Robust food anticipatory activity in BMAL1-deficient mice

Abstract

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Even though nocturnal rodents prefer to forage at night, daytime food anticipatory activity (FAA) is observed prior to short meals presented at a scheduled time of day. Under this restricted feeding regimen, rodents exhibit two distinct bouts of activity, a nocturnal activity rhythm that is entrained to the light-dark cycle and controlled by the master clock in the suprachiasmatic nuclei (SCN) and a daytime bout of activity that is phase-locked to mealtime. FAA also occurs during food deprivation, suggesting that a food-entrainable oscillator (FEO) keeps time in the absence of scheduled feeding. Previous studies have demonstrated that the FEO is anatomically distinct from the SCN and that FAA is observed in mice lacking some circadian genes essential for timekeeping in the SCN. In the current study, we optimized the conditions for examining FAA during restricted feeding and food deprivation in mice lacking functional BMAL1, which is critical for circadian rhythm generation in the SCN. We found that BMAL1-deficient mice displayed FAA during restricted feeding in 12hr light:12hr dark (12L:12D) and 18L:6D lighting cycles, but distinct activity during food deprivation was observed only in 18L:6D. While BMAL1-deficient mice also exhibited robust FAA during restricted feeding in constant darkness, mice were hyperactive during food deprivation so it was not clear that FAA consistently occurred at the time of previously scheduled food availability. Taken together, our findings suggest that optimization of experimental conditions such as photoperiod may be necessary to visualize FAA in genetically modified mice. Furthermore, the expression of FAA may be possible without a circadian oscillator that depends on BMAL1.

Figure 1. The timing of food deprivation affects food anticipatory activity (FAA).

Diagrams of restricted feeding protocols when mice are food deprived starting at ZT3 (A) or ZT12 (C). Wildtype C57BL/6J mice were fed ad libitum and then food availability was gradually reduced (RF) from 8 hrs/day to 6 hrs/day. Then mice were fed 4 hrs/day (from ZT6 to ZT10) for at least 9 days and then provided food ad libitum for 6 days. The time when food was available is indicated by gray shading in A and C. On the seventh day of ad libitum feeding, food was removed at either ZT3 (A, B) or ZT 12 (C, D). Food was replaced 48 hrs later. Group average activity profiles for mice that began fasting at ZT3 (B; n = 4) or ZT12 (D; n = 5) were generated by averaging the number of wheel revolutions per 10-minute bin (black line) and were plotted relative to the light-dark cycle where ZT0 is the time of lights on and ZT12 is lights off. The SEM, which represents the variability among mice, is shown in dark gray shading. The time of previously scheduled food availability is indicated by dotted lines in the activity profiles. The light-dark cycle is indicated by the white and black bars, respectively.

Figure 2. Food anticipatory activity in BMAL1-deficient mice in 12L:12D.

Representative double-plotted actograms (A, C) and group average activity profiles (B, D) of Bmal1 ^+/+ mice (A, B; n = 3) and Bmal1 ^−/− mice (C, D; n = 7) in 12L:12D. The time when food was available is indicated by light gray shading in the activity profiles and on the left half of each actogram. The light-dark cycle is indicated by the white and black bars, respectively. On the left half of each actogram, the dark black line outlines the time of darkness. The black traces in the group average activity profiles represent the mean number of wheel revolutions (in counts/minute) plotted in 10-minute bins relative to the light-dark cycle where ZT0 is lights on and ZT12 is lights off. The SEM is shown in dark gray shading in each activity profile. AL I, FD I, RF, AL II, and FD II labels of the actograms (A, C) indicate the days used to generate the mean activity profiles ad libitum I, food deprivation I, restricted feeding, ad libitum II, and food deprivation II, respectively (B, D).

Figure 3. FAA from individual wildtype and BMAL1-deficient mice during restricted feeding and food deprivation.

FAA during restricted feeding (RF; A) of individual mice in 12L:12D, 18L:6D, and DD was determined by totaling the number of wheel revolutions per minute from 4 hours before feeding time to the end of feeding time (total of 8 hours). FAA for each mouse was averaged over 9 days of restricted feeding. FAA during fasting for Bmal1 ^+/+ (B) and Bmal1 ^−/− (C) mice was defined as the total number of wheel revolutions per minute from 4 hours before feeding time to the end of previous feeding time (total of 8 hours). Wheel-running FAA for each mouse was determined separately for the first (Day 1) or second (Day 2) day of food deprivation.

Figure 4. Food anticipatory activity in BMAL1-deficient mice in 18L:6D.

Representative double-plotted actograms (A, C) and group average activity profiles (B, D) of Bmal1 ^+/+ mice (A, B; n = 10) and Bmal1 ^−/− mice (C, D; n = 10) in 18L:6D. The time when food was available is indicated by light gray shading in the activity profiles and on the left half of each actogram. The light-dark cycle is indicated by the white and black bars, respectively. On the left half of each actogram, the dark black line outlines the time of darkness. The black traces in the group average activity profiles represent the mean number of wheel revolutions (in counts/minute) plotted in 10-minute bins relative to the light-dark cycle where ZT0 is lights on and ZT12 is lights off. The SEM is shown in dark gray shading in each activity profile. AL I, FD I, RF, AL II, and FD II labels of the actograms (A, C) indicate the days used to generate the mean activity profiles ad libitum I, food deprivation I, restricted feeding, ad libitum II, and food deprivation II, respectively (B, D). Food deprivation before restricted feeding (FD 1 or Food deprivation I) was performed in only 3 Bmal1 ^+/+ mice and 4 Bmal1 ^−/− mice. All other mean activity profiles were generated from 10 mice of each genotype.

Figure 5. BMAL1-deficient mice display robust wheel-running activity prior to food availability in constant darkness (DD).

Representative double-plotted actograms of wheel-running activity of Bmal1 ^+/+ (A; n = 6) and Bmal1 ^−/− (B; n = 8) mice in DD. Group mean activity profiles for Bmal1 ^−/− mice (C) were generated by averaging the number of wheel revolutions per 10-minute bin (black line) and were plotted relative to local time (where time 0 was the time of lights on and time 12 was lights off in the light-dark cycle prior to releasing the mice into DD). The SEM, which represents the variability among mice, is shown in dark gray shading. The time when food was available is indicated by light gray shading in the activity profiles and on the left half of each actogram. AL I, FD I, RF, AL II, and FD II labels of the actograms (B) indicate the days used to generate the activity profiles ad libitum I, food deprivation I, restricted feeding, ad libitum II, and food deprivation II, respectively (C).