Nanoscale Imaging Of Protein Molecules At The Postsynaptic Density

The postsynaptic density (PSD) is a large macromolecular machine that lines the postsynaptic membrane at glutamatergic synapses. The PSD is responsible for synaptic transmission, signal transduction, and processing and provides a substrate for synaptic plasticity. Here we describe approaches to unravel the molecular organization of the PSD to gain insights into its synaptic function. Dissociated cultures of rat hippocampal neurons are prepared by high-pressure freezing followed by freeze-substitution and low-temperature embedding to maximize preservation of fragile structures. Dual-axis EM tomographic reconstructions of spine synapses are performed on sections 100-200 nm thick to visualize details obscured by structural overlap prevalent in conventional electron micrographs. EM tomographic analyses show that the PSD proteins are organized as an orthogonal assembly constituting the core of the PSD. Immunogold EM coupled with tomography identifies a class of membrane-associated vertical filaments in the PSD core as PSD-95, the major scaffolding protein. Acute knockdown of PSD-95 with RNAi leads to loss of patches of electrondense PSD material, including loss of vertical filaments and their associated structures demonstrating its key role in the PSD core. The tomographic analyses so far have been limited to sections thinner than the diameter of a PSD, but newly developed scanning transmission electron microscopy tomography permits reconstructions of entire PSDs in dendritic spines in sections 1-2 mu m thick. The overall structure of entire PSD can now be analyzed to delineate how elimination of key PSD proteins might affect its overall structure.