Fife regulates the readily releasable pool of synaptic vesicles. (A and B) Representative traces of EJCs and mean cumulative quantal content (A) in wild type (gray) and Fife^AC/Df (green) during 60-Hz stimulus trains in 1 mM [Ca²⁺]_e. Mean readily releasable vesicle pool size (B) estimated by back extrapolation of a line fitted to the last 10 of 30 cumulative quantal content measurements is significantly reduced in Fife^AC/Df (403 ± 88 vesicles, n = 6 NMJs) compared with wild type (647 ± 67 vesicles, n = 9 NMJs, P = 0.043). (C–E) Representative electron micrographs of ultrathin sections of wild-type (C) and Fife^AC/Df (D) active zones prepared by HPF/FS. Active zone–associated synaptic vesicles are colored gray (C and D, right panels), vesicles in direct contact with or tethered within 5 nm of the presynaptic membrane (marked in white) were quantified and are colored green (C and D, right). (E) Mean number of membrane-associated vesicles at Fife^AC/Df active zones (1.8 ± 0.19 vesicles, n = 30 active zones) is significantly reduced compared with wild-type (2.5 ± 0.19 vesicles, n = 30 active zones, P = 0.017). *, P < 0.05, Student’s t test. Error bars represent SEM.