Figure 1.
PAWW stress desrupted circadian locomotor activities.
Representative double-plot actograms for mice under continuous mild stress (A) and control mice (B) under L:D. The light/dark cycles are shown as white/black bars, respectively, on each actogram. The numbers on the left indicate the days of exposure to PAWW stress and the arrows on the right indicate periods of PAWW stress. The circadian rhythms of locomotor activity in pre-stress mice and in control mice were well organized and became obscure after they were transferred to the cages in which PAWW stress was imposed. (C) Daily total locomotor activity was gradually, but time-dependently, reduced in the mice exposed to PAWW stress (closed circles with a solid line) compared with the non-stressed mice (open circles with a dashed line). Daily total activity was averaged for 7 days within the pre-stress period and after 1, 2, and 3 weeks of PAWW stress. Relative daily total activity is expressed as relative to the averaged activity of the pre-stress period. Values are shown as means ± SEM (n = 12). RM two-way ANOVA revealed a significant main effect of stress on locomotor activity (*P<0.01). (D) Wheel running activity in mice before (pre-stress, open circles) and during 1 week of PAWW stress (closed circles) plotted over 24 h. Wheel activity in 1-h bins was averaged for 6 days before or from days 2–7 of exposure to PAWW stress, and the mean values from individual mice were collated to yield a single 24-h profile. Values are shown as means ± SEM (n = 6). Distribution of diurnal locomotor activity was changed by PAWW stress. RM two-way ANOVA revealed no significant main effect of stress on locomotor activity, while the post hoc Tukey's test indicated significant differences (*P<0.05, **P<0.01). (E) Daytime activity relative to total locomotor activity. Open and closed circles, counts from the non-stressed and continuously stressed mice, respectively. Approximately 10% of the total daily activity occurs during the daytime in the non-stressed mice. Values are shown as means ± SEM (n = 12). Daytime activity was enhanced by PAWW stress by up to 30% (*P<0.001, RM two-way ANOVA).
Figure 2.
PAWW stress affected on body weight, and food intake.
Effect of continuous PAWW stress on body weight (A), food intake (B), and levels of plasma leptin (C). All mice were weighed at ZT3. Values are shown as means ± SEM (n = 12 per group). The stressed mice weighed significantly less than the control mice after 1 week of exposure to PAWW stress (RM two-way ANOVA, *P<0.05) and remained significantly lighter over the next 3 weeks. The food intake of the stressed and control mice (mean ± SEM) differed significantly within 3 weeks (*P<0.01, RM two-way ANOVA). Diurnal plasma leptin levels in mice stressed for 1 week (continuous stress) and control mice (n = 6–8 at each time point). The black and white bars indicate light/dark conditions, respectively. Univariate two-way ANOVA revealed no significant main effect of stress on plasma leptin levels, while the levels were significantly lower during the light period in the stressed mice (**P<0.05, Student's t-test).
Figure 3.
The effects of PAWW stress on plasma level of corticosterone, melatonin and catecholamines.
Diurnal plasma corticosterone (A) and melatonin (B) levels (means ± SEM) in mice under continuous stress (closed circles, solid line) and controls (open circles, dashed line; n = 7 at ZT2 and 14, n = 3 at ZT8 and 20). The white/black bars indicate light/dark conditions, respectively. Univariate two-way ANOVA revealed that the effect of PAWW stress was not significant. Corticosterone levels were significantly increased at ZT2 and decreased at ZT14 (*P<0.05, Student's t-test). The plasma epinephrine (C) and norepinephrine (D) levels were determined in the stressed (solid bar) and control (open bar) mice at ZT2. Data are shown as means ± SEM (n = 6 per group). **P = 0.031, **P = 0.072 for averaged levels in the control mice vs. mice stressed for 7 days (Student's t-test).
Figure 4.
PAWW stress changed diurnal rhythms of core BT.
Diurnal rhythms of core BT of mice on the day before (open circles, dotted line) and at 7 days of PAWW stress (closed circles, solid line). The core BT in 5-min bins was averaged hourly. Data are shown as means ± SEM (n = 6). Black bar, dark period of the day. After 1 week of PAWW stress, the diurnal rhythm of BT was similar to that during the pre-stress period, whereas maintenance of a higher BT during the dark period was disrupted. Averaged BT on day 7 of PAWW stress compared with that at 1 day before exposure to PAWW stress. RM two-way ANOVA indicated a significant interaction between time × stress (F(23,230) = 2.22, P = 0.002), and a significant effect of stress was observed during the nighttime (**P<0.01, *P<0.05, post hoc Tukey's analysis).
Figure 5.
The effect of PAWW stress on diurnal rhythms of sleep.
Hourly progression of time spent by mice in wakefulness (A) or in NREM (B) and REM (C) sleep over 24 h at 1 day before (open circles, dashed line) and on day 7 (closed circles, solid line) of PAWW stress (means ± SEM, n = 8). Black and white bars, light/dark conditions, respectively. Continuous stress reduced the duration of both REM and NREM sleep, especially during the first 6 h after lights on, and during wakefulness at 4 h after lights off. RM two-way ANOVA revealed a significant interaction between time × stress (wakefulness; F(23,230) = 2.22, P = 0.002, NREM; F(23,230) = 2.02, P = 0.005, REM; F(23, 230) = 1.93, P = 0.008). A significant simple main effect of stress treatment was observed (**P<0.01, *P<0.05). Wake/REM/NREM ratio at ZT0–5 (L1), ZT6–11 (L2), ZT12–17 (D1), and ZT18–23 (D2) under pre- and PAWW stress conditions (D). Sleep time distribution––daytime rest and nighttime activity––was rhythmic on the pre-stress days. Continuous stress caused wake epochs to appear frequently during the daytime and significantly increased the duration of NREM sleep during the nighttime, as compared with control mice. G-square test between pre- and post-stress showed significant changes (P<0.01) for the two phases of L1 and D1.
Figure 6.
The effect of PAWW stress on circadian clock.
Continuous stress similarly affects circadian rhythm behavior under constant darkness (D:D) and under light/dark (L:D) conditions (A). Light/dark cycles are shown as white/black bars on the actogram. The numbers and the arrow on the left indicate days of exposure to PAWW stress and periods of PAWW stress, respectively. The arrowhead on the right indicates the transition from L:D to D:D. Circadian oscillation of clock genes, Per2 (B) and Bmal1 (C) in the liver (left) and muscle (right) of the stressed (closed circles) and control mice (open circles). The black and white bars indicate light/dark conditions respectively. Univariate two-way ANOVA did not reveal a significant difference. The asterisks indicate a significant difference between the control and stress conditions by post hoc Tukey's analysis. Relative expression of each gene in the tissues at each time point in 4 mice (means ± SEM).
Table 1.
Comparison of PAWW stress with other conventional stress systems, chronic mild stress, social defeat stress and total sleep deprivation.