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Fig. 6 Mitochondrial metabolism is more active in darkness due to altered SDH activity. (A) Schematic of SDH in the tricarboxylic acid (TCA) cycle and electron transport chain, including COX also assayed in B. White squares represent 12C carbons; green squares,13C carbons for labeling experiments (D–F). (B) Light microscopy images of histochemical staining for SDH (blue) and COX (brown) activities. Shown are frozen sections from albino zebrafish at ZT6 and ZT14 (23:00) from LD and DD groups. Negative controls lacked substrate or contained an inhibitor. Arrowheads indicate corresponding cone subtype and rod (black) mitochondrial clusters. (Scale bars, 10 µm.) (C) Quantification of mean SDH and COX stain intensities in single clusters from LD (open circles) or DD (dark squares) groups; data normalized to ZT0. ‡‡P < 0.001. ***P < 0.0001. (D) Mean incorporation of 13C label from U-13C succinate into fumarate and malate. Whole retinas from light- or dark-adapted zebrafish were incubated in U-13C succinate in light or dark; 13C incorporation was determined with GC/MS. (E) Initial rates of formation of fumarate and malate from U-13C succinate in light and dark from the first 5 min of incubation. In D and E, ‡P < 0.05; ‡‡P < 0.001. (F) Amounts of U-13C-labeled succinate, fumarate, and malate in media after 60-min light or dark incubation with retinas in U-13C succinate. SI Appendix, Table S2 lists P values and Ns from all groups. (G) Schematic of steady-state metabolites inside and outside of retinas after 60-min incubation with labeled succinate. Pie charts denote relative amounts of metabolites in light (unfilled areas) and dark (filled areas).