Ultrastructural and functional fate of recycled vesicles in hippocampal synapses.

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution.

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion.

Fast activity-driven turnover of neurotransmitter-filled vesicles at presynaptic terminals is a crucial step in information transfer in the CNS. Characterization of the relationship between the nanoscale organization of synaptic vesicles and their functional properties during transmission is currently of interest. Here we outline a procedure for ultrastructural investigation of functional vesicles in synapses from native mammalian brain tissue. FM dye is injected into the target region of a brain slice and upstream axons are electrically activated to stimulate vesicle turnover and dye uptake. In the presence of diaminobenzidine (DAB), photoactivation of dye-filled vesicles yields an osmiophilic precipitate that is visible in electron micrographs. When combined with serial-section electron microscopy, fundamental ultrastructure-function relationships of presynaptic terminals in native circuits are revealed. We outline the utility of this protocol for the 3D reconstruction of a recycling vesicle pool in CA3-CA1 synapses from an acute hippocampal slice and for the characterization of its anatomically defined docked pool. This protocol requires 6-7 d.