The post synaptic density (PSD) at excitatory glutamatergic synapses is a complex molecular machine which is known to be a key site of information storage. We have previously shown that the PSD is a dynamic structure, changing its organization during synaptic activity. In order to explore the detailed molecular organization of the PSD, we have developed a method to freeze-substitute hippocampal cultures and then examine them by tomography of thin sections. Tomography reveals that the core of the PSD is a large array of vertically oriented PSD-95 filaments. We also identify AMPA and NMDA receptors, which are both contacted by the PSD-95 filaments. There are also two major types of filaments oriented parallel to the post synaptic membrane. One type contacts the vertical filaments that also contact the NMDA receptors, while the other type contacts all vertical filaments. Most receptors in the spine membrane outside of the PSD are not contacted by filaments. The insight that the central organizing molecules, consisting of PSD-95, are vertically oriented shows how its binding partners can form a stable matrix at the core of the PSD. The PSD-95 matrix can stabilize glutamate receptors and at the same time be remodeled by the addition of new receptors and their binding partners. Ongoing complementary studies with isolated PSDs, using rotary shadowing, have mapped the distributions of PSD-specific scaffolding molecules, including PSD-93 and SAP-97 within the PSD. We affinity purify PSDs from other components of the PSD fraction, thereby allowing independent measurement of CaMKII content, as well as proteomic analysis by mass spectroscopy (with S. Markey). This study was the first proteomic analysis of purified PSDs, thereby eliminating the contributions from abundant contaminating cytoskeletal elements and other proteins in the PSD fraction. Comparison of mass spectrometric data for the conventional and affinity-purified PSD fractions reveals enrichment following affinity purification of specialized scaffolding molecules and glutamate receptors, especially of the AMPA type, accompanied by a notable decrease in certain cytoskeletal and presynaptic proteins formerly regarded as core components of the PSD. These studies will inform further tomographic and immunocytochemical analyses of the organization of the PSD.receptors, especially of the AMPA type, accompanied by a notable decrease in certain cytoskeletal and presynaptic proteins formerly regarded as core components of the PSD.
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