A well-defined readily releasable pool with fixed capacity for storing vesicles at calyx of held

The readily releasable pool (RRP) of vesicles is a core concept in studies of presynaptic function. However, operating principles lack consensus definition and the utility for quantitative analysis has been questioned. Here we confirm that RRPs at calyces of Held from 14 to 21 day old mice have a fixed capacity for storing vesicles that is not modulated by Ca2+. Discrepancies with previous studies are explained by a dynamic flow-through pool, established during heavy use, containing vesicles that are released with low probability despite being immediately releasable. Quantitative analysis ruled out a posteriori explanations for the vesicles with low release probability, such as Ca2+-channel inactivation, and established unexpected boundary conditions for remaining alternatives. Vesicles in the flow-through pool could be incompletely primed, in which case the full sequence of priming steps downstream of recruitment to the RRP would have an average unitary rate of at least 9/s during heav...