Fig. 3: Optogenetic caspase-3 activation induces complement-dependent synaptic phagocytosis.

a Diagram illustrating optic induction of caspase activation. b Images showing cleaved caspase-3 signals at presynapses (arrows) following blue light stimulation of a single presynaptic bouton (arrowhead). The experiment was independently repeated 4 times with similar results. c Timeline showing caspase activation and synaptic complement tagging identification by immunocytochemistry (ICC). d Images showing C1qA and presynapses. The stimulated points are outside the ROI. e Proportion of C1qA area in a presynaptic bouton. LEHD refers to the caspase-9 inhibitor Z-LEHD-FMK TFA. n = 119, 63, 78, 74 presynapses from 3 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. f Timeline showing caspase activation, live imaging of microglial synaptic phagocytosis, and ICC detection of complement receptors. g A representative image of microglia‒neuron interactions. h Magnified images from g (dashed-line square). Top: live images of microglial synaptic phagocytosis. The elapsed time from the start of live imaging is shown in the upper left corner of each image. Bottom: ICC evidence of CR3 surrounding phagocytosed synapses. i Magnified images from h detailing phagocytosed presynapses and CR3. j Comparison of CR3 intensity around phagocytosed synapses (phagocytic site) and at other sites in microglial processes (nonphagocytic site). Plots from the same region are connected with a line. n = 12 presynapses from 7 independent experiments, two-sided paired t test. Source data are provided as a Source Data file.