Fig. 3.

Effect of hypercholesterolemia on behavioral rhythms in constant darkness.

(A, B, and C) Continuous monitoring of locomotor activity of isolated mice was performed in constant darkness. Representative raw activity data of wild-type, wild-type (+ HFD) and Ldlr −/− (+ HFD) are shown as a double plot. After entrainment to regular light and dark cycles, exposure to the HFD was started on day 11, namely the last day before constant darkness. Yellow shadows indicate light periods. Red square brackets represent active phase duration, whose prolongation was visually observed. (D, E, and F) To calculate average activity over several days by correcting phase difference among days, the time of activity onsets was defined as “time 0”, and then average 6-min bin activity was calculated for several-day periods on days 2–10 (before exposure to the HFD), 16–30 (shortly after exposure to the HFD), 56–70, and 96–110. To compare the duration of the active phase among experimental conditions, the peak time of activity concentrated just before the offset was estimated by non-linear Lorentzian curve fitting. Left dots represent hours from onset to the peak time estimated by Lorentzian curve fitting in individual mice. Right dots indicate the average ± SD (n = 6 or 8 mice). A t-test (vs. days 2–10) was performed, and significant prolongation of the active phase duration against days 2–10 (before exposure to the HFD) is indicated with asterisks. (G, H and I) The free-running period and amplitude of circadian behavioral rhythms were calculated with a chi-square periodogram on days 2–10 and days 56–70. Graphs indicate a representative of six or eight mice. Red and black asterisks indicate 12-h and 24-h rhythms, respectively. The numbers shown under the periodograms indicate the average ± SE of 24-h/12-h period and amplitude (n = 6 or 8 mice). (J) To compare the difference in active phase duration between experimental conditions, the average hour from onset to the peak time estimated by Lorentzian fitting on days 2–10, as calculated in D, E and F, was set to 0. A t-test (vs. wild-type) was performed, and a statistically significant difference between wild-type and wild-type (+ HFD) mice, and between wild-type and Ldlr −/− (+ HFD) mice, is indicated as asterisks and pound signs, respectively. (K) To examine statistically significant differences in the magnitude of 12-h rhythmicity among experimental conditions, the ratio of 12-h amplitude to 24-h amplitude was calculated in both the single and group housing condition. Data indicate the average ± SE (single: n = 6 or 8, group: n = 4 or 8). A t-test was performed against wild-type mice, and asterisks indicate statistically significant differences.