Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism1 ensures a constant supply of releasable vesicles at the nerve terminal1. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation2,3,4,5,6,7,8,9,10,11,12,13. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at ≤2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models5,10, including inhibition of endocytosis by Ca2+, could not account for these results—our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency2,3, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.