Aim: 

Neuronal activity requires a constant recycling of synaptic vesicles. Synaptic vesicles reform through clathrin mediated endocytosis in the periphery of the synapse. For many years, a faster mechanism of vesicle recycling has been proposed, in which vesicles might fuse transiently with the presynaptic terminal. This mode of vesicle recycling has been clearly shown to occur in neuroendocrine cells and has been termed kiss-and-run. We aim to study endocytosis in hippocampal neurons.

Methods: 

To study different modes of endocytosis, we have over-expressed in hippocampal neurons in culture synaptic vesicle proteins tagged to GFP in their intravesicular domain. This system allows studying synaptic activity because fluorescence increases as vesicles fuse with the presynaptic terminal and the intravesicular GFP is unquenched by the exposure to normal extracellular pH.

Results and Conclusion: 

Endocytosis and reacidification of endocytosed vesicles appears as a monotonic decay in fluorescence. We have studied the dynamics of hippocampal synaptic buttons at different levels of stimulation induced in current clamp conditions. With minimal stimulation (1 action potential), we have been able to obtain an all-or-none-response, suggesting that the fusion of one synaptic vesicle could be detected with our imaging system. At increasing frequency of stimulation we observed faster mechanisms of endocytosis and recycling. We propose that synaptic vesicles recycle adapts to the demand imposed by stimulation.