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.
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