Fig 1.
Schematic illustration of experimental design time flow.
Animals (n = 42) were acclimatized to the laboratory conditions (7 days). Adaptation to the treadmill was performed in sessions of 1h (5 min ON and 5 min OFF working regime, treadmill belt speed ON = 0.02 m/s, OFF = 0m/s) during the next 3 days. Afterwards, the sleep fragmentation protocol has been applied and animals were divided into three groups (n = 14 in each group) and subjected to one of the three protocols in the treadmill during 6h of the light period: 1) sleep fragmentation (30s OFF: 90 s ON regime, 30 sleep interruptions per hour, SF group), 2) activity control group (10min ON: 30 min OFF regime, 1.5 sleep interruptions per hour, AC group), 3) treadmill control group (constantly OFF regime, 0 sleep interruptions per hour, TC group). Upon completion of sleep fragmentation protocols one cohort of animals (n = 8 from each group) has been subjected to in vivo ethological test of anxiety-like behavior (open filed, light—dart and elevated plus maze test), while another cohort of animals (n = 6 from each group) was sacrificed for blood sampling and further in vitro ELISA determination of hormonal levels (testosterone, progesterone, estradiol, corticosterone). For further details see the Materials and Methods section.
Fig 2.
Representative traces of locomotor activity of animals in the open field test in the (A) treadmill control, (B) activity control and (C) sleep fragmentation groups. Number of rearings (D), thigmotaxis index (E) and time spent in the central area (F) in the open field test registered in animals upon sleep fragmentation method. The number of rearings was calculated as the number of times rat has propped on the hind legs. The index of thigmotaxis was defined as a ratio between the distance of ambulatory movement a rat made in the peripheral areas and the total distance of ambulatory movements in the open field test. Values are mean ± SEM. The statistical difference between groups was estimated by using One-Way ANOVA with Fisher’s LSD post hoc test (*p < 0.05, **p < 0.01 vs. TC; #p < 0.05, ##p < 0.01 vs. AC). For further details see the caption to Fig 1.
Fig 3.
The number of transitions between the opened and closed arm (A) and the time spent in the open arm (B) in elevated plus maze test (EPM) observed in animals from treadmill control (TC), activity control (AC) and sleep fragmentation (SF) group upon sleep fragmentation method. Values are mean ± SEM. The statistical difference between the groups was estimated by using One-Way ANOVA with Fisher’s LSD post hoc test (**p < 0.01, ***p = 0.001 vs. TC, ##p < 0.01, ###p = 0.001 vs. AC). For further details see the caption to Fig 1.
Fig 4.
The time in the light compartment (A) and the number of transitions between the light and the dark compartment (B) in light/dark test observed in experimental and control groups. The statistical difference between groups in the time in the light compartment was estimated by using One-Way ANOVA with Fisher’s LSD post hoc test and in the number of transitions by Kruskall Wallis ANOVA and Mann Whitney U test (*p < 0.05 vs. TC, #p < 0.05 vs. AC). For further details see caption to Fig 1.
Fig 5.
Serum levels of progesterone (A), testosterone (B), estradiol (C) and corticosterone (D) observed in animals from treadmill control (TC), activity control (AC) and sleep fragmentation (SF) group immediately after the experiment. Hormone serum concentrations were determined in separate cohort of animals immediately upon sleep fragmentation method (n = 6 per group) using ELISA method. Values are mean ± SEM. The statistical difference between groups was estimated by using One-Way ANOVA with Fisher’s LSD post hoc test (*p < 0.05 vs. TC, #p < 0.05 vs. AC). For further details see caption to Fig 1.