Influence of diurnal phase on behavioral tests of sensorimotor performance, anxiety, learning and memory in mice | Scientific Reports – Nature.com

To understand the effects of circadian phase on mice behavior, 24 mice were divided into two groups when 4week-old (active phase: mice were maintained in reverse light; inactive phase: mice were maintained in normal light). All mice (8week-old) during the active and inactive phases were subjected to a battery of behavioral tests (initial test), including: rotarod, von Frey test, open field, elevated-O-maze, lightdark box, water maze, T-maze, contextual fear conditioning, and active avoidance. To further confirm the results at a different age, all the behaviors were repeated again three months later (retest, 20week-old) (Fig.1A). Behavioral testing was started two hours after lights on/off; Zeitgeber Time (ZT) 14 for the active group and Zeitgeber Time (ZT) 2 for the inactive group. Ten minutes before the test, tested cages were relocated from animal room to the waiting hallway (red dim light, 15lx) to minimize the effect of light (Fig.1B). Tests were then conducted under the following light intensities: open field (400lx), elevated-O-maze (350lx), lightdark box (440lx), water maze (440lx), T-maze (400lx), contextual fear conditioning (inside the dark box), and active avoidance (inside the dark box).

Effects of diurnal phase on motor coordination and sensory stimulation. (A) Time course of the experimental procedure. Two groups of mice (n=12; 6 males and 6 females in each group) were housed in normal light and reverse light condition. Behavioral tests were conduct 2h after lights on/off (ZT 2 for inactive group, and ZT 14 for active group). (B) The relative position of animal rooms (one reverse light, one normal light) and behavioral testing rooms of the animal house unit. (C) Illustration of rotarod test (left), there is no difference detected in the latency to fall between active group and inactive group in neither the initial test (middle), nor the retest 3months later (right). (D) For von Frey test, mice tested in the active phase exhibit a slightly more sensitive in cutaneoussensory testing than mice tested in the inactive phase in the initial tests. Three months later, mice tested in the active phase still exhibit more sensitivity to filament stimulus compared to mice tested in inactive phase. Blue circles: male mice; Red circles: female mice.

To test the influence of the lightdark cycle on motor coordination and sensory stimuli, mice were subjected to the rotarod and von Frey tests. Our results show that there are no differences on the latency to fall in the rotarod test between inactive and active groups in the initial test (p=0.54, t(22)=0.61), nor the retest three months later (p=0.94, t(22)=0.07) (Fig.1C). Next, to assess sensitivity to sensory stimuli, all mice were subjected to the von Frey test; a mechanicalstimulationbya filament to hind paws. Mice tested in the inactive phase are slightly less sensitive in the initial test (p=0.049, F(1,44)=4.06). For the retest three months later, the results are similar to the initial test, mice tested in inactive phase are still less sensitive than tested in active phase (p=0.0085, F(1,44)=7.59) (Fig.1D). These results suggest that the performance of motor coordination and balance is not influenced by diurnal activity, but cutaneoussensitivity is more responsive in the active phase.

To better understand the effects of diurnal rhythm on anxiety, we chose three common tests associated with anxiety like behavior: the open field test, elevated-O-maze, and lightdark box. All mice were subjected to these three tests. Our results show that there were no significant differences detected between inactive and active groups in the open field test in the initial test (travel distance, p=0.25, t(22)=1.17; time in center zone, p=0.09, t(22)=1.72), nor the retest three months later (travel distance, p=0.89, t(22)=0.13; time in center zone, p=0.57, t(22)=0.56) (Fig.2A). For the elevated-O-maze test, there were no significant difference detected in the initial test (travel distance, p=0.244, t(22)=1.19; during in the open arms, p=0.11, t(22)=1.64). However, mice tested in inactive period showed higher travel distance (p=0.01, t(22)=2.62) and exhibited a trend to stay longer in the open arms (p=0.06, t(22)=1.94) in the retest (Fig.2B). Next, we subjected all the mice to the lightdark box test, in the initial test there were no differences in time spent in the light compartment: p=0.75, t(22)=0.31). Three months later, all mice were subjected to the tests again. Mice tested in the inactive period spent more time in the light compartment (p=0.036, t(22)=2.23) (Fig.2C). The results show that there were no remarkable differences observed in anxiety tests while testing mice during active or inactive periods in the first test. However, three months later upon retesting, we did observe that mice exhibited less travel distance in the elevated-O-maze and spent less time in the light compartment.

Effects of diurnal phase on anxiety tests. (A) There is no significant difference in distance traveled or time spent in the center zone of open field between active and inactive groups in the initial test, nor in the retest 3months later. (B) In the elevated-O-maze, there is no significant difference detected in distance travel or times spent in the open arms between active and inactive groups. However, 3months later, the mice from the inactive group travelled a higher distance, but with no significant difference in time spent in the open arms in the retest. (C) In the lightdark box, there is no significant difference in time spent in the light box in the initial test. In the retest 3months later, mice tested in active phase spent less time in the light compartment. Blue circles: male mice; Red circles: female mice.

It is unclear whether a time-of-day may influence the performance of spatial learning and memory. To investigate the effects of diurnal rhythm on spatial learning and memory, the water maze was used. All mice were subjected to the water maze for 4 days to examine acquisition. The results show there were no differences in escape latency (p=0.24, F(1,22)=1.45) and travel distance (p=0.85, F(1,22)=0.03) in the initial test, nor the retest three months later (escape latency, p=0.53, F(1,22)=0.39; travel distance, p=0.65, F(1,22)=0.2) (Fig.3A). Seven days after the last training, the escape platform was removed, and mice were subjected to the water maze, and spatial memory was evaluated. There were no significant differences detected between the active group and inactive group for the initial test (time spent in target zone, p=0.82, t(22)=0.22; platform crosses, p=0.06, t(22)=1.9), nor the retest three months later (time spent in target zone, p=0.21, t(22)=1.28; platform crosses, p=0.22, t(22)=1.25) (Fig.3B). These data suggest that there are no obvious differences detected in spatial learning and memory between mice tested in active or inactive period.

Effects of diurnal phase on spatial learning and memory. (A) During water maze training, there is no difference in escape latency or travel distance between active and inactive groups, neither in the retest 3months later. (B) For the memory retrieval of the water maze, there is no significant difference detected in time in target zone or platform crosses 7days after the last training, nor in the retest. Blue circles: male mice; Red circles: female mice.

Next, to better assess whether the circadian period affects other types of cognitive behavior, we conducted T-maze, contextual fear, and active avoidance tests. For T-maze alternation, the results show there were no differences in percentage of correct choices in the initial test (p=0.79), nor the second test three months later (p=0.8) (Fig.4A). For contextual fear conditioning, there were no differences detected in freezing time during the pre-shock session (p=0.07, t(22)=1.88), and there was no difference in freezing time detected in the test session 24h after the shock (p=0.15, t(22)=1.47) (Fig.4B). Three months later, all mice were subjected to the footshock chamber again. Mice tested in the active phase exhibited an increase in freezing time during the pre-shock session (p=0.007, t(22)=2.94), but no differences during the test session (p=0.21, t(22)=1.27), suggesting mice tested in the active phase show better long-term memory of the footshock chamber (Fig.4B). For active avoidance, mice tested in the active period exhibited higher escape success rate than those tested in the inactive phase (p=0.0028, t(22)=3.36), suggesting that mice learn active avoidance better during the active phase. Three months later all the mice were subjected to active avoidance testing again, and the results show there was no significant difference (p=0.25, t(22)=1.15) (Fig.4C). Clock genes have been extensively studied and show circadian expression in the brain11,12. To further confirm physiological gene expression pattern of active and inactive periods in these mice, three days after the last behavioral test, we harvested hippocampal tissue for clock gene expression four hours after lights on/off. Our results show that hippocampal tissue harvested in the active period (Zeitgeber Time 16, ZT16) exhibit higher expression of Per1 (p=0.0001, t(8)=6.74) and Per2 (p=0.0013, t(9)=4.57), and lower expression of Bmal1 (p=0.0049, t(9)=3.69) compared to tissue harvested during the inactive phase (Zeitgeber Time 4, ZT4) (Fig.4D). These results confirm the mice had differential gene expression between active and inactive phase.

Effects of diurnal phase on T-maze, contextual fear, active avoidance, expression of clock genes. (A) In the T-maze test, there is no significant difference in the percentage of correct choices observed influenced by diurnal rhythm, nor in the retest. (B) For the contextual fear conditioning, there is no difference in freezing time during the pre-shock session between active and inactive groups. Twenty-four hours after the footshock, both active and inactive groups show increased freezing time during the test session, but no significant difference was detected. Three months later, mice tested in active phase exhibited a higher percentage of freezing in the pre-shock session, but no difference in freezing time during the test session. (C) For active avoidance, mice tested in the active phase feature higher escape success rates compared to mice tested in the inactive phase. In the retest, no significant difference is detected. (D) The phase differences in expression of clock genes (Per1, Per2, and Bmal1) in the hippocampus at ZT4 (inactive group) and ZT16 (active group). Blue circles: male mice; Red circles: female mice.

In order to determine whether differences in sex could be contributing to our results, for each measure we compared a model in which phase predicted the behavior to a model that in addition contained sex as a predictor. We applied a Bonferroni 5% corrected threshold of 0.00156 to take into account the fact that we ran 32 tests (0.05/32=0.00156). Our figures show results for males and females where males are colored blue and females colored red. We found two behavioral results exceeded the 5% threshold; time spent in open arms (p=0.00017) and travel distance (p=0.0012) of elevatedO-maze at 20weeks in our analysis. However, the small sample size and consequent low power means we cannot exclude the presence of a sex difference. Table 1 provides a summary of the sex difference analyses.

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Influence of diurnal phase on behavioral tests of sensorimotor performance, anxiety, learning and memory in mice | Scientific Reports - Nature.com