Fig. 3

Axonal Ca²⁺ increase is required to sustain swellings. A Axonal Ca²⁺, Na⁺ and K⁺ levels during injury measured with the ratiometric probes FURA-2AM, SBFI and PBFI (n = 4–6, scale bar: 10 µm). B AS and axonal Ca²⁺ levels during injury. Mem-mCherry transduced neurons were incubated with Ca²⁺ sensor Fluo-4 AM. Middle: mean axonal Ca²⁺ levels during injury. Right: Mean number of swellings and Ca²⁺ levels during injury (n = 7, scale bar: 20 µm). C AS and Ca²⁺ tracking across the axonal shaft through the injury. Example of a matrix showing detection of swellings (> 150% width) and high Ca²⁺ (> 1.25x) for each segment of the axonal shaft in time. D Percentage of AS per axon that during their duration present always Ca²⁺, part time Ca²⁺ or no Ca²⁺ (left, n = 6, 10–150 swellings/n). Bubble plot shows the relationship between the duration of the AS and the presence of high Ca²⁺ (middle, n = 342 swellings). Pie chart showing the percentage of AS that are preceded (2 s window) by high Ca²⁺ (right, n = 342 swellings). E Schematic representation of the compounds used to block different sources of Ca²⁺. Axons were incubated with compounds, subjected to injury and AS formation and Ca²⁺ levels assessed (n = 4–7). F Comparison of the effects of different compounds on the mean number of AS and Ca²⁺ intensity levels during and after injury (n = 4–7). G Diagram showing the targets of each compound and the mean differences in AS numbers or Ca²⁺ intensity levels compared to the control levels. Data are mean ± SEM (*p < 0.05, **p < 0.01). Statistical comparisons were performed using student t-test (A), one-way ANOVA followed by the Tukey’s multiple comparison test (D) or Dunnett’s multiple comparison test against the control group (F). See also Additional file 7: Fig. S2