Fig. 4

Autophagy suppression in Mushroom body causes age-typical ultrastructural presynaptic phenotypes. Electron micrograph of Kenyon cell bodies in (a) +/atg5-RNAi and (b) ok107/atg5-RNAi animals. Scale bar: 500 nm. Aggregates build up in ok107/atg5-RNAi animals (highlighted in red box). Scale bar: 100 nm. Electron micrograph of Kenyon cell bodies in (c) +/atg9-RNAi and (d) ok107/atg9-RNAi animals. Scale bar: 500 nm. Aggregates build up in ok107/atg9-RNAi animals (highlighted in red box). Scale bar: 100 nm. Electron micrograph showing presynaptic specializations at KC-to-MBON synapses in medial MB lobes of (e) +/atg9-RNAi and (f) ok107/atg9-RNAi animals (outlined as yellow). Scale bar: 100 nm. g Quantification of average T-bar size in +/atg9-RNAi and ok107/atg9-RNAi animals (n = 240 electron micrographs across 3–5 independent animals, with at least 20 T-bars per animals; ***p < 0.001; Mann–Whitney U-test). Electron micrograph showing presynaptic specializations at PN-to-KC synapses in Calyx of (h) +/atg9-RNAi and (i) ok107/atg9-RNAi animals (outlines as yellow). Scale bar: 100 nm. j Quantification of average T-bar size in +/atg9-RNAi and ok107/atg9-RNAi animals (n = 268 electron micrographs across four independent animals, with atleast 20 t-bars per animal; ***p < 0.001; Mann–Whitney U-test). STED images of BRP spots reveal ring-shaped structures (pointed by arrowheads) within the (k, l) Calyx and (n, o) antennal lobe of +/atg9-RNAi and ok107/atg9-RNAi. Scale bar: 500 nm. m Comparison of BRP ring diameter between Calyx of +/atg9-RNAi and Calyx of ok107/atg9-RNAi (total of 2931 BRP rings across 16 independent animals, with at least five rings per animals; *p < 0.05; Mann–Whitney U-test). p Comparison of BRP ring diameter between antennal lobe of +/atg9-RNAi and antennal lobe of ok107/atg9-RNAi (total of 1327 BRP rings across 15 independent animals, with at least five rings per animal; **p < 0.01; Mann–Whitney U-test). In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value