑与大脑切片表面5-10 nm有关的颗粒，标记的空间分辨率应该大大提高。此外，这种方法将大大提高突触结构的分辨率。通过将结构与免疫标记相关联，我希望识别PSD可区分形态成分的化学“特征”。迄今为止的进展是好的。我们完成了6个海马CA1区免疫金标记PSD的层析重建。我们正在分析这些数据。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Excitatory neurotransmission in the brain is mediated mainly via ionotropic glutamate receptors anchored at the postsynaptic membrane by cytoplasmic proteins concentrated in the postsynaptic density (PSD). These PSD proteins help to shape the postsynaptic response to glutamate, but even after the full range of possible biochemical and electrophysiological effects of these molecules has been documented in vitro, their actions in vivo will depend on how they are physically arranged and organized at the synapse. Modem biochemical techniques permit construction of a topological diagram of protein-protein interactions within the macromolecular complex of the PSD, but provide no direct information on the supramolecular architecture of these molecules. My current N111-funded research will perform quantitative immunogold EM in cortex and hippocampus, to map out the relative locations within the PSD of several proteins implicated in organizing receptors, and associated signaling molecules. Using routine techniques, we can localize epitopes to an accuracy of -20 nm; by averagina techniques, we can get estimates of mean position to an accuracy of ¿5 nm. The overall aim of this study is to advance our understanding of the chemical architecture of the synapse. I would like to get more accurate estimates of antigen location than feasible with standard methods. One of the main issues with my current approach arises from section thickness: the gold particles I detect are defined only in relationship to a "smeared" projection of 100 nm section thickness. Electron tomography may provide an elegant solution to this problem. By allowing me to consider particles only in relation to the surface 5-10 nm of the brain section, spatial resolution of the labeling should be considerably improved. Moreover, this approach will considerably improve the resolution of synaptic structure. By correlating structure with immunolabeling, I hope to identify chemical "signatures" of distinguishable morphological components of the PSD. Progress to date has been good. We have completed 6 tomographic reconstructions of immunogold labeled PSD¿s in hippocampal area CA1. We are the process of analyzing this data.