Both stress-induced axonal beading and Ca ²⁺ elevation are spatially restricted. (A) Representative image of non-stressed axonal segments in the soma chamber (Soma) and stressed segments within the injury chamber (Flux) of the same neuron before and after 50 μl/min flux for 180 s. Bracketed regions in the soma (1#) and bracketed regions in flux (2#) are magnified right. Arrows indicate the direction of injecting flux. The two white arrowheads denote a representative region illustrating the flux-induced beading process. Bar = 20 μm. (B) Paired comparison of the number of beads in non-stressed (soma) and stressed segments (fluxed) before and after the flux (N = 6). (C) Axons expressing GCaMP-6f were stressed with 50 μl/min flux for 180 s. [Ca²⁺ ]_axon intensity was color-coded in the non-stressed (Soma) and the stressed (Flux) axonal segment. Arrows indicate the direction of injecting flux. Bar = 20 μm. (D) Paired comparison of the [Ca²⁺ ]_axon in non-stressed (soma) and stressed segments (fluxed) (N = 6). (E) Spatial and temporal changes in [Ca²⁺]_axon before (t1), during (t2), and after (t3) the 180 s of 50 µl/min flux, marked by the red line. The bracketed region is amplified at the bottom, with arrowheads indicating axon beads. Bar = 10 μm. (F) Kymographs of magnified axons in E; x-axis bar = 5 μm, y-axis bar = 20 s. (G) Quantification of the total [Ca²⁺]_axon fluctuation induced by the 50 μl/min flux. The flux span is indicated with shades, with arrows indicating t1, t2, and t3. (H) Quantification of [Ca²⁺]_axon at t1, t2, and t3 in 50 or 200 μl/min fluxed groups. N = 14, 17 (the 200 μl/min subsets of data were also used in Fig. 6 E of Pan et al. [2022]). (I) Time-lapse images showing the kinetics of the [Ca²⁺]_axon along the beading axons, with the beading region indicated with an asterisk and the [Ca²⁺]_axon spreading indicated with an arrow. The kymograph is shown on the right, with a dashed line indicating the spreading of the [Ca²⁺]_axon. Bar = 20 μm (top and bottom left); x-axis bar = 20 μm and y-axis bar = 200 s (bottom right). (J) Quantification of the flux-induced [Ca²⁺]_axon fluctuation in beading and between regions (N = 10). (K) Quantification of the heterogeneity of [Ca²⁺]_axon (N = 115, 150, 110). (L) Comparison of the spreading speeds of the [Ca²⁺]_axon waves along the same axon before and after the 50 μl/min flux, which induced axon beading. Ca²⁺ intensity is color-coded. Arrows indicate the frontier of the wave. Bar = 5 μm. (M) Quantification of the spreading speeds of L (N = 8, 17). (N) Schematic cartoon demonstrating the hindrance of [Ca²⁺]_axon propagation by beading axons, with orange arrows indicating the spreading direction of retrograde [Ca²⁺]_axon. Data represent mean ± SEM; in B and D, paired two-tailed unpaired t test; in H, K, and M, unpaired two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., non-significant.