Figure 4.

Ca²⁺ Enters through Nanoruptures of the Axonal Plasma Membrane

(A) Confocal projection of Thy1-CerTN-L15 axons (top) in healthy (left, control) and EAE (right) spinal cord after subdural injection of Texas-red-labeled dextran 3 kDa (bottom; red arrowhead indicates a dye-positive axon, white arrowhead a dye-negative axon).

(B) Percentage of dye-positive axons after application of fluorescent vital dyes with increasing sizes in control and EAE spinal cord (n = 5–9 animals per group, 78–205 axons per animal, means ± SEM, one-way ANOVA and Dunnett’s multiple comparison test).

(C) In vivo multiphoton projection of spinal cord axons in EAE Thy1-CerTN-L15 mice after subdural injection of Alexa-Fluor594-labeled cadaverine. Red arrowhead indicates dye uptake (left) in a [Ca²⁺]_cyt -high axon (right).

(D) Left: [Ca²⁺]_cyt levels of single dye-negative (gray dots) and dye-positive axons (red dots) in healthy and acute EAE mice, plotted as YFP/CFP channel ratios and normalized to the mean of control axons. Top: percentage of axons with [Ca²⁺]_cyt ≥3 SD above control mean, shown as mean ± SEM (tested per animal in n = 7 control and n = 12 EAE mice, Mann-Whitney U test), Right: percentage dye-positive axons for [Ca²⁺]_cyt-high (yellow area) and [Ca²⁺]_cyt-low (magenta area) axons in the same control and EAE animals (means ± SEM, Mann-Whitney U test).

Scale bars in (A) and (C), 10 μm. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. See also Figures S4 and S5.