Mobility of internalized VAMP2 differs in presynapses and axons. To identify active nerve terminals, hippocampal neurons expressing VAMP2–pHluorin were incubated in low K⁺ buffer (A) followed by 5-min stimulation in high K⁺ buffer (B). (C) Fluorescence intensity of VAMP2–pHluorin as a function of time (s) in the axonal segment (Ax) and presynapse (Ps; dashed-line and continuous-line box indicated in A, respectively). The arrow indicates the time point for high K⁺ stimulation. (D) Normalized mean intensity of VAMP2–pHluorin at axons and presynapses in low K⁺ and high K⁺. n = 20 presynapses and 20 axonal segments from five neurons in individual cultures. Values in C and D were normalized to the first acquired frame. (E) Fluorescence image of internalized Atto647N nanobodies (NB) in the same hippocampal neuron shown in A and B. Corresponding presynapse and axonal segment from A are indicated with a continuous- and dashed-line box, respectively, and shown with higher magnification before (Pre) and after bleaching (Post) in F and G and I and J, respectively. The trajectories from the corresponding regions of the presynapse (H) and axonal segment (K) are shown. Bars: (A, B, and E) 5 µm; (F–K) 1 µm. The mean MSD (L; µm²), AUC (M; arbitrary units [a.u.]), mean frequency distribution of Log₁₀D (N), and the mobile-to-immobile (M/IMM) fraction (O) of SVs in presynapses and axonal segments obtained from sdTIM experiments. n = 113 presynapses (27,700 trajectories) and 80 axonal segments (21,200 trajectories) from 12 hippocampal neurons in individual cultures. Statistical analyses of independent experiments were performed using the Student’s t test (D and O) or Mann–Whitney U test (M). Error bars are ±SEM. See also Fig. S2 C.