Extended Data Fig. 1. Schematic of events at the active zone of a hippocampal bouton within the first 15 ms after an action potential.

Vesicles close to the active zone are proposed to transit between docked and undocked states, with the on- and off-rates resulting in a certain number of vesicles docked and ready to fuse at any given time. Synchronous fusion, often of multiple vesicles, begins throughout the active zone within hundreds of microseconds, and the vesicles finish collapsing into the plasma membrane by 11 ms (note that the high local calcium shown only lasts ~100 microseconds). Between 5 and 11 ms, residual calcium triggers asynchronous fusion, preferentially toward the center of the active zone. Although shown here as taking place in the same active zone, the degree to which synchronous and asynchronous release may occur at the same active zone after a single action potential is unknown. By 14 ms, the vesicles from the peak of asynchronous fusion, which can continue for tens to hundreds of milliseconds, have fully collapsed into the plasma membrane, and new docked vesicles, which may start to be recruited in less than 10 ms, have fully replaced the vesicles used for fusion. These vesicles then undock or fuse within 100 ms. Whether these new vesicles dock at the same sites vacated by the fused vesicles, and whether newly docked vesicles contributed to synchronous and asynchronous fusion during the first 11 ms, remains to be tested.