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. 2021 Mar 2;11(1):4961.
doi: 10.1038/s41598-021-84141-9.

Major oscillations in spontaneous home-cage activity in C57BL/6 mice housed under constant conditions

Karin Pernold  1 , Eric Rullman  1 , Brun Ulfhake  2
Affiliations

Major oscillations in spontaneous home-cage activity in C57BL/6 mice housed under constant conditions

Karin Pernold et al. Sci Rep. .

Abstract

The mouse is the most important mammalian model in life science research and the behavior of the mouse is a key read-out of experimental interventions and genetic manipulations. To serve this purpose a solid understanding of the mouse normal behavior is a prerequisite. Using 14-19 months of cumulative 24/7 home-cage activity recorded with a non-intrusive technique, evidence is here provided for a highly significant circannual oscillation in spontaneous activity (1-2 SD of the mean, on average 65% higher during peak of highs than lows; P = 7E-50) of male and female C57BL/6 mice held under constant conditions. The periodicity of this hitherto not recognized oscillation is in the range of 2-4 months (average estimate was 97 days across cohorts of cages). It off-sets responses to environmental stimuli and co-varies with the feeding behavior but does not significantly alter the preference for being active during the dark hours. The absence of coordination of this rhythmicity between cages with mice or seasons of the year suggest that the oscillation of physical activity is generated by a free-running intrinsic oscillator devoid of external timer. Due to the magnitude of this rhythmic variation it may be a serious confounder in experiments on mice if left unrecognized.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) is a heat map with seven columns corresponding to the days of one calendar week (day 1 to 7) with color coded activations (for conversion see scale to the right) illustrating the daily rhythmicity in activity synchronized to lights-on (4 AM, white stippled line in a,c) and lights-off (4 PM, white stippled line in a and c) in one cage (A04) with 5 male mice. (b) Details of the response to lights-on is shown for each weekday in bc (separate color coded trace for each week day, with dp0 being the day of the cage); followed in bb by the day-time resting pattern (middle graph; each day is color coded as in bc). The cage is changed once a week (day 2 in (a) and dp0 in graph) which instigates a dramatic increase in activity with a carry-over impact on the next following days (see bc). (ba) shows the response to lights off and night time activity and is the average across weeks. In (c) the whole data-set of activations across the 434 days (see Table 1) is shown for the same cage (A04). The ordinate is the same as in (a) while the abscissa is animal age in days (49 to 483 days of age). The elements of the circadian rhythm across day and night in (a) is also evident in (c) (orange arrows to aid navigation). There is an over-all decrease in activity, albeit small yet statistically highly significant (− 4% for this cage; P = 2E−10; see Supportive information Table S1). Inspections of the long term records of activations (c) revealed a marked slow oscillation of peaks (highs; indicated by filled inverted triangles) with intervening lows.
Figure 2
Figure 2
Plot of daily average activations min−1 for each of the cumulative data sets in Group 1; A04 depicted also in Fig. 1 is the top left panel. Cage ID is in each panel in the top left corner. The overall activity decreases with age as indicated by the interrupted black line (for linear regression details see Supportive information Table S1). The continuous black line of each panel represents the moving average using a window of one week (= cage-change cycle). As evident from the distribution of data points in each panel and the moving average trace, highs and lows are present in all cages but the dates for highs and lows are not synchronized across cages (for cage A04 the inverted triangles in Fig. 1c are the first five peaks). Ordinate is average daily activation min−1. Abscissa at the bottom of each column is valid for all its panels and shows the age of the animals.
Figure 3
Figure 3
Superimposed spectrograms of activations clustering at different frequencies of eight cages (color coded; key is to the right in the spectrogram) with male mice in group 1 (see Methods). Note the precise timing across cages of the behaviors triggered by environmental stimuli, while the slow oscillations having a periodicity in the range of 60–90 days are not synchronized across cages. The slow peaks are generated by only 3–6 instances in the cumulative data records while e.g. lights-off occurs 434 times in each of these data sets.
Figure 4
Figure 4
(a) Upper panel shows plot of normalized (see Methods) activations (ordinate is mean and standard deviations of the mean) for cage A04 also shown in Figs. 1c and 2, with highs indicated by inverted triangles (same as in Fig. 1c) and lows by open green circles at the bottom. On top of the slow rhythm and having a similar amplitude is the recurring response to the cage-change which occurs with the period of 1 week (for further details of the plot see Supportive information Fig. S3). The slow rhythmicity is indicated by the red curve and the interrupted blue line is the linear regression showing the over-all decrease in activations with advancing age. The data has been plotted against dates (abscissa bottom), age in days (abscissa middle) and with the seasons of the year as background (abscissa at the top, intervened grey-and-white vertical stripes). (b) Lower panel is the slow rhythmicity (continuous line with different color for each cage) of the complementary 7 cages in Group 1 along with the linear regression of the each cumulative record showing the decrease with advancing age (black line). The abscissa ordinates are the same as in a. (c) shows normalized activations (as in a) up to an age of 699 days in one cage belonging to group 2 (Cage E; activations data available in Supportive information Fig. S2I) where food consumptions (average across one week; see Methods) was measured (green line). The red curve indicates the slow oscillation in activations while the interrupted blue line is the regression of overall change in activity with advancing age. The slow oscillations in food consumption (green) and activations (red) are highly correlated r2 = 0.72. (d) Boxplots visualizing the period of slow oscillations in the three groups of cages. The observation from each cage in each of the groups have been indicated by separate color-filled circles. The average period in group 1–3 is 96, 106 and 96 days, respectively.
Figure 5
Figure 5
Boxplots of weekly average activations during weeks of highs and lows, respectively, followed by peak response in activations (above weekly average) in the responses to lights-off (middle) and lights-on (right) for the three groups of cages during weeks of highs and lows, respectively. Cages belonging to the different groups (Material and Methods) have been indicated with different colors; blue is group 1, grey is group 2 and red is group 3.
Figure 6
Figure 6
Plots of the fraction of the daily home-cage activity that occurs during the dark hours (night) over weeks with high (left panel (a); average 63%) or low (right panel (b); average 60%) level of home-cage activity, respectively. Ordinate is fraction of total daily activations occurring during the dark period and abscissa indicates age in days of the animals. The cumulative data records covers 434 days for group 1, 426 days for group 3, and 565 days for group 2. Key to cohort group at the bottom of the graphs.
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