Two forms of asynchronous release with distinctive spatiotemporal dynamics in central synapses

Asynchronous release is a ubiquitous form of neurotransmitter release that persists for tens to hundreds of milliseconds after an action potential (AP). How asynchronous release is organized and regulated at the synaptic active zone (AZ) remains debatable. Using nanoscale-precision imaging of individual release events in rat hippocampal synapses, we observed two spatially distinct subpopulations of asynchronous events, ~75% of which occurred inside the AZ and with a bias towards the AZ center, while ~25% occurred outside of the functionally defined AZ, i.e., ectopically. The two subpopulations also differed markedly in temporal properties, with ectopic events occurring at significantly longer time intervals from synchronous events. Both forms of asynchronous release did not, to a large extent, utilize the same release sites as synchronous events. Both asynchronous event subpopulations also differ from synchronous events in some aspects of exo-endocytosis coupling. Specifically, for synchronous but not asynchronous events, coupling with the fast calcium-dependent endocytosis had a gradient decreasing from the AZ center towards its periphery. These results identify two distinct subpopulations of asynchronous release events with distinctive spatiotemporal organization and coupling to endocytic mechanisms. ### Competing Interest Statement The authors have declared no competing interest.