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. Author manuscript; available in PMC: 2026 Mar 5.
Published in final edited form as: Am J Physiol Gastrointest Liver Physiol. 2026 Jan 27;330(3):G270–G279. doi: 10.1152/ajpgi.00368.2025

Elucidation of daily timed–palatable meal–anticipatory activity in the circadian system of mice

S K Tahajjul Taufique 1,#, Averey Eischeid 1,2,#, Isabel Magaña 3,#, David E Ehichioya 1, Sofia Farah 1, Yuuki Obata 1,3,4, Shin Yamazaki 1,4,*
PMCID: PMC12959641  NIHMSID: NIHMS2143516  PMID: 41592555

Abstract

Anticipation of daily recurring changes in the environment is critical for survival. When food access is limited to a few hours during the daytime, nocturnal rodents exhibit food-anticipatory activity, which appears a few hours before scheduled mealtime. The rodents are also known to exhibit anticipatory activity for time-restricted palatable meals under ad libitum access to chow. When 1 h of chocolate chip access was given during the day, mice exhibited robust anticipatory activity. In contrast, despite the peanut butter–fed mice eating two times the calories of peanut butter than the chocolate-fed mice did of chocolate chips, we observed only negligible anticipatory activity for daily 1 h peanut butter administration. In ex vivo explants, the phase of the liver in mice subjected to timed–chocolate chip access was significantly advanced, similarly to that in mice subjected to 4 h restricted feeding during the day. Similar to anticipatory activity, negligible phase changes in the liver were observed in the mice given 1 h of peanut butter access during the day. Therefore, robustness of palatable meal–anticipatory activity and phase advance in the liver are unlikely to be in direct response to increased calorie intake during the day. We measured food-seeking nose-poking behavior during food deprivation following daily 1 h chocolate chip access. Mice expressed anticipatory food seeking around the time that they had previously been given daily chocolate chips. This suggests that the time of chocolate chip access is encoded to the same circadian pacemaker that controls food-anticipatory activity.

Keywords: obesity, restricted feeding, junk food, night eating

NEW & NOTEWORTHY

Anticipatory activity for daily chocolate chip access is stronger than that for daily peanut butter access. Mice given daily chocolate chip access, but not peanut butter access, exhibited altered circadian organization among peripheral clocks. During food deprivation, mice exhibited anticipatory food-seeking behavior at the time they had previously been given chocolate chip access, suggesting that the time of palatable meals is encoded to the circadian pacemaker controlling food-anticipatory activity.

Graphical Abstract

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INTRODUCTION

Circadian rhythms are 24-hour rhythms in physiology and behavior controlled by autonomous circadian pacemakers, allowing organisms to anticipate daily recurring changes in the environment (1). The primary central circadian pacemaker is located in the suprachiasmatic nucleus (SCN) (2–7). The SCN entrains to the environmental light-dark cycle through photoreceptors located in the retina and orchestrates the ensemble of circadian rhythms in peripheral tissues (8–10). The molecular mechanism for circadian rhythm generation in the SCN and peripheral organs is based on transcription-translation feedback loops of circadian genes (11–13).

Although light is the most prominent environmental factor that entrains circadian rhythms, food availability also often exhibits daily changes and can be a potent signal for the entrainment of circadian rhythms. In fact, animals exhibit anticipatory activity for daily food availability. When food availability is restricted to a few hours during the day (restricted feeding), rodents typically express food-anticipatory activity starting a few hours before the daily scheduled mealtime (14–17). The rodents can anticipate 2–3 mealtimes per day (18, 19) by time-stamping mealtimes to a pacemaker located outside of the SCN (20). When conflicting light-dark and feeding-fasting cycles are given, some peripheral circadian oscillators entrain to the feeding cycle rather than the light-dark cycle, suggesting that the feeding-fasting cycle is the primary cue entraining these peripheral circadian oscillators (21, 22). Although a few brain areas have been proposed as candidates for the pacemaker controlling food-anticipatory activity, other studies have demonstrated that these areas are not essential for the expression of food-anticipatory activity (23–37); as such, despite extensive efforts of chronobiologists, the neuroanatomical location of the circadian pacemaker controlling food-anticipatory activity has not yet been identified (28, 38). In fact, it is proposed that this pacemaker is network oscillators located multiple areas in the body rather than a single locus in the brain (15, 39). Additionally, despite several papers reporting that circadian gene knockout mice do not express proper food-anticipatory activity (29, 31, 40), there is a consensus that the rhythm of the pacemaker controlling food-anticipatory activity is independent of the circadian genes that are essential for the SCN and peripheral circadian oscillators (17, 20, 30, 41–46). Therefore, the food-anticipatory activity is controlled by a non-canonical circadian pacemaker, and the molecular mechanism that generates circadian food-anticipatory activity rhythms is yet to be discovered.

Rodents can anticipate scheduled palatable meals even under ad libitum access to food. When nocturnal rodents, who are normally asleep during the day, are given 1 h access to a palatable meal during the daytime, they become active a few hours before the scheduled palatable meal (47–52). Although researchers have consistently observed anticipatory activity for daily palatable meals in rats, similar studies in mice are somewhat controversial. One study reports that the mice didn’t exhibit anticipatory activity for daily chocolate, chocolate-flavored Slimfast, or Fruit Crunchies (49). In contrast, other studies demonstrated that mice exhibit anticipatory activity for daily peanut butter(50, 52). Therefore, we conducted experiments to measure anticipatory activity for peanut butter or chocolate chips under ad libitum feeding. We also determined how these palatable meals alter the phase of peripheral clocks. Moreover, we conducted experiments to observe how timed palatable meal access affects food-seeking behavior and food intake rhythm. We observed mice exhibit anticipatory activity for both chocolate chips and peanut butter, but anticipatory activity for chocolate chips is much stronger and comparable with food-anticipatory activity for daytime restricted feeding. We observed that daytime chocolate chip access, but not peanut butter access, advanced the phase of the circadian oscillator in the liver in a similar way as daytime restricted feeding did. This phase shift is unlikely to be caused by changes in the daily calorie intake pattern but instead potentially controlled by the central pacemaker located outside of the SCN. Our data also suggest that timed palatable meal access is time-stamped to the circadian pacemaker controlling food-anticipatory activity.

MATERIALS AND METHODS

Mice

Fifty-two heterozygous PERIOD2::LUCIFERASE (PER2::LUC) knock-in mice (10) (9–46 weeks old, backcrossed C57BL/6J for 13 generations in Takahashi lab or 36–38 generations in Yamazaki lab) were used in the current study. Ten male and 10 female mice were assigned as controls. Six male and 6 female mice were assigned to the chocolate chip administration group. Four male and 2 female mice were assigned to the peanut butter administration group. Three male and 3 female mice were assigned to the restricted feeding group. Four males and 4 females were assigned for measurement of food-seeking behavior during and after chocolate chip administration. Both males and females were used. Sex is indicated in the individual actograms presented in the supplemental figures (Fig. S1–5). Sexes were initially analyzed separately. As no significant sex differences were observed, data from both sexes were analyzed together (except Fig. S6). All experiment procedures were carried out following the National Institutes of Health Guidelines regarding the care and use of animals for experimental procedures and were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center (Protocol #: 2016–10376-G).

Behavior measurement

Mice were individually housed in cages (32.5 cm in length, 14.5 cm in width, and 13 cm in height, equipped with an 11-cm diameter running wheel) with woodchip bedding (Sani-Chips, PJ Murphy Forest Products, Montville, NJ, USA) and unlimited access to chow and water throughout the experiment, except when chow was removed from food hopper during the fasting period. The cages were placed in light-tight ventilated cabinets. Temperature, humidity, and light intensity inside the cabinet were recorded every 5 min by Chamber Controller software (ver. 4.104, Actimetrics, Lafayette, IN, USA). Mice were under a 12 h light (~200 lux at cage level, generated by white LEDs at the top of the light-tight box) and 12 h dark condition controlled by the Chamber Controller software. The wheel-running activity (via micro-switches mounted to the sides of the running wheel cages) was continuously recorded every 1 minute by the ClockLab acquisition system (version 3.604, Actimetrics, Lafayette, IN, USA; RRID: SCR_014309) for each mouse. Cages and water bottles were changed every 3 weeks.

Pellet intake and food-seeking behavior measurement

Mice were kept under the aforementioned conditions, except food was delivered by a cage-side attached open-source operant feeder, the Feeding Experimentation Device version 3 (FED3 (53)) filled with 20 mg food pellets (F0163, Bos-Serv, Flemington, NJ, USA) (53, 54). The feeder was set to dispense one pellet by a single nose-poke to left nose-poke hole. The right nose-poke hole was disabled; however, right nose-poking was recorded as exploratory activity. The times of pellet intake, right nose-poke, and left nose-poke events were recorded on the SD card inside the FED3. Pellets were filled every 3 days.

Restricted Feeding

Restricted feeding was done manually by experimenters (D.E.E. and I.M.). In the first 3 days of the experiment, mice had ad libitum access to chow. On day 4, starting at Zeitgeber time 12 (ZT12, time of lights-off), mice were placed on an overnight fast for a duration of 16 h. The next 2 days, food access was restricted to 8 h (ZT4–12), then 6 h (ZT4–10) for the 2 days after that. This was followed by 4 h of restricted feeding (ZT4–8) for a period of 17 days. Mice were then euthanized for tissue explants.

Daytime peanut butter administration

After 12 days of baseline recording, mice were placed on an overnight fast for a duration of 16 h starting at ZT12. Then mice were given access to peanut butter for 8 hours (ZT4–12) on the first day, and on subsequent days for 1 hour (ZT4–5). Daily peanut butter feeding was done manually. An experimenter (D.E.E.) placed a 35 mm petri dish containing 3 g (first day) or 1 g (subsequent days) of peanut butter (Jif Creamy peanut butter; J.M. Smucker Company, Orrville, OH, USA; containing 48% fat, 21% protein, and 24% carbohydrates by weight) at the bottom of cage and removed a dish 8 h later at the first day and 1 h later in subsequent days. The initial 8 h peanut butter access was given as this increased the mice’s peanut butter consumption in subsequent days and resulted in more consistent peanut butter consumption. Mice had ad libitum access to chow and water during peanut butter feeding. After 12–20 days of daily peanut butter, mice were euthanized for tissue explants.

Daytime chocolate chip administration

After 2 days of baseline recording, mice were placed on an overnight fast for a duration of 16 h starting at ZT12. Then, mice were given access to 1 h of daily chocolate chips. Experimenters (S.K.T.T. and A.E.) placed a 35 mm petri dish containing 1 g of chocolate chips (Milk Chocolate Baking Chips, Kroger, Cincinnati, Ohio, USA; containing 26% fat, 6% protein, and 66% carbohydrates by weight) at the bottom of cage and removed the dish 1 h later. In our preliminary experiment, mice consistently consumed chocolate chips with 1 h chocolate chip access each day without being given 8 h access on the first day. Therefore, mice were given 1 h access to chocolate chips on the first day. Mice had ad libitum access to chow and water during chocolate chip feeding. After 12–27 days of daily chocolate chip feeding, mice were euthanized for tissue explants. Body weight was measured at day 0 (baseline) and day 14 (11th days of daily chocolate chip access). In a separate experiment, with FED3 feeders, mice were given ad libitum access to pellets by nose-poking, except overnight fasting a day before chocolate chip feeding and 48 hour fasting period at the end of the experiment. After 14 days of baseline, mice were given 1 h chocolate chip access at ZT4 for 17 days. Five days later, mice were food deprived for 48 hours. To avoid providing an additional time cue to the mice, body weight was not measured during the experiment.

Tissue explants and bioluminescence recording

Mice were euthanized without anesthesia by cervical dislocation followed by decapitation. Euthanasia was done ~2–3 hours after lights-on or ~1 h before lights-off (see actual time in Fig. S7–8). The brain and peripheral organs were quickly removed and placed in cold HEPES-buffered HBSS with antibiotics (H9394–500ML, H0887, P0781, Sigma-Aldrich, St. Louis, MO, USA). Tissue explants were made as previously described (55), except that CellGro (cat. no. 90–013PB, Corning, Glendale, AZ, USA) with L-glutamine (G7513, Sigma-Aldrich) with 10% FBS (21922, HyClone, Cytiva, Wilmington, DE, USA) recording medium was used. For intestine explants, the longitudinal muscle–myenteric plexus (LMMP) layer from the ileum and proximal colon was used. To isolate the LMMP layer, a tip of 1 mL pipette was inserted into the lumen to extend the intestinal wall, and the LMMP was gently separated from the mucosa using cotton swabs, as previously described (56). The LMMP layer was washed three times in cold HBSS to remove luminal debris observed under a stereomicroscope. The SCN, pituitary, and arcuate complex were cultured on Millicell culture inserts (PICM ORG50, MilliporeSigma, Burlington, MA, USA) with 1.2 mL of recording medium. The liver, ileum, and colon were cultured on 0.5 mm Woven Filters (146418 Spectra/Mesh, Repligen, Waltham, MA, USA) with 1.5 mL of recording medium. Other tissues were cultured on 1 mm Woven Filters (146446 Spectra/Mesh, Repligen, Waltham, MA, USA) with 1.5 mL of recording medium. Bioluminescence was monitored in real-time with the LumiCycle 32 (Actimetrics, Lafayette, IN, USA) for ~3–7 days.

QUANTIFICATION AND STATISTICAL ANALYSIS

Wheel-running activity, nose-poke, and pellet intake data were analyzed by ClockLab analysis software (version 6.1.18 Actimetrics, Lafayette, IN, USA). Double-plotted ethograms were generated in 6 min bins using the percentile plot with quantile 50. Group average 24-h profiles were generated with 30 min bins. The individual 24-h daily profiles were first generated by averaging 3 days (control: days 11–13; RF, PB, CC: days 8–10 of each feeding condition), then mean and SEM among the individuals were determined. Therefore, SEM represents individual variability.

Bioluminescence was analyzed by LumiCycle software (version 3.101, Actimetrics, Lafayette, IN, USA). First, the baseline was subtracted from the raw luminescence data using a 24-hour moving average, and the data was smoothed by 0.5-hour adjacent averaging. Since early peaks, including the first peaks for AM cultures, were often masked by rapid changes in the baseline, the circadian phase was defined for AM cultures as the second peak and for PM cultures as the first peak after 12 h from the time the explants were made.

All statistical analyses were done by GraphPad Prism (version 10.6.1; Dotmatics, Boston, MA, USA ; RRID: SCR_002798). Mann-Whitney test, Kruskal-Wallis test followed by Dunn’s multiple comparison test, and 2-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparison test were used, p < 0.05, p < 0.01, p < 0.0001 are considered significant. For daily total wheel-running, pellet intake, and calorie intake analysis, daily mean of baseline (days 7–13) and chocolate chip administration (days 26–32) were compared by a two-tailed paired t-test. Daily pattern of calorie intake during baseline and chocolate chip administration was analyzed by 2-way ANOVA followed by Bonferroni’s multiple comparison test.

All the statistical tests and results are reported in the results or in the figure legends.

RESULTS

Mice exhibited robust anticipatory activity for timed–chocolate chip access

In the initial few days of 1 h access to chocolate chips given at ZT4, the mice became active at the time of chocolate chip access and continued to be active for a few hours after termination of chocolate chip access (Fig. S3). Within a few days, most mice began expressing anticipatory activity starting a few hours prior to chocolate chip access (Fig. 1A). This anticipatory activity was comparable to the food-anticipatory activity observed in mice given 4 h daily scheduled meals (Fig. 1A, B, Fig. S5). In contrast to food-anticipatory activity, which immediately started declining at the onset of the mealtime, anticipatory activity for chocolate chips continued to increase and reached its peak at the end of the 1 h chocolate chip access and gradually decreased over the following few hours (Fig. 1A). There was no sex difference in the robustness of the chocolate chip–anticipatory activity (Fig. S6). Mice also exhibited anticipatory activity for peanut butter (Fig. S4). However, despite the mice eating ~2 times more calories of the palatable meal during 1 h access to peanut butter than mice with 1 h access to chocolate chips, anticipatory activity for peanut butter was much weaker than that for chocolate chips (Fig. 1B, C). Total amounts of daily wheel-running activity were comparable across all 4 groups (ad libitum, 1 h daytime chocolate chip access, 1 h daytime peanut butter access, and daytime restricted feeding, Fig. 1D). Because daytime activity was significantly increased in mice with 4 h daytime restricted feeding and 1 h chocolate chip access, nighttime activity in those mice were significantly decreased (Fig. 1D). The 11 days of chocolate chip administration didn’t alter the body weight (Fig. S9).

Figure 1.

Figure 1.

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Anticipatory activities for time-restricted feeding and palatable meal access. A: 24 h group-averaged activity profiles under different feeding conditions. Three days of activity in each feeding condition (control: days 11–13; RF, PB, CC: days 8–10 of each feeding condition) were averaged by individual with 30 min bins, then averaged among individuals. Black (Control): ad libitum control (n = 17, 3 control mice which had only 7 days of behavior recording were excluded from the analysis), Green (RF): ZT4–8 time-restricted feeding (n = 6), Orange (PB): peanut butter access ZT4–5 under ad libitum access to chow (n = 6), Brown (CC): chocolate chip access ZT4–5 under ad libitum access to chow (n = 12). All individual actograms are shown in Fig. S1–5. B: Total wheel revolutions in the 3 hours preceding the onset of food or palatable mealtime (anticipatory activity). Black (Control): ad libitum control (n = 17, 3 control mice which had only 7 days of behavior recording were excluded from the analysis), Green (RF): ZT4–8 time-restricted feeding (n = 6), Orange (PB): peanut butter access ZT4–5 under ad libitum access to chow (n = 6), Brown (CC): chocolate chip access ZT4–5 under ad libitum access to chow (n = 12). **p < 0.01, ****p < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparison test). C: Daily calorie intake from palatable meal (ZT4–5) during 1 h access of palatable meal (as peanut butter access was 8 h on the first day, peanut butter daily intake was plotted from day 2). Mean ± SEM, *p < 0.05 (2-way RM-ANOVA followed by Bonferroni’s multiple comparison test). D: Daily total wheel revolutions during day and night. Three-day average (the days used in A) was analyzed. Total amounts of activity during the day and during the night were compared in each feeding condition. Black (Control): ad libitum control (n = 17, 3 control mice which had only 7 days of behavior recording were excluded from the analysis), Green (RF): ZT4–8 time-restricted feeding (n = 6), Orange (PB): peanut butter access ZT4–5 under ad libitum access to chow (n = 6), Brown (CC): chocolate chip access ZT4–5 under ad libitum access to chow (n = 12). Different letters and symbols indicate significant difference, p < 0.05 (2-way ANOVA followed by Tukey’s multiple comparison test).

Liver phase was advanced by daily access to chocolate chips

To observe how daily scheduled palatable meal access alters the circadian organization, we constructed phase maps by estimating the in vivo phases from PER2::LUC rhythms of ex vivo tissue explants. One caveat of phase map construction by ex vivo tissue explants is that the culture procedure may reset the phase of the circadian rhythm in the ex vivo tissue so that the phase of the ex vivo tissue no longer reflects the in vivo phase. To address this potential issue, we explanted tissues at two different time points, approximately 10 h apart (just after lights-on and just before lights-off). If the morning (AM) and evening (PM) cultures express a similar phase relative to culture time, we concluded that the phases of those tissues were reset by the culture procedure and excluded those explants from the analysis (Fig. S7, S8). For instance, when plotted relative to light-dark cycle, the phases of ileum and colon were similar between the AM and PM cultures in ad libitum control, chocolate chip, and peanut butter groups (Fig. 2). When the same data was plotted relative to culture time, the phases of AM and PM cultures were separated by ~10 h (Fig. S7 and S8), suggesting that culture procedures had negligible influence on the phases in ex vivo explants. In contrast, the phases of the ileum and colon explanted from mice under 4 h restricted feeding overlapped when plotted relative to culture time (Fig. S8B). This strongly suggests that the culture procedures completely reset the phases of the ileum and colon explanted from mice under 4 h daytime restricted feeding. As low-amplitude oscillators are more sensitive to external stimuli (57), we interpreted that phase resetting by culture procedure as due to a reduction of circadian amplitude of the tissue in vivo.

Figure 2.

Figure 2.

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Phase map of central and peripheral circadian oscillators estimated by ex vivo tissue explants. A: 3 representative examples of PER2::LUC rhythms from the SCN, liver, and abdominal fat from control mice and mice given 1 h chocolate chip access at ZT4. AM: tissue was explanted ~ZT2–3, PM: tissue was explanted ~ZT10–11. Each data point was normalized by the first peak that occurred 12 h after the tissue explant. B: The phase of ex vivo tissue was determined for AM cultures by the second peak and for PM cultures by the first peak detected after 12 h from the time of explants were made.

Mean ± SEM is shown for the tissues that weren’t reset by the culture procedure (SEM is smaller than the size of the symbol in the SCN and CC colon). The phases of all tissues are plotted for the tissues reset by culture procedure (open circle: AM cultures explanted at ~ZT2–3, closed circle: PM cultures explanted at ~ZT10–11). As phases of abdominal fat tissues explanted from chocolate chip-administered mice are scattered all over the cycle, individual phases are plotted. *p < 0.05, **p < 0.01, ****p < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparison tests to compare with controls). The sample size is shown (number of rhythmic tissues/number of tissues tested). The same data plotted relative to culture time and tissues excluded from the analysis are shown in Fig. S7 and S8.

We compared the phase maps of mice under 4 different feeding conditions (Fig. 2). The phases of the SCN, ileum, colon, lung, and spleen were not affected by daily access to palatable meals. Among the peripheral organs, the liver underwent noticeable phase changes. The phase of the liver was advanced by 7.6 h in mice under restricted feeding. The phase of the liver also advanced by 4.4 h in mice given daily chocolate chip administration. No statistically significant phase advance was observed in mice under peanut butter administration. This was surprising: even though the mice given peanut butter ate 2 times the calories of palatable meal than mice given chocolate chips did (Fig. 1C), the mice given peanut butter didn’t show robust anticipatory activity and expressed only a negligible advance of the liver phase (Fig. 1A, B). This suggests that the phase advance of the liver clock in the mice given chocolate chips was not the result of increased daytime caloric intake. Another noticeable effect of daytime chocolate administration is the phase of abdominal fat. Phases of abdominal fat from mice under daytime chocolate administration were spread throughout the circadian time. This was not caused by phase-resetting from the culture procedure, as AM and PM cultured abdominal fat didn’t show distinct phase clusters either when phases were plotted relative to culture time or ZT time (Fig. 2, Fig. S7B). Interestingly, this effect was not seen in mice under daytime peanut butter access, suggesting this effect was also unlikely to be caused by increased calorie intake during the day (Fig. 2). Finally, the phases of pituitary and colon were slightly advanced in mice with daily chocolate chip access.

Time of access to chocolate chip is likely time-stamped to the circadian pacemaker controlling food-anticipatory activity.

To reveal the circadian clock mechanism underlying palatable meal anticipation, we measured food-seeking behavior and pellet intake patterns during daytime chocolate chip administration. We have previously shown both that food-seeking nose-poking activity is a better proxy behavior output and that the time of food-seeking nose-poking behavior is time stamped to the circadian oscillator that controls food-anticipatory activity (20, 54, 58). We used an open-source operant feeding device, the FED3 (53). We programmed the FED3 to dispense pellets by single pokes in the left nose-poke hole but not to dispense pellets by pokes in the right nose-poke. We measured pellet intake, rewarded left pokes, and unrewarded right pokes during ad libitum feeding, ad libitum feeding with 1 h chocolate chip access at ZT4–5, subsequent ad libitum feeding, and 48-hour food deprivation (Fig. 3). With this protocol, our previous study showed that food deprivation reveals time-memory encoded to the circadian oscillator: exploratory nose-poking behavior coincides with the time of previous feeding in the absence of the feeding cue (20, 54, 58). Due to one mouse (#11309) not eating chocolate chips during the experiment, and an SD card issue in the FED3 used for mouse #11560, we excluded the data collected from these two mice from the analysis (Fig. S10). Mice expressed anticipatory activity for daily 1 h chocolate chip access (Fig. 3A). We noticed that the observed chocolate chip–anticipatory activity in this cohort (Fig. S10, S11) is slightly weaker than the chocolate chip–anticipatory activity observed in the initial experiment (Fig. 1A, S3). It is possible that ad libitum access to food from an operant feeder weakens anticipatory activity for chocolate chips. One hour of daily access to chocolate chips slightly altered the nocturnal food intake pattern, with a reduction of pellet intake at the end of night (Fig. 3B, S11). In contrast, there was a slight increase in pellet intake in the few hours before the time of chocolate chip administration (Fig. 3B, S11). As mice needed to poke the left nose-poke hole to dispense pellets, the rewarded left nose-poke pattern was nearly identical to the pellet intake pattern (Fig. 3C, S11). There was no notable change in unrewarded right pokes with daily chocolate chip access (Fig. 3D, S11). Interestingly, during subsequent food deprivation, food-seeking left nose-poking and unrewarded right poking behavior were observed occurring at the same time as the previous chocolate chip administration (Fig. 3C, D, S11). Mice also exhibited elevation of both the rewarded left pokes and unrewarded right pokes during the night, which corresponds to previous habitual feeding at night. This suggests that daily chocolate timing is encoded to the circadian pacemaker controlling food-anticipatory activity, in the same way that mealtimes are encoded. Total daily wheel-running activity remained constant throughout the experiment (Fig. 4A). Daily average wheel-running between baseline (mean: 28864.3 ± SEM: 2701.4 during days 7–13) and chocolate chip administration (mean: 23110.1 ± SEM: 4140.2, during day 26–32) was statistically no different (p = 0.09 by two-tailed paired t-test). Daily total pellet intake was slightly reduced during daily 1 h chocolate chip access; as a result, daily total calorie intake remained constant (Fig. 4B-C). Daily average pellet intake between baseline (mean: 206.1 ± SEM: 10.5 during days 7–13) and chocolate chip administration (mean: 150.7 ± SEM: 10.9, during days 26–32) was statistically significant (p = 0.009 by two-tailed paired t-test). Daily average calorie intake between baseline (mean: 14.4 ± SEM: 0.6 during days 7–13) and during chocolate chip administration (mean: 13.6 ± SEM: 0.6, during days 26–32) was statistically no different (p = 0.36 by two-tailed paired t-test). Daily pattern of calorie intake was also slightly changed during daily 1 h chocolate chip access at ZT4 (Fig. 4D). Reduction of pellet intake was prominent during the last half of the dark period. As consistent with previous studies (47–52), anticipatory activity continued during subsequent ad libitum feeding and food deprivation.

Figure 3.

Figure 3.

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Group-averaged ethograms of mice with chocolate chip access at ZT4–5. Lighting and feeding conditions are indicated in the left half panel of the double-plotted actogram. Gray: dark, Brown: chocolate chip access, Red: food deprivation. All individual ethograms are shown in Fig. S10. 24-h group averaged profiles in each feeding condition are shown in Fig. S11.

Figure 4.

Figure 4.

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Mice self-adjusted total daily calorie intake during 1 h access to chocolate chip. A: Daily total running-wheel activity. B: Daily total pellet intake. C: Daily total calorie intake includes calories from both pellets and chocolate chip. D: 24-h group averaged calorie intake during baseline (Black) and 1 h daily access of chocolate chip (Brown). 3-day averages of the last 3 days of baseline (ad libitum feeding) and daytime chocolate chip access were averaged individually. Mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.001 (2-way ANOVA followed by Bonferroni’s multiple comparison test)

DISCUSSION

The mice exhibited anticipatory activity for daily 1 h access to palatable meals with ad libitum access to chow. Despite the mice that were given peanut butter consuming more calories of peanut butter than the mice that were given chocolate chips consumed of chocolate, anticipatory activity for chocolate chips was much stronger than that for peanut butter. The amount of phase advance of PER2::LUC rhythm in the liver was also larger in mice with chocolate chip access compared to that in mice with peanut butter access. The amounts of both phase advance in the liver and robustness of anticipatory activity did not correlate with the amount of calorie intake from a palatable meal, suggesting that the phase advance is less likely to be a result of increased calorie intake during the day but instead may be due to differences in the reward value of the palatable meal. This is unexpected as it is thought that the phase of the circadian oscillator in the liver directly entrains to the feeding cycle. Hsu and her colleagues reported that mice didn’t express anticipatory activity for daytime 2 h access to chocolate chip (Hershey’s Milk Chocolate Chips, containing 33% fat, 6% protein, and 53% carbohydrates) (49). Our current study clearly demonstrates that mice anticipate 1 h daily access to a chocolate chip containing 26% fat, 6% protein, and 66% carbohydrates. Mice also exhibited weak anticipatory activity for daily 1 h access to peanut butter containing 48% fat, 21% protein, and 24% carbohydrates. It is possible that a palatable meal with higher sugar, but not fat, content has a higher reward value for mice. It is also possible that the duration of daily palatable meal access (1 h vs. 2 h) affects the response in the mice. It is also worth pointing out that our current study shows ad libitum access to food through the FED3 (operant feeder) seems to reduce the robustness of anticipatory activity for daily chocolate chips. It is possible that dispensing a pellet by nose-poke may change the internal reward state of mice. Future studies are necessary to reveal the mechanism of anticipation for palatable meal reward under ad libitum access to food. Our results also raised the intriguing possibility that the phase of the liver is at least partially controlled by the circadian pacemaker controlling food/palatable meal–anticipatory activity. In fact, a previous study of daytime chocolate chip access in rats showed that c-fos expression in the brain areas of the limbic system were increased prior to daily chocolate chip access in a similar manner to under daytime restricted feeding (51). Phase of PER2::LUC rhythm in abdominal fat was unaffected by daily access to peanut butter. In contrast, the phases of abdominal fat in mice under daily chocolate chip access were scattered across the day. Neither time of chocolate chip access, light-dark cycle, nor time when tissue explants were made was correlated with the phase of ex vivo circadian rhythm in the abdominal fat. Although it is clear that the culture procedure didn’t reset the phase of ex vivo abdominal fat explants, culture procedure might still shift the phase of the rhythm. However, if this were the case, we would expect the phases of abdominal fat explants to still be clustered together. Therefore, it is likely that the in vivo phase of abdominal fat in mice with chocolate chip access was randomly distributed across the day.

We observed that the daily pattern of pellet intake is slightly altered by daytime chocolate chip access. Pellet intake slightly increased in the hours before daytime chocolate chip access. Pellet intake during night was slightly decreased. Interestingly, mice exhibited food-seeking nose-poking behavior during subsequent food deprivation, and the times of this food-seeking behavior matched with those of the previous daytime chocolate chip access and nighttime habitual feeding time. These data suggest that daytime palatable meal intake is time-stamped to the same oscillator as controls food-anticipatory activity and food-seeking nose-poking. Calorie intake during from the increased pellet intake in the few hours before chocolate chip access is very small compared to the subsequent calorie intake from chocolate chips. Therefore, it is less likely that the slight increase of pellet intake before chocolate chip access is what was encoded as a time-memory to the pacemaker controlling anticipatory food-seeking behavior.

A limitation of the current study is that we estimated the in vivo phase by the phase of reporter luciferase activity from cultured tissue (ex vivo). We carefully assessed and excluded the tissues in which the circadian phase was reset by culture procedures, and in certain tissues under specific conditions, the phases of all tissue explants were reset by culture procedures. This implies that the amplitude of the in vivo rhythm was damped and is itself an interesting result, but regardless, we were not able to estimate in vivo phase in these tissues. Another limitation of the current study is that we only measured the rhythm of PER2::LUC activity. To overcome these limitations, a future study that measures the mRNA expression rhythm of several circadian genes in vivo tissue sampling is necessary. To avoid providing an additional time cue to the mice, we only measured body weight at the baseline and 11th day of chocolate chip administration (Fig. S9). The long-term effect of daily palatable meal administration on body weight needs to be evaluated in the future.

Timed access for palatable meals during inactive time may have a detrimental effect on circadian clock system by disrupting normal circadian organization. In the current study, we conducted timed access of palatable meal only during the daytime when nocturnal mice are typically inactive and asleep. Further investigation is necessary to determine if palatable meal access during the active period has any beneficial effects. It has been shown that time-restricted feeding scheduled during the active period has beneficial effects by reducing body weight gain in high-fat diet–fed mice, extending lifespan, and improving age-related health decline (59–61). Timed eating is beneficial for treating cardiometabolic disorders in humans (61–63). Our current study indicates that timed palatable meals and time-restricted feeding at least partially have a similar effect to the circadian system. As people have often expressed difficulty continuing time-restricted eating for the long term (64), a timed palatable meal may be a possible alternative way to achieve health benefits. Understanding the mechanisms that control the time of eating, including of palatable meals, and how this affects circadian organization in central and peripheral clocks could provide novel chronobiology-based therapeutic strategies for the treatment of obesity and diabetes.

Supplementary Material

Supplemental Figures S1–11: doi.org/10.17632/9st5gv7zjz.3

ACKNOWLEDGMENTS

We thank Melody Shen for discussions, language editing, and illustrations for the graphical abstract and Dr. Bhaskar Thakur for helping with the statistical analysis.

GRANTS

This work was supported by grants from the National Institutes of Health R01NS114527 and the National Science Foundation IOS-1931115 awarded to S.Y. Y. O. is supported by the Nancy Cain and Jeffrey A. Marcus Scholar Award in Medical Research (in honor of B. S. Vowell), the Pew Scholars Program in Biomedical Sciences (00036890), and the Welch Foundation Research Grant (I-2153–20230405). A.E. was an undergraduate student supported by the Multi-Institutional Summer Undergraduate Research Program to Promote Diversity and Excellence in Sleep and Circadian Research Careers (National Institutes of Health R25 NS 125603).

DATA AVAILABILITY

All raw ClockLab, LumiCycle data, analyzed phase have been deposited to Mendeley Data [https://data.mendeley.com/datasets/9st5gv7zjz/3].

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All raw ClockLab, LumiCycle data, analyzed phase have been deposited to Mendeley Data [https://data.mendeley.com/datasets/9st5gv7zjz/3].

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