The overall goal of these studies is to determine the molecular mechanisms for activity-dependent regulation of postsynaptic cytoskeletal organization. These studies will focus on microtubules, actin filaments, and the dendritic microtubule-associated protein MAP2. Cultured neurons will be labeled for MAP2, F-actin, and/or their associated proteins, and examined using either two-photon fluorescence microscopy or HVEM. Labeling will be accomplished using eosin conjugated reagents followed by photo-oxidation for electron microscopic examination. The organization of actin filaments in dendritic spines will be examined in cultures incubated in the absence or presence of glutamate agonists. Preliminary experiments performed at the NCMIR have shown that photo-oxidation of phalloidin-eosin provides beautiful and selective delineation of dendritic spines in slices of cerebellum. We will use tomographic reconstructions and IVEM to correlated light microscopic observations on the effects of glutamatergic stimulation on spine structure.. We will also use this technique to examine the 3-dimensional organization of the actin cytoskeleton itself under different conditions. The interaction of MAP2 in situ with actin filaments or the RII regulatory subunit of PKA will be examined using FRET combined with high resolution two-photon imaging. These data will be integrated into a developing model of the protein-protein interactions that occur among cytoskeletal elements in the postsynaptic compartment of neurons and their regulation by neural activity. Preliminary experiments to determine optimal specimen preparation procedures are underway.
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