Feeding Time Entrains the Olfactory Bulb Circadian Clock in Anosmic PER2::LUC Mice.

Circadian rhythms in many brain regions and peripheral organs can be entrained by daily feeding schedules. The set of feeding-related signals that entrain peripheral clocks are tissue specific and include nutrients, metabolic hormones and temperature. Signals that entrain neural circadian clocks to mealtime have yet to be established for any brain region. The olfactory bulb (OB) contains a robust circadian clock that can cycle independently of the suprachiasmatic nucleus (SCN) master pacemaker. We used PER2::LUC mice to evaluate the suitability of the OB for analysis of inputs that mediate entrainment of neural clocks by feeding schedules. Explants of SCN and OB from mice fed ad-libitum exhibited robust circadian rhythms of bioluminescence for three or more days in vitro, with rhythm peaks occurring late in the day and night, respectively. Mice restricted to a 4 h meal/day in the light period exhibited food anticipatory activity and a 6.3 h advance of the OB PER2::LUC rhythm. The cumulative phase shift of the OB rhythm increased gradually in OB explants harvested after 2-7 days of restricted feeding. In mice anosmic after nasal irrigation with zinc sulfate and fed ad-libitum for one week, OB phases were desynchronized. Group synchrony in anosmic mice was restored by restricted feeding. The OB circadian clock is food-entrainable, entrains gradually to a mid-day meal, and requires neither olfaction nor circadian signaling from olfactory sensory neurons. The OB can be used as a model system for analysis of input pathways by which circadian clocks in the brain entrain to daily mealtimes.

Ultrasonic vocalizations in rats anticipating circadian feeding schedules.

Rats readily learn to anticipate a reward signaled by an external stimulus. Anticipatory behaviors evoked by conditioned stimuli include 50 kHz ultrasonic vocalizations (USVs), a proposed behavioral correlate of positive affect and activation of midbrain dopamine pathways. Rats can also anticipate a reward, such as food, provided once daily, without external cueing. Anticipation of a daily reward exhibits formal properties of a circadian rhythm. The neural circuits that regulate the timing and amplitude of these rhythms remain an open question, but evidence suggests a role for dopamine. To gain further insight into the neural and affective correlates of circadian food anticipatory rhythms, we made 2h and 24h USV recordings in rats fed 2h/day in the light period, a procedure that induces robust anticipation 2-3h before mealtime. Potential interactions between internal and external time cues in USV production were evaluated by inclusion of a 3 kHz tone 15 min before mealtime. Prior to scheduled feeding, spontaneous 50 kHz USVs were rare during the light period. During scheduled feeding, flat and frequency modulated (FM) 50kHz USVs occurred prior to and during mealtime. FM USVs were more closely related to anticipation, while flat USVs were more dependent on food access. USVs also occurred during spontaneous waking at other times of day. The tone did not evoke USVs but did modulate activity. Behavioral anticipation of a daily meal is accompanied by USVs consistent with a positive affective state and elevated dopamine transmission.

Circadian mechanisms of food anticipatory rhythms in rats fed once or twice daily: clock gene and endocrine correlates.

Circadian clocks in many brain regions and peripheral tissues are entrained by the daily rhythm of food intake. Clocks in one or more of these locations generate a daily rhythm of locomotor activity that anticipates a regular mealtime. Rats and mice can also anticipate two daily meals. Whether this involves 1 or 2 circadian clocks is unknown. To gain insight into how the circadian system adjusts to 2 daily mealtimes, male rats in a 12∶12 light-dark cycle were fed a 2 h meal either 4 h after lights-on or 4 h after lights-off, or a 1 h meal at both times. After 30 days, brain, blood, adrenal and stomach tissue were collected at 6 time points. Multiple clock genes from adrenals and stomachs were assayed by RT-PCR. Blood was assayed for corticosterone and ghrelin. Bmal1 expression was quantified in 14 brain regions by in situ hybridization. Clock gene rhythms in adrenal and stomach from day-fed rats oscillated in antiphase with the rhythms in night-fed rats, and at an intermediate phase in rats fed twice daily. Corticosterone and ghrelin in 1-meal rats peaked at or prior to the expected mealtime. In 2-meal rats, corticosterone peaked only prior the nighttime meal, while ghrelin peaked prior to the daytime meal and then remained elevated. The olfactory bulb, nucleus accumbens, dorsal striatum, cerebellum and arcuate nucleus exhibited significant daily rhythms of Bmal1 in the night-fed groups that were approximately in antiphase in the day-fed groups, and at intermediate levels (arrhythmic) in rats anticipating 2 daily meals. The dissociations between anticipatory activity and the peripheral clocks and hormones in rats anticipating 2 daily meals argue against a role for these signals in the timing of behavioral rhythms. The absence of rhythmicity at the tissue level in brain regions from rats anticipating 2 daily meals support behavioral evidence that circadian clock cells in these tissues may reorganize into two populations coupled to different meals.

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.

Scheduled daily mating induces circadian anticipatory activity rhythms in the male rat.

Daily schedules of limited access to food, palatable high calorie snacks, water and salt can induce circadian rhythms of anticipatory locomotor activity in rats and mice. All of these stimuli are rewarding, but whether anticipation can be induced by neural correlates of reward independent of metabolic perturbations associated with manipulations of food and hydration is unclear. Three experiments were conducted to determine whether mating, a non-ingestive behavior that is potently rewarding, can induce circadian anticipatory activity rhythms in male rats provided scheduled daily access to steroid-primed estrous female rats. In Experiment 1, rats anticipated access to estrous females in the mid-light period, but also exhibited post-coital eating and running. In Experiment 2, post-coital eating and running were prevented and only a minority of rats exhibited anticipation. Rats allowed to see and smell estrous females showed no anticipation. In both experiments, all rats exhibited sustained behavioral arousal and multiple mounts and intromissions during every session, but ejaculated only every 2-3 days. In Experiment 3, the rats were given more time with individual females, late at night for 28 days, and then in the midday for 28 days. Ejaculation rates increased and anticipation was robust to night sessions and significant although weaker to day sessions. The anticipation rhythm persisted during 3 days of constant dark without mating. During anticipation of nocturnal mating, the rats exhibited a significant preference for a tube to the mating cage over a tube to a locked cage with mating cage litter. This apparent place preference was absent during anticipation of midday mating, which may reflect a daily rhythm of sexual reward. The results establish mating as a reward stimulus capable of inducing circadian rhythms of anticipatory behavior in the male rat, and reveal a critical role for ejaculation, a modulatory role for time of day, and a potential confound role for uncontrolled food intake.