Fig. 3.

Newly tethered events contribute to the sustained release at cMF terminals. (A) Representative traces for Ca²⁺ current and capacitance in double pulse experiments. Two 0-mV depolarization pulses for 30 ms were applied with an interstimulus interval of 100 ms. (B) Ca²⁺ currents and capacitance jumps by the first and the second depolarization pulses, and the slope of the capacitance increase after the pulses were measured (n = 32; mean ± SEM). ***P < 0.001, Student’s t test. (C) (Top) Peristimulus time histograms for “vanish” and “tethering” events observed by TIRF imaging from 80 terminals. The stimulation periods of the pulses are indicated by gray. (Bottom) Cumulative numbers of events for the first response were fitted with a single exponential curve (red). Cumulative numbers for the second response were fitted with an exponential curve plus a line (blue). (D) Comparison of the time course of fluorescent spots disappearing between total (Top) “vanish” events after the second pulse and (Bottom) “tethering−vanish” events. In “tethering−vanish” events, the events visibly appearing between 0 ms and 130 ms were analyzed. The cumulative number of vanish events that tether between pulses was fitted with a single exponential curve (red). (E) (Left) Representative and (Right) averaged (n = 7) time courses of the fluorescent intensity within a center (347 nm diameter; filled squares) and concentric annulus (347 nm inner and 867 nm outer diameter; open squares) for “diffuse” events that are tethered between pulses. Average images are taken at the times indicated by numbers. Error bars show SEM. (Scale bar, 500 nm.)