(A) Live confocal imaging of a calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles, and individual SV tracking sorted according to trajectory lengths (Blue <2 µm, Green 2–4 µm and Red >4 µm). (B) Speed variation profiles during short (Blue), medium (Green) or long (Red) trajectories. (C) Displacement modalities. Representative displacement curves showing different modes of movements. Each representative curve for diffusive motion and active motion (facilitated and impeded) was calculated and plotted from an average of 12 different curves extracted from displacement plots similar to Figure 2D. (D) Confocal imaging of a giant calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles. White circles and black lines represent swelling and finger areas, respectively. (E) Comparison of the diffusion coefficient of SVs in swellings or fingers in various conditions (Control, 30 µM nocodazole, 2.5 µM OA, 65 mM KCl, 500 mM sucrose or 1 Hz electrical stimulation, n = 6 terminals for each condition). (F) 3D tracking of SVs labeled with Q655-Syt2 in an individual swelling. Left panel: SV trajectories (time color-coded), right panel: Displacement vectors of SV trajectories. (G) Schematic diagram showing the proportion of SVs with displacement vectors going toward the synaptic cleft (Green), away from the synaptic cleft (Red), or moving laterally (Blue) in an individual calyceal swelling. (H) Comparison of SV mobilities between 2D and 3D tracking. Trajectory length analysis (Left panel) and diffusion coefficient (Right panel) in single 2D confocal section (Blue, n = 3) and 3D confocal z-stack (Red, n = 3). Two-tailed unpaired t-test (*p<0.05; ns, not significant).