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Figure 6 dmist mutants have altered sodium homeostasis. (A) Brain sodium levels are significantly elevated after exposure to pentylenetetrazol (PTZ) in both atp1a3aΔ19/Δ19 (two independent experiments) and dmisti8/i8 (four independent experiments) fish relative to wild type and heterozygous mutant siblings, as measured by fluorescence intensity of Sodium Green, normalised to the sample mean intensity. Crosses show mean ± SEM. n indicates the number of animals. Below are example images of brains stained with Sodium Green. *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post hoc test. (B) Under baseline conditions, brain sodium levels are significantly elevated in dmisti8/i8 fish at night but not during the day, as measured by fluorescence intensity with Sodium Green. Crosses show mean ± SEM. *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post hoc test. (C) dmisti8/i8 larvae have increased rebound sleep compared to wild-type siblings following exposure to 5 mM PTZ. Representative sleep traces of dmist+/+ (no drug, water vehicle controls in black; PTZ exposed in blue) and dmisti8/i8 (no drug in purple; PTZ exposed in red) following 1 hr exposure to 5 mM PTZ (black bar) in the morning. Data are mean ± SEM. dmisti8/+ animals are not plotted for clarity but are included in panel (D). (D) Rebound sleep after exposure to 5 mM PTZ, calculated from the experiment in (C). Each dot represents a single fish, grey lines show mean ± SEM. (E) Effect size of change in sleep after 1 hr treatment with 5 mM PTZ (and washout) compared to vehicle-treated controls (error bars show 95% confidence intervals). *p<0.05, one-way ANOVA, Tukey’s post hoc test. (F) Effect sizes (and 95% confidence interval) relative to wild types (dotted line) on sleep at night in larvae from dmist+/-; atp1a3a+/- in-crosses from three independent experiments. p-Values are assigned by an F-test on the fixed effects coefficients from the linear mixed effects model relative to dmist+/+; atp1a3a+/+ animals. For all sleep-wake parameters, see Figure 6—figure supplement 1. *p<0.05, **p<0.01, ***p<0.0001, ns p>0.05..