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Figure 5 Mutation of the Na+/K+ pump alpha subunit atp1a3a reduces sleep at night. (A, B) Sleep and waking activity traces (± SEM) of wild-type larvae following exposure to 1 µM ouabain. Arrows indicate time the drug was added. (C, D) At night, sleep is significantly reduced and waking activity is significantly increased after ouabain exposure. Student’s t-test, one-tailed. (E) Alignments of Na+/K+ pump alpha subunits around the ouabain binding sites. Red indicates residues that are critical for higher sensitivity to ouabain, both of which are present in mouse Atp1a3 but not Atp1a1. (F) In situ hybridisation of atp1a3a at 24 hpf (whole animal) and 5 dpf brain (ventral view). Anterior is to the left. Scale bar = 0.5 mm (24 hpf); 0.1 mm (5 dpf). A, anterior; P, posterior; D, dorsal; V, ventral (G) CRISPR-Cas9 targeting of the atp1a3a resulted in a 19 bp deletion that eliminates the start codon (blue) and splice junction. Guide RNA target sequence and PAM sequence are shown as black bars. Sequence that is deleted in the mutant is indicated with a red bar. (H, I) Representative behavioural experiment showing atp1a3aΔ19/Δ19 fish are hyperactive throughout the day-night cycle and have decreased sleep at night. Mean ± SEM are shown. (J) atp1a3aΔ19/ Δ19 larvae sleep less at night due to shorter sleep bouts. Plotted are the genotype effect sizes (95% confidence interval) on each parameter relative to wild type. Shading indicates day (white) and night (grey). p-Values are assigned by an F-test on the fixed effects coefficients from the linear mixed effects model. *p<0.05, **p<0.01, ***p<0.001, ns p>0.05.