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. 2022 Feb 15:11:e70271.
doi: 10.7554/eLife.70271.

Assessing the effects of stress on feeding behaviors in laboratory mice

Marie Francois  1 , Isabella Canal Delgado  1 , Nikolay Shargorodsky  1 , Cheng-Shiun Leu  1   2 , Lori Zeltser  1   3
Affiliations

Assessing the effects of stress on feeding behaviors in laboratory mice

Marie Francois et al. Elife. .

Abstract

Stress often affects eating behaviors, increasing caloric intake in some individuals and decreasing it in others. The determinants of feeding responses to stress are unknown, in part because this issue is rarely studied in rodents. We focused our efforts on the novelty-suppressed feeding (NSF) assay, which uses latency to eat as readout of anxiety-like behavior, but rarely assesses feeding per se. We explored how key variables in experimental paradigms - estrous and diurnal cyclicity, age and duration of social isolation, prandial state, diet palatability, and elevated body weight - influence stress-induced anxiety-like behavior and food intake in male and female C57BL/6J mice. Latency to eat in the novel environment is increased in both sexes across most of the conditions tested, while effects on caloric intake are variable. In the common NSF assay (i.e., lean mice in the light cycle), sex-specific effects of the length of social isolation, and not estrous cyclicity, are the main source of variability. Under conditions that are more physiologically relevant for humans (i.e., overweight mice in the active phase), the novel stress now elicits robust hyperphagia in both sexes . This novel model of stress eating can be used to identify underlying neuroendocrine and neuronal substrates. Moreover, these studies can serve as a framework to integrate cross-disciplinary studies of anxiety and feeding related behaviors in rodents.

Keywords: circadian rhythm; emotional eating; mouse; neuroscience; novelty suppressed feeding; sex differences; social isolation; stress.

Plain language summary

In times of heightened anxiety – say, during a global pandemic – many of us will reach for donuts or a particularly appetizing pizza for comfort. Others, however, will tend to shun food. What underlies these differences, and, in fact, the neural and hormonal pathways at play during stress eating (when people eat without being hungry due to emotional reasons), remain unclear. This is partly because scientists lack good animal models in which to study these behaviors. In particular, female rodents are usually excluded from studies under the assumption that their hormonal cycles will disrupt the results. Yet, women are overrepresented in studies on feeding habits. Modeling human behaviors using rodents is harder than it may appear. These animals are most active at night – yet most experiments are performed during the day. The same stressors also have different outcomes in males and females. François et al. therefore explored better ways to induce anxiety and evaluate feeding behavior in mice, hoping to reliably elicit stress eating. The starting point was a common type of experiments known as novelty-suppressed feeding. First, mice are kept alone in a cage for up to two weeks on a normal diet so that they are used to experimental conditions. Then they are deprived of food overnight, before being given free access to food in the morning in a new environment. This stressful experience normally causes mice to take longer to eat than in their home cage. In rodents, the delay is thought to reflect stress as it is reliably reversed by anti-anxiety compounds approved for human use. In the novelty-suppressed feeding assay, both male and female animals exhibit signs of anxiety, but how much females eat is variable. François et al. showed that this variability is not due to hormonal changes, but instead to how long female mice had been kept alone. Crucially, the test could be adapted so that mice would consistently exhibit behavior similar to human stress eating, whereby they eat more during the test without having fasted the night before. The changes included running the experiment at night, when the animals are normally most active, and using overweight mice (which captures the fact that, in humans, being overweight is associated with being prone to stress eating). Stress eating is an important clinical issue, hindering weigh loss in people with obesity. The new model developed by François et al. could be adopted by other laboratories, enabling better research into this behavior.

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Conflict of interest statement

MF, IC, NS, CL, LZ No competing interests declared

Figures

Figure 1.
Figure 1.. Effects of sex and estrous cyclicity in the standard novelty-suppressed feeding (NSF) assay.
(A) Experimental paradigm for the standard NSF assay performed on the bench, in the morning following an overnight fast. (B) Latency to eat in home (H) and novel (N) tests in males (blue, n = 10, paired t-test: t = 4.094; df = 9; p = 0.027) and females (red, n = 17, Wilcoxon test: W = 141; p = 0.0002). (C) Latency to eat in the home test in males (blue, n = 10) and females (red, n = 17) categorized by estrous cycle stage (D: diestrus; P: proestrus; E: estrus) (one-way analysis of variance [ANOVA]: F(3, 23) = 22.52; p < 0.0001). (D) Latency to eat in the novel test in males (blue, n = 10) and females (red, n = 17) categorized by estrous cycle stage (Kruskal–Wallis test: H(3) = 16.80; p = 0.0008). (E) Food intake in home (H) and novel (N) tests in males (blue, n = 10, Wilcoxon test: W = −36; p = 0.0078) and in females (red, n = 17, Wilcoxon test: W = −67; p = 0.0562). (F) Food intake in males (blue, n = 10) and females (red, n = 17) categorized by estrous cycle stage in the home test (Kruskal–Wallis test: H(3) = 15.86; p = 0.0012). (G) Food intake in males (blue, n = 10) and females (red, n = 17) categorized by estrous cycle stage in the novel test (one-way ANOVA: F(3, 23) = 2,370; p = 0.0968). Significant differences denoted by different letters. **p < 0.01 and ***p < 0.001 between home and novel tests. SI, social isolation; n.s., not significant. See Figure 1—source data 1.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Evaluation of the estrous phase with cytology and hormone level measurements.
(A) Representative pictures of vaginal cytology used to characterize phases of the estrous cycle. (B) Circulating levels of luteinizing hormone (LH) across the estrous cycle (Kruskal–Wallis test: H(2) = 0.1165; p = 9434). (C) Circulating levels of follicle-stimulating hormone (FSH) across the estrous cycle (Kruskal–Wallis test: H(2) = 17.20; p = 0.0002). (D) Circulating levels of estradiol across the estrous cycle (one-way analysis of variance [ANOVA]: F(2, 45) = 0.02807). Significant differences denoted by different letters. n.s., not significant. See Figure 1—source data 2.
Figure 2.
Figure 2.. Effects of minimal environmental stress on the novelty-suppressed feeding (NSF) assay in females.
(A) Experimental paradigm for the automated NSF assay performed in the automated recording system, in the morning following an overnight fast, in adult female mice socially isolated 2 weeks before the tests (n = 21). (B) Latency to eat in the home test in females categorized by estrous cycle stage (D: diestrus; P: proestrus; E: estrus) (Kruskal–Wallis test: H(2) = 1.269; p = 0.5479). (C) Latency to eat in the novel test in females categorized by estrous cycle stage (one-way analysis of variance [ANOVA]: F(2, 18) = 1.385; p = 0.2757). (D) Latency to eat in home (H) and novel (N) tests (Wilcoxon test: W = 151; p = 0.0071). (E) Food intake in the home test in females categorized by estrous cycle stage (one-way ANOVA: F(2, 18) = 0.0701; p = 0.9326). (F) Food intake in the novel test in females categorized by estrous cycle stage (one-way ANOVA: F(2, 18) = 0.2336; p = 0.7941). (G) Food intake in home (H) and novel (N) tests (paired t-test: t = 1.810; df = 20; p = 0.0854). **p < 0.01 between home and novel tests. SI, social isolation; n.s., not significant. See Figure 2—source data 1.
Figure 3.
Figure 3.. Effects of adolescent social isolation on the novelty-suppressed feeding (NSF) assay in males and females.
(A) Experimental paradigm for the NSF assay performed in the morning following an overnight fast, in adult mice socially isolated at 5 weeks of age. (B) Latency to eat in home (H) and novel (N) tests in males (blue, n = 11, Wilcoxon test: W = 66; p = 0.001) and in females (green, n = 8, Wilcoxon test: W = 36; p = 0.0078). (C) Food intake in home (H) and novel (N) tests in males (blue, n = 11, paired t-test: t = 2.193; df = 10; p = 0.0531) and in females (green, n = 8, paired t-test: t = 6.347; df = 7; p = 0.0004). **p < 0.01, ***p < 0.001 between home and novel tests. SI, social isolation. See Figure 3—source data 1.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Parsing the effects of length vs. timing of social isolation on the standard novelty suppressed feeding (NSF) assay in females.
(A) Experimental paradigm for the manual novelty-suppressed feeding (NSF) assay in adolescent and adult females. (B) Latency to eat in home (H) and novel (N) tests in adult females socially isolated from 8 weeks of age for 7 weeks (red, n = 9, Wilcoxon test: W = 43; p = 0.0078) and in 7-week-old female mice socially isolated at 5 weeks of age (green, n = 13, Wilcoxon test: W = 85; p = 0.0012). (C) Food intake in home (H) and novel (N) tests in adult females socially isolated at 8 weeks of age for 7 weeks (red, n = 9, paired t-test: t = 2.211; df = 8; p = 0.058) and in 7-week-old female mice socially isolated at 5 weeks of age (green, n = 13, Wilcoxon test: W = 18; p = 0.2656). **p < 0.01. SI, social isolation; n.s., not significant. See Figure 3—source data 2.
Figure 4.
Figure 4.. Effects of diurnal factors on the novelty-suppressed feeding (NSF) assay in males and females.
(A) Experimental paradigm for the automated NSF assays performed at the onset of the dark phase in adult mice. (B) Food intake per g body weight across the light cycle (white bar) and dark cycle (black bar) of female mice fed ad libitum (AL) or on a 7 pm to 7 am schedule (n = 7, paired t-test: t = 1.777; df = 6; p = 1259). (C) Latency to eat in home (H) and novel (N) tests in males socially isolated 2 weeks before the tests (blue, n = 15, Wilcoxon test: W = 92; p = 0.0067), females socially isolated during adolescence (green, n = 16, Wilcoxon test: W = 120; p = 0.0008), and females socially isolated 2 weeks before the tests (red, n = 12, Wilcoxon test: W = 126; p = 0.0003). (D) Food intake in home (H) and novel (N) tests in males socially isolated 2 weeks before the tests (blue, n = 15, Wilcoxon test: W = −4.0; p = 0.9229), females socially isolated during adolescence (green, n = 16, paired t-test: t = 2.166; df = 15; p = 0.0468), and females socially isolated 2 weeks before the test (red, n = 12, Wilcoxon test: W = 106; p = 0.0038). *p < 0.05, **p < 0.01, ***p < 0.001 between home and novel tests. SI, social isolation; n.s., not significant. See Figure 4—source data 1.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Influences of the estrous cycle when the stress assay is performed in the dark phase of the diurnal cycle.
(A) Home test latency across the estrous cycle (D: diestrus; P: proestrus; E: estrus) when the assay is performed at the onset of the dark phase (n = 28, Kruskal–Wallis test: H(2)= 0.5602; p = 0.7557). (B) Novel test latency across the estrous cycle when the assay is performed at the onset of the dark phase (n = 28, one-way analysis of variance [ANOVA]: F(2, 25) = 1.197; p = 0.0053). (C) Home test latency across the estrous cycle when the assay is performed at the onset of the dark phase (n = 28, Kruskal–Wallis test: H(2) = 7.649; p = 0.0218). (D) Novel test latency across the estrous cycle when the assay is performed at the onset of the dark phase (n = 28, Kruskal–Wallis test: H(2) = 0.4612; p = 0.7941). Significant differences denoted by different letters. SI, social isolation; n.s., not significant. See Figure 4—source data 1.
Figure 4—figure supplement 2
Figure 4—figure supplement 2. Parsing the effects of the time of day vs. prandial state on the effects of novel environment stress on feeding behavior.
(A) Experimental paradigm for the automated novelty-suppressed feeding (NSF) assays performed at the onset of the dark phase following 90% calorie restriction in adult females (n = 8). (B) Representation of how 7 am to 7 pm average intake preceding an overnight fast was matched on a 7 pm to 7 am food access schedule. (C) Latency to eat in home (H) and novel (N) tests (paired t-test: t = 2.932, df = 7; p = 0.022). (D) Food intake in home (H) and novel (N) tests (paired t-test: t = 2.104, df = 7; p = 0.0734). *p < 0.05. SI, social isolation; n.s., not significant. See Figure 4—source data 2.
Figure 5.
Figure 5.. Effects of chronic exposure to high-fat diet (HFD) on the novelty-suppressed feeding (NSF) assay in males and females.
(A) Experimental paradigm for the automated NSF assay performed at the onset of the dark phase in adult chronically exposed to HFD mice socially isolated 2 weeks before the tests. (B) Body weights before exposure to HFD and at the time of the tests, in males (blue, n = 13, paired t-test: t = 11.25; df = 12; p < 0.0001) and females (red, n = 14, paired t-test: t = 13.53; df = 13; p < 0.0001). (C) Blood glucose levels in males (blue, n = 13) and females (red, n = 14, unpaired t-test: t = 3.460; df = 24; p = 0.002). (D) Latency to eat in home (H) and novel (N) tests in adult males (blue, n = 13, paired t-test: t = 4.751; df = 12; p = 0.0005) and females (red, n = 14, Wilcoxon test: W = 5; p = 0.9032). (E) Food intake in home (H) and novel (N) tests in adult males (blue, n = 13, Wilcoxon test: W = 89; p = 0.0005) and in adult females (red, n = 14, Wilcoxon test: W = 105; p = 0.0001). (F) Correlation between change in food intake in N vs. H and change in body weight before and after chronic exposure to HFD. *p < 0.05, ***p < 0.001, ****p < 0.0001. SI, social isolation; n.s., not significant. See Figure 5—source data 1.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Effect of novel envrionment stress assessed in the dark phase of the light cycle in lean mice acutely exposed to high-fat diet (HFD).
(A) Experimental paradigm for the automated novelty-suppressed feeding (NSF) assay performed at the onset of the dark phase in lean adult socially isolated 2 weeks before the tests, when high-fat diet (HFD) was presented during the test. (B) Body weights before acclimation to HFD and at the time of the tests, in males (blue, n = 11, paired t-test: t = 1.266; df = 10; p = 0.2343) and females (red, n = 15, paired t-test: t = 1.389; df = 15; p = 0.1865). (C) Latency to eat in home (H) and novel (N) tests in males (blue, n = 11, Wilcoxon test: W = 46; p = 0.04) and females (red, n = 15, paired t-test: t = 0.6366; df = 14; p = 0.5347). (D) Food intake in home (H) and novel (N) tests in adult males (blue, n = 11, paired t-test: t = 3.956; df = 10; p = 0.0027) and in adult females (red, n = 15, paired t-test: t = 0.5385; df = 14; p = 0.5987). *p < 0.05; **p < 0.01. SI, social isolation; n.s., not significant. See Figure 5—source data 2.
Figure 6.
Figure 6.. Summary.
Combining experimental variables that influence stress-induced food intake in the same direction yields consistent and reproducible effects in males and females. Sex-specific models of stress-induced hypophagia (top panel). The lean state and performing the assay in the light phase promote hypophagic responses. A short period of social isolation in adult (≥10 weeks) males elicits hypophagic responses, but a prolonged (~6 weeks) period of social isolation starting in adolescence (5 weeks) is needed to produce the same effect in females. Sex-independent model of stress-induced hyperphagia (bottom panel). Chronic exposure to high fat diet and performing the assay in the dark phase promote hyperphagic responses in both sexes.
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