Conclusions from a behavioral aging study on male and female F2 hybrid mice on age-related behavior, buoyancy in water-based tests, and an ethical method to assess lifespan

Abstract

Due to strain-specific behavioral idiosyncrasies, inbred mouse strains are suboptimal research models for behavioral aging studies. The aim of this study is to determine age-related behavioral changes of F2 hybrid C57BL/6NxBALB/c male and female mice. Lifespan was followed (n_males=48, n_females=51) and cohorts of mature adult (7 months), middle-aged (15 months), and old mice (22 months of age; n=7-12 per group) were assessed regarding open-field activity, exploration, passive avoidance learning/memory, and depressive-like behavior. We found that both males and females demonstrated decreased exploratory behavior with age, while memory and depressive-like behavior were maintained. Females exhibited enhanced depressive-like behavior compared to males; however, a correlation between fat mass and swimming activity in the test directly accounted for 30-46% of this behavioral sex difference. In addition, we suggest a method to qualitatively estimate natural lifespan from survival analyses in which animals with signs of pain or severe disease are euthanized. This is, to our knowledge, the first behavioral study to consider both sex and aging in hybrid mice. We here define decreased exploratory behavior as a conserved hallmark of aging independent of sex, highlight the effect of buoyancy in water tests, and provide a method to assay lifespan with reduced animal suffering.

Keywords: F2 hybrid mice; aging; exploratory activity; sex comparison; water-based behavioral tests.

Figure 1

Survival of C57BL/6N×BALB/c F2 hybrid male and female mice presented as intervals of natural lifespan. The lower limit of the lifespans, referred to as “Survival Curve A” in Table 2, was obtained by considering euthanization of animals as the same fate as natural death and the upper limit of the lifespans, referred to as “Survival Curve B” in Table 2, by considering euthanized animals as healthy upon removal (censored) and only animals that died of intrinsic causes were counted as deaths. Animals in the cohorts for behavioral assessment were included in the survival analysis until the time of their first test (7, 15, or 22 months of age). Timepoints are indicated by dotted vertical lines. Total number of animals: n_males=48, n_females=51.

Figure 2

Physiological parameters of mice in the cohorts for behavioral assessment. (A) There was no difference in body weight between the cohorts, but (B) DEXA analysis showed that (p_M7-F7=0.0078, p_M15-F15=0.0011; and lean mass increased with age in males p_M7-22=0.040, p_M15-22=0.036; Mann Whitney), whereas (C) females exhibited increased fat mass as compared to males (p_M7-F7=0.0023, p_M15-F15=0.0012; Welch’s t-test). Values are mean ± SEM; n_M=7, n_M15=10, n_M22=10, n_F7=9, n_F15=12, n_F22=7.

Figure 3

Horizontal and rearing activity per minute in an activity box open field test. (A) Activity in the first 5 minutes – the exploratory phase – decreased with aging for both males and females. The decline in horizontal (p_M7-22=0.0065, p_F7-22=0.0006; Student’s t-test) and rearing activities (p_M7-22=0.0003, p_F7-22=0.068; Mann-Whitney) indicated that exploratory behavior was highly affected by age. (B) Activity in 1-hour sessions of the same test. Horizontal activity was not altered from 7 to 22 months of age, whilst rearing activity declined for both female and male hybrids, thus illustrating an aging effect (p_F7-22=0.043, p_M7-22=0.043; Mann-Whitney). The decrease in horizontal activity observed in 15-month-old females was likely caused by different handling of this particular group due to technical reasons (see Methods). Values are mean ± SEM; n_M7=7, n_M15=10, n_M22=10, n_F7=10, n_F15=12, n_F22=7.

Figure 4

Learning and memory assessed by shuttle box passive avoidance test. Data are presented as the time it took to enter the conditioned area on the testing day (Day 2) subtracted by the time to enter on the conditioning day (Day 1), calculated for each individual animal, and indicate no major age or sex difference (p_M7-F7=0.055, p_M15-F15=0.97, p_M22-F22=0.74, p_M7-15=0.11, p_M7-22=0.23, p_M15-22=0.19, p_F7-15=0.62, p_F7-22=0.30, p_F15-22=0.60; Mann-Whitney). Values are mean ± SEM; n_M7=7, n_M15=10, n_M22=10, n_F7=9, n_F15=12, n_F22=7.

Figure 5

Forced swim test to assay depressive-like behavior. (A) Immobility in the forced swim test is considered an indicator of depressive-like behavior, and F2 hybrid females were found to be more immobile (p=0.0013, 2-way ANOVA), as compared to males. Values are mean ± SEM. (B) Due to no age effect found, all timepoints for each sex were pooled to demonstrate a 20% difference in immobility between females and males (p=0.0002, Mann-Whitney). Values are mean ± SEM. (C) Immobility plotted against fat mass for each animal. Females exhibited increased fat mass as compared to males, and immobility was found to correlate to percent fat mass. The correlation slopes were constrained to be equal since they were not significantly different (p=0.58, ANCOVA). (D) Bayesian mediation analysis separated the total effect of the difference between males and females in the forced swim test into effect mediated by fat mass or by unknown mechanisms. The sex difference in fat mass accounts for 30% of the sex difference in immobility (Bayesian p=0.96) and other mechanisms account for the remaining 70% (Bayesian p=0.99; Bayesian mediation analysis). Values are mean ±50% (thick lines) and 95% confidence interval (thin lines); n_M7=7, n_M15=10, n_M22=10, n_F7=9, n_F15=12, n_F22=7.