Disease-associated KCNMA1 variants decrease circadian clock robustness in channelopathy mouse models

Figures

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  Figure 1.. Evaluation of circadian rhythm from wheel running behavior. (A) Representative double-plotted running wheel actograms for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice. White and black bars denote 12:12-h LD cycle for first 10 d and the grey shaded area denotes DD (last 15 d). (B) χ2 periodograms for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice, generated from DD activity data in A. The dotted line denotes a 0.01 confidence interval. (C–F) Average circadian period (C; τ; WT versus Kcnma1D434G/D434G, P = 0.0068, t test, n = 11, 11, respectively), (D) χ2 amplitude (WT versus Kcnma1N999S/WT, P = 0.0163, t test, n = 11, 14, respectively; WT versus Kcnma1D434G/D434G, P = 0.0192, MWU, n = 11, 11, respectively), (E) the relative power of the dominant circadian component of a Fourier transform (WT versus Kcnma1N999S/WT, P = 0.0073, t test; WT versus Kcnma1D434G/D434G, P = 0.0352, MWU, n = 11, 11, respectively), and (F) average active period (α) of DD activity data from A for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice as indicated. Data is presented as individual data points with median and interquartile range.

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  Figure 2.. Analysis of locomotor activity bouts. (A) Activity profiles of average counts per minute for Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice and WT littermates as indicated. Grey shaded area denotes times of lights off. (B–E) Average number of counts during consolidated subjective night activity (B; α counts; WT versus Kcnma1N999S/WT, P < 0.0001, t test, n = 11, 14, respectively; WT versus Kcnma1D434G/D434G, P = 0.0002, t test, n = 11, 11, respectively), (C) average number of bouts per day (WT versus Kcnma1H444Q/H444Q, P = 0.0292, t test, n = 9, 9, respectively), (D) average duration of bouts, and (E) average number of counts per bout (WT versus Kcnma1N999S/WT, P = 0.0090, t test, n = 11, 14, respectively; WT versus Kcnma1D434G/D434G, P = 0.0105, t test, n = 11, 11, respectively), for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice housed in DD, as indicated. Data are presented as individual data points with median and interquartile range.

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  Figure 3.. Analysis of responses to phase-shifting light stimuli. (A) Representative double-plotted running wheel actograms for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice. A 30-min light pulse was delivered 4 h into the subjective night (yellow circle indicates the day the light pulse was administered). (B) Number of hours of delay for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice are presented as individual data points with median and interquartile range (WT versus Kcnma1N999S/WT, P = 0.0398, MWU, n = 11, 12, respectively; WT versus Kcnma1D434G/D434G, P = 0.0409, t test, n = 11, 11, respectively). (C) Representative double-plotted running wheel actograms for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice. Mice were entrained to a LD cycle until 7 d of stable re-entrainment (indicated by bars at the top). The LD cycle was then advanced 6 h as indicated by the bar at the bottom. Yellow shaded area denotes the number of days for each mouse to re-entrain to the new LD cycle. (D) Number of days to stable re-entrainment for WT, Kcnma1N999S/WT, Kcnma1D434G/D434G, and Kcnma1H444Q/H444Q mice are presented as individual data points with…

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  Figure 4.. Evaluation of action potential firing in Kcnma1N999S/WT SCN neurons. (A) Relative proportion of WT and Kcnma1N999S/WT neurons that were silent and active. (B) Firing rate of all neurons was recorded, including silent cells. Data presented as individual values with mean ± SEM. (C) Baseline membrane potential for silent and active neurons. Data presented as individual values with mean ± SEM (WT silent versus active neurons, P = 0.0010, t test, n = 6 neurons, 17 neurons from 12 slices, respectively; Kcnma1N999S/WT silent versus active neurons, P = 0.0003, t test, n = 9, 23 neurons from 15 slices, respectively). (D and E) Representative spontaneous action potentials from WT and Kcnma1N999S/WT silent and active cells, as indicated, dotted line is −60 mV. (F) Spontaneous action potential frequency distribution. The histogram shows the percent of total events at each instantaneous frequency for WT (black) and Kcnma1N999S/WT (orange).