Analysis of Molecular Assembly of Synaptic Molecules during Development

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Transition of excitatory synapses from dendritic shafts to spines takes place during the period of 10-20 days in culture of hippocampal pyramidal neurons. This culture system provides a good model for the study of formation and remodeling of synaptic structure. To analyze dynamic properties of multiple postsynaptic density (PSD) proteins, we generated green fluorescent protein (GFP)-tagged PSD proteins, such as PSD-95 and PSD-Zip45 (also known as Homer Ic), together with presynaptic marker synaptophysin. The first set of experiments aimed at revealing time-course of morphogenesis of dendritic spines, assembly of PSD proteins, and accumulation of synaptic vesicles at single synaptic junctions. We performed dual-wavelength, time-lapse fluorescence microscopy of living hippocampal neurons and revealed temporal correlation among these three events. Furthermore, synaptogenesis was a rapid process, which took place less than an hour. In the second research project we analyzed dynamic redistr … More ibution of PSD-Zip45 after stimulation of cultured neurons. In contrast to the stable nature of PSD-95 after stimulation, PSD-Zip45 changed its distribution bidirectionally after increase of the intracellular calcium. This result indicates the presence of molecular mechanism that drives PSD-Zip45-specific translocation by the neuronal activity. In the third set of experiments we generated transgenic mice expressing either GFP-tagged PSD-95 or PSD-Zip45 to achieve stable expression of probe molecules in the hippocampus. Using cultured neurons isolated from the transgenic hippocampus, we characterized slow remodeling of PSD-95 and PSD-Zip45 clusters for >1 week. The results indicate the presence of cAMP-dependent signaling system that regulates the average density of PSD clusters within a single cell. In summary, these three sets of experiments revealed novel molecular dynamics of PSD proteins in living neurons and regulation of the distribution of PSD proteins by complex signaling system that can be differentially activated by various neuronal activities. Less