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Mechanisms of presynaptic release and postsynaptic development in cortical neurons Prange, Oliver


Electrophysiology and imaging techniques have been applied to study presynaptic vesicle release and postsynaptic development in vitro at cortical synapses. The amphiphilic dye FM1-43 was used to evaluate vesicle turnover at synaptic terminals in response to action potential (AP) stimulus evoked- and spontaneous miniature synaptic activity. Loading and unloading of FM1-43 into and out of synaptic vesicles was visualized in real-time using laser scanning confocal microscopy. Under conditions that maximized release probability at synapses, we found that the amounts of AP-evoked vesicular FM1-43 release could not be described by a normal distribution, but were positively skewed. A significant, albeit relatively small, fraction of all synapses (13 – 17 %) released FM1-43 amounts that could be attributed to the release of more than one vesicle per AP stimulus. When utilizing miniature synaptic activity for FM1-43 uptake, we found a large variability in the amount of FM1-43 loading among presynaptic terminals, indicating a non-uniform probability for miniature release among synapses. Imaging of AP-stimulated vesicle release showed that release rates were significantly elevated at synapses with high rates of miniature activity compared to control synapses. In addition, we found a significant correlation between miniature activity and AP-evoked release probability at single synaptic sites. Lastly, real-time confocal imaging of filopodial postsynaptic spine precursors was employed in cortical neurons during two stages of early in vitro development. Particle mediated gene transfer was used to overexpress a green fluorescent protein (GFP)-labeled form of the postsynaptic density protein PSD-95, which can mediate the clustering of postsynaptic receptors and signal transduction proteins. Neurons overexpressing PSD-95/GFP were additionally labeled with the red dye sulforhodamine and two-channel confocal data acquisition was used. We found that preformed PSD-95 clusters could be rapidly translocated into and out of filopodia and spines. Moreover, filopodia and spines that were associated with clusters of PSD-95 were significantly more stable (i.e. less likely to turn-over) during 1 hour of imaging than filopodia and spines without PSD-95 clusters. In summary, these findings suggest the occurrence of multivesicular release at cortical synapses, indicate a physiological association between spontaneous miniature and evoked synaptic release, and implicate PSD-95 in the stabilization of filopodial spines during early development of cortical neurons.

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