The post synaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine of molecular weight greater than one billion Daltons. The PSD is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we have developed a new method to freeze-substitute hippocampal cultures and then examine them by EM tomography in thin sections. Tomography reveals that the core of the PSD is an array of vertically oriented filaments that contain PSD-95, and contact two types of transmembrane structures at PSDs matching AMPA and NMDA receptors. The C-terminal ends of the vertical filaments are linked by horizontally oriented filaments. One class of horizontal filaments is ordered to form hexagons cross-linking the vertical filaments which are concentrated under the NMDA receptors. These findings show how the PSD-95 matrix can stabilize glutamate receptors, and at the same time could be remodeled by the addition of new receptors at the edges of the PSD. The idea that the scaffold stabilizes the PSD has been explored by using EM tomography to determine the effects of knocking down PSD-95. After PSD-95 is knocked down, patches of PSD unravel, demonstrating the central role of the PSD-95 scaffold. Examination of the effects of knocking down other (MAGUK) proteins is now in progress. A newly developed electron microscopic method high voltage STEM tomography is being applied to examine the effects of knocking down MAGUKS. HVST is compatible with sections up to two m thick, revealing detailed reconstructions of many whole synapses. A method for high resolution EM tomography of isolated PSDs became available with the discovery of a negative stain compatible with tomography. Using this stain makes it possible accurately to map the distribution of CaMKII in isolated PSDs. A second pool of CaMKII is found embedded in the matrix of the PSD, which may be functionally distinct from the soluble pool of CaMKII that binds to the PSD during activity.
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