Dynamic Structural Properties of Synapses

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Movements of PSD components during activity were studied by pre-embedding Nanogold labeling of dissociated hippocampal neurons in culture, using strategies similar to those employed in previous work with CaMKII. Depolarization with high K+ is typically used to depolarize and activate neuronal cultures, but other protocols closer to physiological stimulation, such as application of NMDA and synaptic activation with glycine in the absence of Mg2+, are being tested. Now that antibodies for nearly all major components of the PSD have been obtained and tested for compatibility for immuno-electron microscopy, a general picture of activity-induced re-organization of the PSD at the molecular level begins to emerge. The main PSD scaffold, consisting of PSD-95 and GKAPs, appears to be relatively stable during short-term activity, while Shanks, especially Shank1, move closer to the PSD. While we have previously described movement of CaMKII towards the PSD during activation, we now show that another major regulatory protein, SynGAP, moves away from the PSD upon stimulation. These stimulation induced changes coincide with a marked increase of AMPA receptor labeling at the PSD, compatible with enzymatic as well as structural roles of CaMKII and SynGAP in regulating AMPA receptor trafficking at the PSD. EM tomography of isolated PSDs is being used to describe with more precision the re-localization of proteins at the PSD following activity. Two technical breakthroughs, the isolation of PSD fractions from hippocampal slice cultures, and a negative staining method compatibile with EM tomography, have been essential. PSDs isolated from depolarized hippocampal slices show elevated levels of CaMKII confirming that activity-induced changes at the PSD are preserved during isolation. CaMKII association domains appear as rings that are readily recognizable in tomograms, allowing accurate localization of individual CaMKII molecules within the PSD. Mapping the distribution of CaMKII will show exactly where in the PSD CaMKII is bound at rest, and after stimulation protocols with defined physiological consequences. This is an essential question to explore because CaMKII has multiple phosphorylation targets at multiple locations within the PSD. Localization of CaMKII after depolarization with high K+ will be followed by stimulation protocols known to induce long-term changes, such as chemLTP and chemLTD. Quantitative mass spectrometry protocols are in place to track parallel changes in protein composition and phosphorylation.