Standards of Evidence in Chronobiology: Critical Review of a Report that Restoration of Bmal1 Expression in the Dorsomedial Hypothalamus is Sufficient to Restore Circadian Food Anticipatory Rhythms in Bmal1-/- Mice

Daily feeding schedules generate food anticipatory rhythms of behavior and physiology that exhibitcanonical properties of circadian clock control. The molecular mechanisms and location of foodentrainablecircadian oscillators hypothesized to control food anticipatory rhythms are unknown.In 2008, Fuller et al reported that food-entrainable circadian rhythms are absent in mice bearing anull mutation of the circadian clock gene Bmal1 and that these rhythms can be rescued by virallymediatedrestoration of Bmal1 expression in the dorsomedial nucleus of the hypothalamus (DMH)but not in the suprachiasmatic nucleus (site of the master light-entrainable circadian pacemaker).These results, taken together with controversial DMH lesion results published by the samelaboratory, appear to establish the DMH as the site of a Bmal1-dependent circadian mechanismnecessary and sufficient for food anticipatory rhythms. However, careful examination of themanuscript reveals numerous weaknesses in the evidence as presented. These problems aregrouped as follows and elaborated in detail: 1. data management issues (apparent misalignments ofplotted data), 2. failure of evidence to support the major conclusions, and 3. missing data andmethodological details. The Fuller et al results are therefore considered inconclusive, and fail toclarify the role of either the DMH or Bmal1 in the expression of food-entrainable circadian rhythmsin rodents

Food Anticipation in Bmal1-/- and AAV-Bmal1 Rescued Mice: A Reply to Fuller et al

Evidence that circadian food-anticipatory activity and temperature rhythms are absent in Bmal1knockout mice and rescued by restoration of Bmal1 expression selectively in the dorsomedialhypothalamus was published in 2008 by Fuller et al and critiqued in 2009 by Mistlberger et al. Fulleret al have responded to the critique with new information. Here we update our critique in the lightof this new information. We also identify and correct factual and conceptual errors in the Fuller etal response. We conclude that the original results of Fuller et al remain inconclusive and fail toclarify the role of Bmal1 or the dorsomedial hypothalamus in the generation of food-entrainablerhythms in mice

Palatable Meal Anticipation in Mice

The ability to sense time and anticipate events is a critical skill in nature. Most efforts to understand the neural and molecular mechanisms of anticipatory behavior in rodents rely on daily restricted food access, which induces a robust increase of locomotor activity in anticipation of daily meal time. Interestingly, rats also show increased activity in anticipation of a daily palatable meal even when they have an ample food supply, suggesting a role for brain reward systems in anticipatory behavior, and providing an alternate model by which to study the neurobiology of anticipation in species, such as mice, that are less well adapted to "stuff and starve" feeding schedules. To extend this model to mice, and exploit molecular genetic resources available for that species, we tested the ability of wild-type mice to anticipate a daily palatable meal. We observed that mice with free access to regular chow and limited access to highly palatable snacks of chocolate or “Fruit Crunchies” avidly consumed the snack but did not show anticipatory locomotor activity as measured by running wheels or video-based behavioral analysis. However, male mice receiving a snack of high fat chow did show increased food bin entry prior to access time and a modest increase in activity in the two hours preceding the scheduled meal. Interestingly, female mice did not show anticipation of a daily high fat meal but did show increased activity at scheduled mealtime when that meal was withdrawn. These results indicate that anticipation of a scheduled food reward in mice is behavior, diet, and gender specific

Dopaminergic Regulation of Circadian Food Anticipatory Activity Rhythms in the Rat

Circadian activity rhythms are jointly controlled by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) and by food-entrainable circadian oscillators (FEOs) located elsewhere. The SCN mediates synchrony to daily light-dark cycles, whereas FEOs generate activity rhythms synchronized with regular daily mealtimes. The location of FEOs generating food anticipation rhythms, and the pathways that entrain these FEOs, remain to be clarified. To gain insight into entrainment pathways, we developed a protocol for measuring phase shifts of anticipatory activity rhythms in response to pharmacological probes. We used this protocol to examine a role for dopamine signaling in the timing of circadian food anticipation. To generate a stable food anticipation rhythm, rats were fed 3h/day beginning 6-h after lights-on or in constant light for at least 3 weeks. Rats then received the D2 agonist quinpirole (1 mg/kg IP) alone or after pretreatment with the dopamine synthesis inhibitor α-methylparatyrosine (AMPT). By comparison with vehicle injections, quinpirole administered 1-h before lights-off (19h before mealtime) induced a phase delay of activity onset prior to the next meal. Delay shifts were larger in rats pretreated with AMPT, and smaller following quinpirole administered 4-h after lights-on. A significant shift was not observed in response to the D1 agonist SKF81297. These results provide evidence that signaling at D2 receptors is involved in phase control of FEOs responsible for circadian food anticipatory rhythms in rats

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

Midday Meals Do Not Impair Mouse Memory

Nocturnal mice fed in the middle of the light period exhibit food anticipatory rhythms of behavior and physiology under control of food-entrainable circadian clocks in the brain and body. This is presumed to be adaptive by aligning behavior and physiology with predictable mealtimes. This assumption is challenged by a report that daytime feeding schedules impair cognitive processes important for survival, including object memory and contextual fear conditioning assessed at two times of day. To further evaluate these effects, mice were restricted to a 6 h daily meal in the middle of the light or dark period and object memory was tested at four times of day. Object memory was not impaired by daytime feeding, and did not exhibit circadian variation in either group. To determine whether impairment might depend on methodology, experimental procedures used previously to detect impairment were followed. Daytime feeding induced food anticipatory rhythms and shifted hippocampal clock genes, but again did not impair object memory. Spontaneous alternation and contextual fear conditioning were also not impaired. Hippocampal memory function appears more robust to time of day and daytime feeding schedules than previously reported; day-fed mice can remember what they have seen, where they have been, and where it is dangerous

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

Standards of evidence in chronobiology: critical review of a report that restoration of Bmal1 expression in the dorsomedial hypothalamus is sufficient to restore circadian food anticipatory rhythms in Bmal1-/- mice

Daily feeding schedules generate food anticipatory rhythms of behavior and physiology that exhibit canonical properties of circadian clock control. The molecular mechanisms and location of food-entrainable circadian oscillators hypothesized to control food anticipatory rhythms are unknown. In 2008, Fuller et al reported that food-entrainable circadian rhythms are absent in mice bearing a null mutation of the circadian clock gene Bmal1 and that these rhythms can be rescued by virally-mediated restoration of Bmal1 expression in the dorsomedial nucleus of the hypothalamus (DMH) but not in the suprachiasmatic nucleus (site of the master light-entrainable circadian pacemaker). These results, taken together with controversial DMH lesion results published by the same laboratory, appear to establish the DMH as the site of a Bmal1-dependent circadian mechanism necessary and sufficient for food anticipatory rhythms. However, careful examination of the manuscript reveals numerous weaknesses in the evidence as presented. These problems are grouped as follows and elaborated in detail: 1. data management issues (apparent misalignments of plotted data), 2. failure of evidence to support the major conclusions, and 3. missing data and methodological details. The Fuller et al results are therefore considered inconclusive, and fail to clarify the role of either the DMH or Bmal1 in the expression of food-entrainable circadian rhythms in rodents

Sleep Timing and Duration in Indigenous Villages with and Without Electric Lighting on Tanna Island, Vanuatu

It has been hypothesized that sleep in the industrialized world is in chronic deficit, due in part to evening light exposure, which delays sleep onset and truncates sleep depending on morning work or school schedules. If so, societies without electricity may sleep longer. However, recent studies of hunter-gatherers and pastoralists living traditional lifestyles without electricity report short sleep compared to industrialized population norms. To further explore the impact of lifestyles and electrification on sleep, we measured sleep by actigraphy in indigenous Melanesians on Tanna Island, Vanuatu, who live traditional subsistence horticultural lifestyles, in villages either with or without access to electricity. Sleep duration was long and efficiency low in both groups, compared to averages from actigraphy studies of industrialized populations. In villages with electricity, light exposure after sunset was increased, sleep onset was delayed, and nocturnal sleep duration was reduced. These effects were driven primarily by breastfeeding mothers living with electric lighting. Relatively long sleep on Tanna may reflect advantages of an environment in which food access is reliable, climate benign, and predators and significant social conflict absent. Despite exposure to outdoor light throughout the day, an effect of artificial evening light was nonetheless detectable on sleep timing and duration

Enhanced Food Anticipatory Activity Associated with Enhanced Activation of Extrahypothalamic Neural Pathways in Serotonin2C Receptor Null Mutant Mice

The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin2C receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enhanced food anticipatory activity compared to wild-type littermates, without phenotypic differences in the impact of restricted feeding on food consumption, body weight loss, or blood glucose levels. Moreover, we show that the enhanced food anticipatory activity in 5-HT2CR null mutant mice develops independent of external light cues and persists during two days of total food deprivation, indicating that food anticipatory activity in 5-HT2CR null mutant mice reflects the locomotor output of a food-entrainable oscillator. Whereas restricted feeding induces c-fos expression to a similar extent in hypothalamic nuclei of wild-type and null mutant animals, it produces enhanced expression in the nucleus accumbens and other extrahypothalamic regions of null mutant mice relative to wild-type subjects. These data suggest that 5-HT2CRs gate food anticipatory activity through mechanisms involving extrahypothalamic neural pathways