A study of the inter-bouton exchange of synaptic vesicles at central synapses
Neurotransmitter release at central synapses is sustained by the synaptic vesicle cycle. It has been assumed that vesicle replenishment operates autonomously at individual presynaptic terminals. In this study the classical model of a compartmentalized synaptic vesicle cycle was tested by using a novel combination of FRAP (fluorescence recovery after photobleaching) and CLEM (correlative light and electron microscopy) in cultured hippocampal neurons. The stability of vesicle clusters labelled with fluorescent styryl dye at individual synapses were assessed by photobleaching using a confocal laser microscope and monitoring fluorescence recovery over time. The observed fluorescence recovery which was abolished by inhibitors of vesicular transport, suggested that synaptic vesicles recycled at sites outside the bleach region were transported along axons into bleached synapses. These newly-imported vesicles could undergo exocytosis upon stimulation, demonstrating that they formed part of the functional recycling pool. The spatial organization of imported vesicles in presynaptic boutons was examined using CLEM and FM dye photoconversion techniques. Imported vesicles were distributed throughout the native vesicle cluster, indicating that they become morphologically integrated into the synapse. The ability of imported vesicles to mix well with native vesicles highlights the dynamic nature of vesicle clusters at resting synapses. The departure of fluorescent packets from boutons into axons was observed by time- lapse microscopy. Ultrastructural analysis confirmed the mature state of donor synapses and showed these mobile packets to be loose aggregates of synaptic vesicles. Mobile vesicle clusters were comprised of vesicles from both the recycling and resting pools of the synapse, thus demonstrating no preference for mobility of any one vesicle population.