(A) Sample traces for paired-pulse stimulations using channelrhodopsin recorded from an adult body muscle (acr-16(ok789); oxSi91[Punc17::ChIEF::mCherry]). The second pulses are indicated by colors: yellow, 100 ms; green, 300 ms; emerald, 1000 ms; blue, 3000 ms; and dark blue, 10000 ms. (B) Recovery of evoked responses in paired pulse stimulations. The ratios of the second current peak (Pi) to the first current peak (P1) were calculated and plotted relative to the recovery intervals. There are two time constants for recovery: the first tau was 0.1 s, and the second tau was 2.2 s. The rapid recovery (τ = 100 ms) is likely due to a transition of docked vesicles from an incompetent to competent state (Zenisek et al., 2000), rather than the translocation and docking of vesicles. (C) Average number of vesicles per profile docked in the active zone is plotted relative to the intervals between stimulation and freezing. The time constant for the recovery was 2.4 s. (D) Average number of vesicles docked per profile at various time points after the stimulus. Docked vesicles were reduced at time points before 3 s. (E) Total number of synaptic vesicles, averaged per profile. A slight reduction in the total number of synaptic vesicles was observed after stimulation. (F) Number of vesicles per profile tethered in the active and perisynaptic zones. The size of the tethered pool drops initially after stimulation. The numbers in (D) and (F) include vesicles in both the active and perisynaptic zones. The numbers in (D), (E) and (F) are normalized by the area of the profiles to account for differences in the size of varicosities (‘Materials and methods’). All the p values were calculated against no stimulation control. Bonferroni correction was applied for multiple comparisons. For detailed numbers and statistical analysis, Figure 4—source data 1. ***, ** and * indicate p-values of <0.0001, <0.001 and <0.007, respectively. n.s., ‘not significant’. N values: not-stimulated: 102 profiles; no retinal 50 ms: 51 profiles; 20 ms: 50 profiles; 50 ms: 104 profiles; 100 ms: 83 profiles; 300 ms: 89 profiles; 1 s: 111 profiles; 3 s: 91 profiles; 10 s: 121 profiles. The standard error of the mean is shown in each graph.