Extended Data Fig. 5: Sleep history alters the morphology of dFBN mitochondria.
a, Mean volumes (left y-axes) and volume ratios (right y-axis) of dFBN and PN mitochondria determined by volume electron microscopy (EM), optical photon reassignment microscopy (OPRM), and confocal laser-scanning microscopy (CLSM). b, Correlation between dFBN and PN mitochondrial volume estimates obtained by EM, OPRM, and CLSM (residual s.d. = 0.0041). EM data are from the hemibrain connectome39; OPRM and CLSM measurements of mitochondrial volumes in flies expressing mito-GFP are re-plotted from Fig. 3b and Extended Data Fig. 6b, and from f and Extended Data Fig. 6d, respectively. c, d, Sleep deprivation via thermogenetic activation of arousing dopaminergic neurons (c) causes mitochondrial fragmentation detected by OPRM (d, number of mitochondria: P = 0.1812, volume: P = 0.0010, sphericity: P = 0.0192, branch length: P = 0.2013, two-sided t-test). Experimental and control flies (n = 30 and 33, respectively) were reared and maintained at 21 °C and shifted to 29 ˚C between zeitgeber times 12 and 24 on day 2. The arrowhead marks the time point when 11 experimental and 13 control flies were removed and dissected for mitochondrial morphometry. e, f, Maximum intensity projections (e) and morphometric parameters (f) of automatically detected mitochondria in CLSM image stacks of dFBN dendrites in rested flies, sleep-deprived flies, flies allowed to recover for 24 h after sleep deprivation, and rested and sleep-deprived flies co-expressing R23E10-GAL4-driven AOX or TrpA1, which was activated at 29 °C. Sleep history-dependent changes in mitochondrial volume (P = 0.0025, Holm-Šídák test after ANOVA), sphericity (P = 0.0001, Holm-Šídák test after ANOVA), and branch length (P = 0.0414, Holm-Šídák test after ANOVA) are occluded by the co-expression of AOX (P ≥ 0.1515, two-sided t- or Mann-Whitney test) or the simultaneous activation of TrpA1 (P ≥ 0.2002, two-sided t- or Mann-Whitney test) and overcorrected after recovery sleep (all parameters: P < 0.0001, Holm-Šídák test after ANOVA). The number of mitochondria is unchanged by sleep deprivation (P > 0.9999) but elevated after recovery sleep (P < 0.0001, Dunn’s test after Kruskal-Wallis ANOVA). Two data points exceeding the y-axis limits are plotted as triangles at the top of the graphs; mean and s.e.m. are based on the actual values. g, Volumetric renderings of automatically detected mitochondria in OPRM image stacks of dFBN dendrites in rested and sleep-deprived flies co-expressing R23E10-GAL4-driven AOX or TrpA1, which was activated at 29 °C. h, Sleep in flies expressing R23E10-GAL4-driven split-GFP-based contact site sensors (SPLICS) or fluorescent fusion proteins located in the outer mitochondrial (OMM), endoplasmic reticulum (Sec61β), or plasma membrane (CD4) (P = 0.0648, ANOVA). Data are means ± s.e.m. or ratios of means ± error-propagated s.e.m. (a, light gray); n, number of cells (a, b, EM), dendritic fields (a, b, OPRM and CLSM, d, f), or flies (c, h); asterisks, significant differences (P < 0.05) in planned pairwise comparisons. Scale bars, 10 µm (e,g). For statistical details see Supplementary Table 2.
