Circadian Clocks for All Meal-Times: Anticipation of 2 Daily Meals in Rats

Anticipation of a daily meal in rats has been conceptualized as a rest-activity rhythm driven by a food-entrained circadian oscillator separate from the pacemaker generating light-dark (LD) entrained rhythms. Rats can also anticipate two daily mealtimes, but whether this involves independently entrained oscillators, one ‘continuously consulted’ clock, cue-dependent non-circadian interval timing or a combination of processes, is unclear. Rats received two daily meals, beginning 3-h (meal 1) and 13-h (meal 2) after lights-on (LD 14∶10). Anticipatory wheel running began 68±8 min prior to meal 1 and 101±9 min prior to meal 2 but neither the duration nor the variability of anticipation bout lengths exhibited the scalar property, a hallmark of interval timing. Meal omission tests in LD and constant dark (DD) did not alter the timing of either bout of anticipation, and anticipation of meal 2 was not altered by a 3-h advance of meal 1. Food anticipatory running in this 2-meal protocol thus does not exhibit properties of interval timing despite the availability of external time cues in LD. Across all days, the two bouts of anticipation were uncorrelated, a result more consistent with two independently entrained oscillators than a single consulted clock. Similar results were obtained for meals scheduled 3-h and 10-h after lights-on, and for a food-bin measure of anticipation. Most rats that showed weak or no anticipation to one or both meals exhibited elevated activity at mealtime during 1 or 2 day food deprivation tests in DD, suggesting covert operation of circadian timing in the absence of anticipatory behavior. A control experiment confirmed that daytime feeding did not shift LD-entrained rhythms, ruling out displaced nocturnal activity as an explanation for daytime activity. The results favor a multiple oscillator basis for 2-meal anticipatory rhythms and provide no evidence for involvement of cue-dependent interval timing

Behavioral and Neural Correlates of Acute and Scheduled Hunger in C57BL/6 Mice

In rodents, daily feeding schedules induce food anticipatory activity (FAA) rhythms with formal properties suggesting mediation by food-entrained circadian oscillators (FEOs). The search for the neuronal substrate of FEOs responsible for FAA is an active area of research, but studies spanning several decades have yet to identify unequivocally a brain region required for FAA. Variability of results across studies leads to questions about underlying biology versus methodology. Here we describe in C57BL/6 male mice the effects of varying the ‘dose’ of caloric restriction (0%, 60%, 80%, 110%) on the expression of FAA as measured by a video-based analysis system, and on the induction of c-Fos in brain regions that have been implicated in FAA. We determined that more severe caloric restriction (60%) leads to a faster onset of FAA with increased magnitude. Using the 60% caloric restriction, we found little evidence for unique signatures of neuronal activation in the brains of mice anticipating a daily mealtime compared to mice that were fasted acutely or fed ad-libitum–even in regions such as the dorsomedial and ventrolateral hypothalamus, nucleus accumbens, and cerebellum that have previously been implicated in FAA. These results underscore the importance of feeding schedule parameters in determining quantitative features of FAA in mice, and demonstrate dissociations between behavioral FAA and neural activity in brain areas thought to harbor FEOs or participate in their entrainment or output

Photic and Pineal Modulation of Food Anticipatory Circadian Activity Rhythms in Rodents

Restricted daily feeding schedules entrain circadian oscillators that generate food anticipatory activity (FAA) rhythms in nocturnal rodents. The location of food-entrainable oscillators (FEOs) necessary for FAA remains uncertain. The most common procedure for inducing circadian FAA is to limit food access to a few hours in the middle of the light period, when activity levels are normally low. Although light at night suppresses activity (negative masking) in nocturnal rodents, it does not prevent the expression of daytime FAA. Nonetheless, light could reduce the duration or magnitude of FAA. If so, then neural or genetic ablations designed to identify components of the food-entrainable circadian system could alter the expression of FAA by affecting behavioral responses to light. To assess the plausibility of light as a potential mediating variable in studies of FAA mechanisms, we quantified FAA in rats and mice alternately maintained in a standard full photoperiod (12h of light/day) and in a skeleton photoperiod (two 60 min light pulses simulating dawn and dusk). In both species, FAA was significantly and reversibly enhanced in the skeleton photoperiod compared to the full photoperiod. In a third experiment, FAA was found to be significantly attenuated in rats by pinealectomy, a procedure that has been reported to enhance some effects of light on behavioral circadian rhythms. These results indicate that procedures affecting behavioral responses to light can significantly alter the magnitude of food anticipatory rhythms in rodents

Group mean (± SEM, N = 15 rats) anticipatory wheel running onsets in minutes prior to meal 1 (m1; red closed circles, dotted curve) and meal 2 (open squares, black solid curve) during each day of restricted feeding (RF) in

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031772#s3" target="_blank"><b>Experiment 1</b></a><b>.</b> Mealtime 1 and 2 began 3-h and 13-h after lights on (LD 14∶10). Meal 1 was omitted on day 13, both meals were omitted and the lights were left off on day 16, meal 1 was delivered 3-h early on day 20 and both meals were omitted on day 23.</p

Wheel running activity of four representative rats in Experiment1.

<p>Each line represents 24 h, with time of day plotted left to right in 10 min bins. Time bins during which wheel counts were registered are denoted by heavy bars. Meals are indicated by opaque bars. Experimental conditions are numbered to the left of each panel: 1 = 37-h food deprivation, 2 = restricted feeding days, 3 = meal 1 omitted, 4 = both meals omitted in constant dark, 5 = meal 1 provided 3-h early.</p

Wheel running of representative rats in

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031772#s5" target="_blank"><b>Experiment 3</b></a><b>. Panels A–C. Rats from cohort A.</b> Experimental conditions are numbered to the left of each actogram: (1) 37-h food deprivation. (2) Two daily meals, denoted by opaque vertical bars. (3) Meal 1 omitted. (4) Both meals omitted. (5) Both meals omitted for 2 days in DD. (6) Meal 1 advanced by 3-h. Panels D–F. Rats from cohort B. Experimental conditions: (1) 37-h food deprivation. (2) Meal 2 omitted. (3) Meal 1 omitted. (4) Both meals omitted for 2 days in DD. (5) Both meals omitted in LD.</p

Group mean waveforms of wheel running activity in rats from Cohort A (panels A–C) and Cohort B (panels D–F).

<p>Panels A,D: ad-lib food access (grey shading) and 37-h food deprivation prior to restricted feeding (red curve). Panels B,E: Restricted feeding (RF) days (black line) prior to 2 days food deprivation (FD, red line) in constant dark (DD). Panels C,F: RF days (black line) prior to omission of both meals for one day (red line) in LD. Other plotting conventions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031772#pone-0031772-g001" target="_blank">Figure 1</a>.</p

Group mean (± sem) waveforms of normalized wheel running activity in

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031772#s3" target="_blank"><b>Experiment 1</b></a><b>.</b> Time is plotted in 10 min bins from lights-on (Hour 0, yellow bar). Meal times are denoted by green stripped vertical bars (hollow when meals were skipped). A. Last 4 days of ad-lib food access (dashed line) and 37-h food deprivation (red solid line, shaded) prior to initiation of 2-meal restricted feeding (RF) schedule. B. Ad-lib (dashed line) and RF days 8–12 (blue, solid line). C. RF day 12 (blue line) and Meal 1 omission day (red line, shaded), n = 9 rats showing delayed onset of anticipation to Meal 2. D. RF day 12 (blue line) and Meal 1 omission day (red line, shaded), n = 6 rats showing no change in anticipation onset. E. RF day 15 (blue line) and total food deprivation day with lights-off (DD, red line, shaded). F. Total food deprivation day in DD (red line, shaded) and first day of RF after food deprivation (blue line). G. RF day 19 (blue line) and 3-h shift of Meal 1 (red line, shaded). H. RF day 19 (black line), 3-h shift of Meal 1 (red line, shaded), and day after 3-h shift of Meal 1 (RF21, blue line).</p

Effects of dark exposure on food anticipation ratios of rats entrained to a full photoperiod (FPP).

<p>Ratios are plotted for restricted feeding days 10–14 (block 3), day 15 (DD, no light from ZT0-6 prior to mealtime), day 16 (normal full photoperiod), day 17 (D-pulse, lights off for 90 min prior to mealtime) and day 18 (full photoperiod).</p