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

Abstract

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%.

Keywords: antibiotics; circadian; food-entrainable oscillator; gut microbiome; locomotor activity; restricted feeding; reward; sex differences.

FIGURE 1

The experimental procedure for microbiota depletion and feeding schedule. The days of beginning of ad libitum feeding (ALI, ALII), timed restricted feeding (TRF), food deprivation (FD) and collection of cecum and feces are indicated with the arrows.

FIGURE 2

Group average double-plotted actograms of antibiotics-treated and control mice. Group average actograms of control females (A), control males (B), antibiotics-treated females (C), antibiotics-treated males (D) were plotted with 10 min bin and scale format (0–100 counts/min). The light and dark cycle is indicated with white and black bars on top of each actogram. Time of food availability is indicated with the light orange shading only on the left half of each actogram. ALI (ad libitum before RF), TRFI (first 4 days of timed restricted feeding), TRFII (last 7 days of timed restricted feeding) and ALII (ad libitum after RF) labeled on right side of the actograms indicates the days used to generate the daily activity profiles for before, during and after timed restricted feeding shown in Figure 4. Individual actograms of all mice are shown in Supplementary Figure S1.

FIGURE 3

Quantitative analysis of FAA. Daily changes of wheel running revolution in FAA windows (mean ± SEM) in females (A) and males (B) were presented. Steady state FAA, wheel revolution in FAA windows of the last 7 days of 4 h restricted feeding schedule were shown in (C) (females) and (D) (males). Daily changes of FAA duration in females (E) and males (F) and FAA duration in the last 7 days of 4 h restricted feeding in females (G) and males (H) were shown. Plots represent mean ± SEM. Two-way repeated measures ANOVA detected a significance only in (F) * represents p < 0.05 by Tukey’s post hoc multiple comparison test.

FIGURE 4

Group average activity profiles and nighttime activity before, during and after timed restricted feeding. Group average activity profiles in females (A): ALI, (C) TRFI, (E) TRFII, (G) ALII) and males (B): ALI, (D) TRFI, (F) TRFII, (H) ALII) were generated during the days indicated in Figure 2. Averaged total number of wheel revolution at night were plotted in (I–P) (I: female ALI, (J) male ALI, (K) female RFI, (L): male RFI, (M): female TRFII, (N): male TRFII, (O): female ALII, (P): male ALII). Each plot represents mean ± SEM.

FIGURE 5

Relative abundances of microbiota taxa from control and antibiotic-treated mice. Plot representing bacterial load in control (A) and antibiotics-treated (B) mice. Antibiotics depleted majority of gut microbiota. The “Mouse proportion” represents the number of reads that map to the One Codex database mouse genome divided by the total number of reads, while the “Bacterial proportion” represents the number of reads that map to bacteria at the taxonomic rank divided by the total number of reads. The “Other” category includes any reads classified as non-bacterial microorganisms. Taxonomic analysis showing gut microbiota population shifts at Family level. Relative abundance of top 15 families of microbial taxa in control (C), and antibiotics-treated (D) are shown. Lachnospiraceae (indicated as green) was present in all control mice but depleted in antibiotics treated mice. Erysipelotrichaceae was not detected in both control and antibiotics-treated mice. Top 15 orders and genus were shown in Supplementary Figure S5A–D. The number in the brackets represents NCBI taxonomic ID.