Investigating the dynamics of vesicle pool traffic in hippocampal synapses. (360G-Wellcome-084357_Z_07_Z)

Hippocampal synapses are now known to trade vesicles constitutively via an extrasynaptic mobile vesicle pool. This proposal will examine the regulation of this process in supporting the maintenance of synaptic structures, and examine whether vesicle trading can be utilized in a dynamic, activity-dependent manner to modulate synaptic efficacy. Vesicles will be labelled using GFP-based, photoswitchable, and activity-dependent fluorescent probes, and import/export dynamics examined using localized photobleaching/photoswitching techniques. Experiments will characterize cellular/molecular factors contributing to constitutive vesicle sharing under steady-state conditions. Activity-dependence of vesicle exchange will be tested by combining imaging with global or localized synapse-specific stimulation. These approaches will also be used to evoke forms of LTP-like long-term plasticity to test whether presynaptic vesicle pools locally recruit vesicles to undergo long-term remodelling, or build n ew functional release sites. The detailed changes associated with activity-dependent remodelling will also be examined by correlative fluorescence-ultrastructural analysis. Experiments will be performed in culture, but also in acute slices to establish the functional relevance of this process in native tissue. With these direct indices of presynaptic function, targeted at individual synapses and visualized down to their participating vesicle pools, this study should reveal important new insights into vesicle dynamics associated with presynaptic regulation and modulation.