Restimulation increases the mobility of internalized VAMP2 in presynapses but not in axons. (A) Hippocampal neurons expressing VAMP2–pHluorin were subjected to sdTIM. After the first 5-min high K⁺ stimulation (B) and 10-min chase, the neurons were subjected to a second stimulation (restimulation) with high K⁺ (C), and the fluorescence of VAMP2–pHluorin was recorded. The boxed presynapse in A is shown at higher magnification before and after the first (B) and second (C) stimulation. The corresponding fluorescence intensity of VAMP2–pHluorin during the first and second high K⁺ stimulation is shown in B and C, respectively. The intensities were normalized to the first acquired frame. Bars: (A) 5 µm; (B and C) 2 µm. (D–L) The SV mobility in resting and restimulated neurons subjected to sdTIM was compared in neuronal, presynaptic, and axonal regions of interest. The mean MSD (D, G, and J; µm²), AUC (E, H, and K; arbitrary units [a.u.]), and mean frequency distribution of Log₁₀D and the M/IMM fraction (F, I, and L) after restimulation from indicated regions. n_internalized = 17 hippocampal neurons (45,200 trajectories) and n_restimulated = 23 neurons (33,000 trajectories) (D–F), n_internalized = 74 presynapses from 17 hippocampal neurons (13,600 trajectories) and n_restimulated = 113 presynapses from 23 hippocampal neurons (7,800 trajectories) (G–I), and n_internalized = 108 axonal segments from 17 hippocampal neurons (23,600 trajectories) and n_restimulated = 148 axonal segments from 23 hippocampal neurons (21,500 trajectories) (J–L), all in individual cultures. Statistical analyses of independent experiments were performed using the Student’s t test (F, I, and L, insets) or Mann–Whitney U test (E, H, and K). Error bars are ±SEM. See also Fig. S2 (D and E).