Simulation of P_D(S_i) plots with two-step release model. (A–C) Plots of P_D(S_i) as a function of i from Monte Carlo simulations performed at 25 (top) and 50 Hz (bottom) using the two-step model (dotted lines). Two parameters were fixed: r′ and s. Two parameters changed with the presynaptic holding potential: at −50 mV, p = 1 and ρ = 0.2 (A, orange); at −60 mV, p = 0.95 and ρ = 0.65 (B, gray); and at −80 mV, p = 0.85 and ρ = 0.9 (C, blue). Finally, δ was taken at 0.45 at −50 mV and at 0.50 at both −60 and −80 mV. Corresponding experimental results are depicted by shaded areas (mean ± SEM; n = 12 pairs at 25 Hz and 8 pairs at 50 Hz). Both experimental data and simulations indicate the following: at −50 mV, a depression/facilitation sequence (A); at −60 mV, an abrupt fall between first and second stimulations followed by a flat P_D(S_i) curve for i ≥ 2 (B); and at −80 mV, a monotonic depression (C). Top: At 25 Hz, the values of r′ and s were r′ = 0.15 and s = 0.35. Bottom: At 50 Hz, the values of r′ and s were corrected for the change in frequency. This was done in two ways. In one variant, transition rates R′ and S were the same as for the 25-Hz simulations, giving r′ = 0.08 and s = 0.2 at 50 Hz (light-colored dashed lines). In the other variant, r′ and s were increased to 0.1 and to 0.24, respectively, to take into account the increased speed of vesicle replenishment with calcium concentration (dark-colored dashed lines). δ, p, and ρ were as before. The second variant offers a slightly better fit than the first.