Fig. 1: Increased neuronal activity triggers local caspase-3 activation at presynapses.

a Representative images of cultured neurons expressing synaptophysin-mCherry (left) 6 hours after drug application. The middle and right panels are magnified images of the dashed-line square in the left panel. Arrowheads indicate caspase-3 immunosignals in presynapses. b Cleaved caspase-3 volume in a presynaptic bouton 6 hours after drug application. DEVD refers to the caspase-3 inhibitor Z-DEVD-FMK. Mean ± SD, n = 121, 188, 124 presynapses in 20 regions from 2 independent experiments, Dunn’s test after one-way ANOVA on ranks. Box plots are defined as follows: the central line within the box represents the median (50th percentile), while the bounds of the box represent the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. All individual data points are plotted. c Representative images of cleaved caspase-3 in presynapses (arrowheads) and βIII-tubulin-immunostained axons (arrows) 6 hours after drug application. d Cleaved caspase-3 signals in H2B-GFP-positive soma, βIII-tubulin-immunostained axons and synaptophysin-mCherry-positive presynapses 6 hours after drug application. Plots from the same region are connected with a line. n = 20 regions from 2 independent experiments, Tukey’s test after two-way repeated-measures ANOVA. e Diagram illustrating the real-time caspase-3 activity assay using mSCAT3. f Images displaying cleaved caspase-3 detected by immunocytochemistry (left, center) and the mECFP/mVenus ratio for synaptophysin-mSCAT3 (right). Arrows indicate cleaved caspase-3-positive presynapses. The experiment was independently repeated 2 times with similar results. g Frequency distribution of maximum mECFP/mVenus ratios in presynapses, based on the presence (magenta bars) or absence (gray bars) of cleaved caspase-3 signals. The inset image shows the mECFP/mVenus ratio in a single presynapse, and arrows point to the maximum and minimum ratios. n = 143 (cleaved caspase-3 positive), 1576 (cleaved caspase-3 negative) presynapses in 3 regions from 2 independent experiments. h, j Images showing the mECFP/mVenus ratio for synaptophysin-mSCAT3 before and after drug application. Arrows indicate presynapses whose mECFP/mVenus ratio is 1 or higher. i, k Proportion of presynapses whose mECFP/mVenus ratio is 1 or higher. n = 20 regions from 3 independent experiments, two-sided Wilcoxon signed-rank test (i), two-sided paired t test (k). Source data are provided as a Source Data file.