A single incidental dark pulse during daytime attenuated food anticipatory behavior.

Using an open-source operant feeding device (FED3), we measured food-seeking nose poking behavior in mice. When the mice were exposed to 4 h restricted feeding at night, all mice exhibited robust food anticipatory nose poking starting ~4 h before scheduled mealtime. When the light-dark cycle was advanced by 6 h, mice exhibited two distinct bouts of anticipatory poking, one corresponding to actual mealtime which continued at the same time of day, and one corresponding to predicted mealtime which shifted parallel with the light-dark cycle. Likewise, two similar bouts of food-seeking behavior appeared when the light-dark cycle was delayed for 9 h. These data suggest that food anticipatory behavior is encoded to a circadian oscillator that entrains to the light-dark cycle. Two weeks after advancing the light-dark cycle, mice incidentally received a 3.5 h dark pulse in the middle of the day. This single dark pulse had a negligible effect on running wheel behavior but caused a temporary attenuation of both food anticipatory poking and pellet intake. These results suggest that the circadian oscillator controlling food anticipatory poking is sensitive to light disruption and that proper food anticipation is critical for sufficient food intake during temporally restricted feeding.

Protocol to study circadian food-anticipatory poking in mice using the feeding experimentation device version 3.

Food-anticipatory nose poking is a unique food-seeking behavior driven by the food-entrainable oscillator. Here, we present a protocol to record a novel food-seeking nose poking behavior in mice under temporally restricted feeding followed by food deprivation using the open-source feeding experimentation device version 3 (FED3). We describe steps for setting up the FED3 and cage, training, and habituation. We then detail procedures for setting up the schedule for time-restricted feeding and food deprivation and for generating ethograms from FED3 data. For complete details on the use and execution of this protocol, please refer to Ehichioya et al.1.

Gut microbiota depletion minimally affects the daily voluntary wheel running activity and food anticipatory activity in female and male C57BL/6J mice.

Emerging evidence has highlighted that the gut microbiota plays a critical role in the regulation of various aspects of mammalian physiology and behavior, including circadian rhythms. Circadian rhythms are fundamental behavioral and physiological processes that are governed by circadian pacemakers in the brain. Since mice are nocturnal, voluntary wheel running activity mostly occurs at night. This nocturnal wheel-running activity is driven by the primary circadian pacemaker located in the suprachiasmatic nucleus (SCN). Food anticipatory activity (FAA) is the increased bout of locomotor activity that precedes the scheduled short duration of a daily meal. FAA is controlled by the food-entrainable oscillator (FEO) located outside of the SCN. Several studies have shown that germ-free mice and mice with gut microbiota depletion altered those circadian behavioral rhythms. Therefore, this study was designed to test if the gut microbiota is involved in voluntary wheel running activity and FAA expression. To deplete gut microbiota, C57BL/6J wildtype mice were administered an antibiotic cocktail via their drinking water throughout the experiment. The effect of antibiotic cocktail treatment on wheel running activity rhythm in both female and male mice was not detectable with the sample size in our current study. Then mice were exposed to timed restricted feeding during the day. Both female and male mice treated with antibiotics exhibited normal FAA which was comparable with the FAA observed in the control group. Those results suggest that gut microbiota depletion has minimum effect on both circadian behavioral rhythms controlled by the SCN and FEO respectively. Our result contradicts recently published studies that reported significantly higher FAA levels in germ-free mice compared to their control counterparts and gut microbiota depletion significantly reduced voluntary activity by 50%.

A time memory engram embedded in a light-entrainable circadian clock.

A longstanding mystery in chronobiology is the location and molecular mechanism of the food-entrainable oscillator (FEO).1,2,3 The FEO is an enigmatic circadian pacemaker that controls food anticipatory activity (FAA). The FEO is implicated as a circadian oscillator that entrains to feeding time. However, the rhythmic properties of the FEO remain a mystery in part due to technical limitations in distinguishing FAA from locomotor activity controlled by the primary circadian pacemaker in the suprachiasmatic nucleus (SCN). To overcome this limitation, we used the Feeding Experimentation Device version 3 (FED3) to measure food-seeking, nose-poking behavior. When food availability was limited to 4 h at night, mice exhibited strong anticipatory nose-poking behavior prior to mealtime. When food availability was moved to the daytime, mice quickly expressed daytime anticipatory nose pokes without displaying transients. Unexpectedly, the mice also maintained nighttime anticipatory nose pokes, even though food pellets were no longer dispensed at night. We next tested if food anticipation was directly encoded on a light-entrainable oscillator by shifting the light-dark cycle without changing mealtime. Anticipatory behavior shifted in parallel with the light-dark cycle, although meal timing was unchanged. Next, we tested whether encoding meal timing for anticipatory nose pokes required a functional SCN by studying Period 1/2/3 triple knockout mice with disabled SCN. Food anticipatory nose poking of Period knockout mice shifted in parallel with the light-dark cycle independent of a functional SCN clock. Our data suggest that food anticipation time is embedded in a novel, extra-SCN light-entrainable oscillator.