SUPRAMOLECULAR ORGANIZATION OF POSTSYNAPTIC DENSITY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脑内兴奋性神经传递主要由突触后密度(PSD)中的胞浆蛋白锚定在突触后膜上的离子型谷氨酸受体介导。这些PSD蛋白有助于塑造对谷氨酸的突触后反应，但即使在体外记录了这些分子可能产生的各种生化和电生理效应后，它们在体内的作用仍将取决于它们在突触处的物理排列和组织方式。现代生化技术允许在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，但不提供关于这些分子的超分子结构的直接信息。我目前由N111资助的研究将在大脑皮层和海马区进行定量免疫金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，我们可以定位表位的精度为-20纳米；通过平均技术，我们可以获得平均位置的估计，精度为？5纳米。 这项研究的总体目的是促进我们对突触的化学结构的理解。我希望获得比标准方法更准确的抗原位置估计。我目前的方法的主要问题之一是截面厚度：我检测到的金粒子只与100 nm截面厚度的“涂抹”投影相关。电子断层扫描可能为这个问题提供了一个很好的解决方案。通过允许我只考虑与大脑切片表面5-10纳米相关的颗粒，标记的空间分辨率应该会显著提高。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可区分的形态成分的化学“特征”。

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.