Temperature-dependent differences in mouse gut motility are mediated by stress

The study investigates how ambient housing temperature affects mouse gastrointestinal motility and the mechanisms underlying these effects. Standard laboratory housing (~22°C) is not evidence-based for mice and likely induces chronic cold stress, whereas thermoneutrality for mice is ~29–33°C (commonly 30°C). Cold stress at 22°C alters metabolism, cardiovascular parameters, immune function, and tumor growth and may undermine translational relevance and reproducibility. Basic aspects of gut physiology, including motility, have not been characterized at thermoneutrality. Prior work links cold exposure to activation of the hypothalamic-pituitary-adrenal (HPA) axis and shows that stress hormones (e.g., CRH, corticosterone) modulate gut motility. The authors hypothesize that ambient temperature influences gut motility via stress pathways and aim to delineate whether differences arise from compartment-specific motility, the gut microbiota, or HPA axis signaling, and whether these changes are reversible by acclimation in adulthood.

- Standard housing temperature (~22°C) for mice is based on human comfort, not mouse physiology; multiple studies indicate 22°C induces chronic cold stress affecting metabolism, cardiovascular physiology, immunity, and tumor biology. - Thermoneutrality for mice (29–33°C) is argued to improve translational fidelity; many studies show phenotypes at 30°C better align with humans in diverse contexts. - Prior studies in non-model organisms found conflicting effects of cold on GI motility (faster gastric emptying in dogs; slower GI motility in young chickens except esophagus/crop). - Stress-gut axis: Cold exposure elevates corticosterone in rodents; restraint stress or exogenous CRH alters GI motility in rats and raises corticosterone with dysmotility in mice, implicating the HPA axis in motility control. - Ambient temperature influences mouse gut microbiota composition; motility itself correlates with microbiome features in humans (e.g., reduced Akkermansia muciniphila associates with faster transit). Group housing can mitigate cold stress effects on food intake compared with single housing.

Animal models and housing: C57BL/6NJ and 129×1/SvJ mice were bred and reared under SPF conditions at either 22°C (standard) or 30°C (thermoneutral) in Innovive filter-top cages with ad libitum chow and water; group-housed (2–5 per cage), both sexes, aged 10–18 weeks. C57BL/6J-Crh+ (CRH-deficient) mice were included for genetic studies. Mice acclimated to any new environment for at least 10 days. Experiments were performed in the morning (ZT2–ZT5). Gut motility assays: - Whole gut transit time (WGTT): Following a 16 h fast, mice were gavaged with 300 µl of 6% carmine red in 0.5% methylcellulose; time to first red fecal pellet was recorded. - Gastric emptying: Following 16 h fast, gavage of 100 µl 25 ng/µl FITC-dextran (70 kDa) in 0.5% methylcellulose; after 30 min, stomach and small intestine (SI) collected, homogenized, and fluorescence quantified; gastric emptying expressed as stomach fluorescence fraction of total. - Small intestinal transit: FITC-dextran geometric center calculated across 8 equal SI segments; complementary “leading edge” method used 200 µl of 10% activated charcoal in 0.5% methylcellulose and measured bolus distance/total SI length after 30 min. - Colon transit: Standard distal colon bead expulsion assay after 16 h fast: 3 mm glass bead inserted 2 cm into distal colon under isoflurane; expulsion time recorded. Temperature acclimation (swap) experiment: WGTT measured at baseline; mice either stayed at their original temperature (controls) or were transferred to the alternate temperature (22↔30°C) for 2 weeks; WGTT re-measured and within-mouse change calculated. Microbiome profiling and manipulation: - Shotgun metagenomics of fecal pellets using Illumina Nextera XT libraries and HiSeq 4000; reads classified with Kraken2/Bracken against the Mouse Gastrointestinal Bacteria Catalogue; beta diversity (Bray–Curtis, PCoA, PERMANOVA), alpha diversity (Simpson, Shannon), and species enrichment (ALDEx2, BH correction). - Antibiotic depletion: 2-week drinking-water cocktail (vancomycin 1 g/l, ampicillin 1 g/l, neomycin 1 g/l, metronidazole 0.5 g/l) in flavored vehicle; microbiota depletion verified by culture-based CFU reduction and fecal DNA quantitation. WGTT assessed in vehicle vs antibiotic-treated cohorts at both temperatures. - Serotonin ELISA on feces to assess microbiome-regulated hormone related to motility. Ex vivo colon motility: Isolated colons with attached cecum mounted in 37°C Krebs’ solution (oxygenated with 95% O2/5% CO2); after 10 min acclimation, high-resolution video recorded and analyzed (Scribble 2.0/Matlab plugin Analyse 2.0) for propulsive contraction velocity, duration, and length. HPA axis assessments: - Plasma corticosterone and ACTH measured by ELISA at ~ZT3 following CO2 euthanasia and cardiac puncture. - CRH expression: RNAscope fluorescent in situ hybridization for Crh mRNA in paraventricular hypothalamus (PVH); quantified total fluorescence intensity in ROI. Pharmacologic and genetic modulation of CRH: - Astressin (CRH receptor antagonist) administered intraperitoneally at 200 µg/kg vs PBS control, followed by colon bead expulsion assay. - CRH genetic deletion: Crh−/− mice compared with controls at 22°C and 30°C for effects on gut motility. Food intake and body mass: Average food consumption per mouse per day measured in group-housed cages over 3 days (basket weights at ZT10). Longitudinal body mass tracked; sex stratification examined. Statistics: R v4.2.1; two-sided tests at α=0.05. Shapiro-Wilk to assess normality; Student’s t-test for normal data; Wilcoxon rank-sum otherwise. Sample sizes and exact statistics provided per figure; outliers excluded per predefined criteria (e.g., hemolyzed plasma, distress).

- Ambient temperature robustly alters gut motility: - WGTT was roughly 2× faster in 22°C mice versus 30°C; effect observed in both C57BL/6 and 129×1/SvJ strains; no sex-dependent effect on WGTT. - Compartment-specific effects: Gastric emptying showed no difference (P=0.655). Small intestinal transit was modestly slower at 22°C versus 30°C (FITC-dextran geometric center t=−2.30, P=0.0293; leading-edge charcoal corroborated). Colon transit was ~3× faster at 22°C than 30°C (significantly different). - Microbiome differences by temperature but not causal for motility differences: - Fecal microbiota beta diversity differed by housing temperature (PERMANOVA F=13.710, P=0.001). Alpha diversity higher at 22°C (Simpson t=5.24, P=1.80×10−5; Shannon t=4.94, P=3.11×10−5). Twenty-nine species differentially enriched; Akkermansia muciniphila reduced at 22°C (Wilcoxon P=0.0331), consistent with signatures of faster transit. - Antibiotic depletion did not abolish the WGTT difference: 22°C remained faster than 30°C under both vehicle and antibiotic treatment (vehicle+Abx 22°C vs 30°C t=−5.19, P=9.66×10−6). Antibiotics slightly slowed WGTT at 30°C but not at 22°C. Fecal serotonin concentrations were similar between temperatures (P=0.725). - Acclimation in adulthood is sufficient: Switching 30→22°C decreased WGTT versus 30→30°C controls (W=304.5, P=6.27×10−6); switching 22→30°C increased WGTT versus 22→22°C controls (t=3.82, P=6.59×10−3). Control groups showed no change. - Colon-intrinsic properties are not significantly different ex vivo: Velocity, duration, and length of propulsive contractions in isolated colons trended higher at 22°C but were not statistically significant, implying extrinsic regulation. - HPA axis activation at 22°C correlates with motility differences: - Plasma corticosterone elevated at 22°C in both sexes (males t=3.07, P=0.00662; females t=2.80, P=0.0159). ACTH trended higher at 22°C (not statistically significant). PVH Crh mRNA expression significantly elevated at 22°C (t=2.63, P=0.0275). - Pharmacologic CRH inhibition (astressin) or genetic CRH deletion reduced motility in 22°C mice to 30°C levels, with little to no effect in 30°C mice, indicating CRH-dependent mediation of temperature effects on motility. - Food intake and body mass: No significant differences in average food intake between 22°C and 30°C in group-housed mice; similar weight gain trajectories, with only slightly higher body mass in female 30°C mice.

The findings demonstrate that housing temperature is a potent modulator of mouse gut motility, with 22°C producing a markedly faster whole-gut transit driven by colonic acceleration and a slight slowing in small intestinal transit. The consistent temperature effect across strains and its rapid reversibility with a 2-week acclimation suggest an adult, colon-extrinsic regulatory mechanism rather than developmental programming. Ex vivo assays showed no significant intrinsic differences in colon contractile properties, pointing to systemic signals. Microbiome composition differed substantially by temperature, including reduced A. muciniphila at 22°C, but antibiotic ablation and unchanged fecal serotonin indicate that microbiota are not the primary drivers of the motility phenotype. Instead, elevated HPA axis activity at 22°C (higher corticosterone and PVH Crh expression) and the loss of temperature-dependent motility differences upon CRH blockade or deletion support a model where chronic cold stress increases gut motility via CRH-dependent stress signaling. These results have implications for translational research: shifting facilities to thermoneutrality may alter drug absorption and disease model outcomes, influence microbiome composition, and reduce stress-related variability, potentially improving reproducibility. Researchers working on gut immunity, infection models, or pharmacokinetics should consider temperature-driven motility differences when designing and interpreting experiments.

This study identifies ambient temperature as a key environmental factor shaping mouse gut motility and establishes the HPA axis—specifically CRH signaling—as the central mediator of temperature-dependent differences. Mice housed at 22°C exhibit approximately twofold faster whole-gut transit than thermoneutral (30°C) mice, driven by a threefold acceleration in colonic transit. Although gut microbiota composition differs by temperature, it is not the primary cause of motility changes. The motility phenotype is reversible with brief acclimation, colon-extrinsic, and abrogated by pharmacologic or genetic CRH inhibition. These insights underscore the importance of carefully controlling and reporting housing temperatures to enhance reproducibility and translational relevance in mouse studies. Future directions include: defining upstream cold-sensing pathways (e.g., TRPA1) and social factors driving HPA activation; characterizing specific in vivo motility patterns (e.g., giant migrating contractions) altered by temperature; testing linearity or thresholds in the temperature–motility relationship; using germ-free mice to conclusively exclude microbiome causality; assessing contributions from metabolism and water intake; and evaluating how thermoneutral housing influences outcomes in stress-related, immunological, infection, and pharmacological models.

- Microbiome causality: Although antibiotics markedly reduced microbial load, residual or uncultured taxa may remain; germ-free validation was not performed. - Brief procedural exposure: Thermoneutral (30°C) mice experienced short exposures to ~22°C during procedures (except colon transit assay), introducing potential confounding stress. - Metabolic and hydration factors: Metabolic rates were not measured (group housing precluded indirect calorimetry), and differential water intake was not assessed; both could influence motility. - Motility mechanisms: Specific in vivo colonic motility patterns (e.g., giant migrating contractions) responsible for faster transit at 22°C were not directly measured. - Stress paradigm scope: The study did not induce or compare different stress types/timings; ACTH differences trended but were not significant, and sex-dependent corticosterone differences at 22°C did not translate to sex differences in WGTT. - Ex vivo sensitivity: Ex vivo colon assays showed non-significant trends; subtle intrinsic differences or neural inputs may not be fully captured in the preparation.