Development of behavioral patterns in young C57BL/6J mice: a home cage-based study

Abstract

Evidence exists that behavioral patterns only stabilize once mice reach adulthood. Detailed information about the course of behavioral patterns is of particular relevance for neuroscientific research and for the assessment of cumulative severity in genetically modified mice. The analysis considered five age groups focusing on behavioral assessments in the animals' familiar home cage environment during the adolescence phase. We confirmed age- and sex-specific differences for several of the behavioral parameters and fecal corticosterone metabolites. Interestingly, an age-dependent decline in saccharin preference was detected in female mice. Regardless of sex, relevant levels of burrowing activity were only observed during later developmental phases. The development of nest complexity following the offer of new material was affected by age in female mice. In female and male mice, an age-dependency was evident for wheel running reaching a peak at P 50. A progressive increase with age was also observed for Open field activity. The data sets provide guidance for behavioral studies and for development of composite measure schemes for evidence-based severity assessment in young mice. Except for the burrowing test, the different behavioral tests can be applied in different age groups during post-weaning development. However, age- and sex-specific characteristics need to be considered.

Figure 1

Saccharin preference and burrowing performance. (a) Age-dependent differences in saccharin preference, shown as total saccharin intake, were only detected in female mice: preference for saccharin in prepubescent and pubescent mice exceeded that of the adult control group, and prepubescent mice showed a higher preference than the respective sexually mature mice (F(4,45) = 6.495, p = 0.0003). Regardless of sex, burrowing performance overnight (first test) increased significantly in mice reaching sexual maturity (b, first test; interaction p = 0.5912, age phase p < 0.0001, sex p = 0.0051) (c, second test; interaction p = 0.6044, age phase p < 0.0001, sex p < 0.0020). A difference between sexes was only detected in the second burrowing test during the light phase in young adult animals (d) (interaction p = 0.2394, age phase p = 0.0068, sex p = 0.0119). One-way ANOVA, followed by Bonferroni multiple comparison tests for (a), and two-way ANOVA, followed by Bonferroni multiple comparison tests for (b–d). *p < 0.05. Colored dots refer to the respective age groups. Error bars indicate the standard error of the mean (SEM).

Figure 2

Nest building performance and nest complexity. The development of nest complexity scores differed between age phases in (a) female animals (F(4, 290) = 2.75991, p = 0.02804), but not in (b) male animals (F(4, 290) = 1.07471, p = 0.3692). In male animals, the nest scores in P50 animals exceeded that of P120 animals on the second day (b, #) (F(4, 45) = 2.885, p = 0.0329). Sex differences were evident (c) on the first day in all age groups except for P25 animals (interaction p = 0.3161, age phase p = 0.0205, sex p < 0.0001). In contrast, sex differences in P25 mice became evident (d) only on the last day (interaction p = 0.6905, age phase p = 0.0504, sex p < 0.0001). Linear regression for (a) and (b); one-way ANOVA, followed by Bonferroni multiple comparison tests for #; two-way ANOVA followed by Bonferroni multiple comparison tests for (c) and (d), *p < 0.05, median. Colored dots/lines refer to the respective age groups.

Figure 3

Voluntary wheel running. The total distance moved during the entire observation period (a) increased until animals reached sexual maturity (interaction p = 0.1802, age phase p < 0.0001, sex p < 0.0001). Analysis of the increase in activity during the testing phase did not reveal age-related differences in female (b) (F(4,190) = 1.02147, p = 0.3974) and male animals (c) (F(4,190) = 0.367026, p = 0.8319). Assessment of running activity at separate days (day 1–4) (d–g) demonstrated that running activity in P50 animals exceeded that of the sex-matched P25 group on all test days. Two-way ANOVA followed by Bonferroni multiple comparison tests for (a,d–g); linear regression for (b) and (c); *p < 0.05. Colored dots/lines refer to the respective age groups. Error bars indicate the standard error of the mean (SEM).

Figure 4

Open field test. In the first 5 min of the Open field test, illustrated in (a–c), prepubescent and pubescent animals of both sexes spent more time in the ‘wall’ zone than adult mice (a) (interaction p = 0.0239, age phase p < 0.0001, sex p < 0.0095). Male prepubescent animals moved a shorter distance (b) (interaction p = 0.0733, age phase p = 0.0122, sex p = 0.1613) and moved more slowly (c) (interaction p = 0.0734, age phase p = 0.0125, sex p = 0.1618) than the sex-matched adult group. Analysis of activity during the total test duration of 15 min, illustrated in (d–f), revealed that the distance moved (d) and velocity (e) in P50 and P65 mice exceeded that of the respective prepubescent animals (d, interaction p = 0.9626, age phase p < 0.0001, sex p = 0.0441, e, interaction p = 0.9628, age phase p < 0.0001, sex p = 0.0440). Independent of the sex of the animals, rearing frequency (f) proved to be lower in prepubescent animals as compared to sexually mature, young adult, and adult animals (interaction p = 0.2458, age phase p < 0.0001, sex p = 0.7122). Two-way ANOVA, followed by Bonferroni multiple comparison tests, *p < 0.05. Colored dots refer to the respective age groups. Error bars indicate the standard error of the mean (SEM).

Figure 5

Irwin score. As compared to female adult animals, handling-associated vocalization (a) and defecation (c) scores were increased in prepubescent animals. Handling-associated urination (b) in sexually mature female animals exceeded that of female adult animals (a, F(4,95) = 3.387, p = 0.0123; b, F(4,95) = 3.437, p = 0.0114; c, F(4,95) = 3.474, p = 0.0108). Rectal body temperatures (d) in female mice exceeded those in male mice in all age groups except for P25, and body temperatures in pubescent animals of both sexes were higher as compared to those in young adult animals (interaction p < 0.0001, age phase p < 0.0001, sex p < 0.0001). One-way ANOVA, followed by Bonferroni multiple comparison tests for (a–c), and two-way ANOVA, followed by Bonferroni multiple comparison tests for (d); *p < 0.05. Colored dots refer to the respective age groups. Error bars indicate the standard error of the mean (SEM).

Figure 6

Fecal corticosterone metabolite concentrations (FCM) (a) were higher in prepubescence than in pubescence and adulthood in both female and male animals (interaction p = 0.0130, age phase p < 0.0001, sex p < 0.0001). Moreover, concentrations in prepubescent male animals were significantly higher than in all other age groups. Two-way ANOVA, followed by Bonferroni multiple comparison tests, *p < 0.05. Error bars indicate the standard error of the mean (SEM). The age-dependent body weight increase in the different age groups is illustrated in (b). Colored dots refer to the respective age groups. (c) Illustrates an overview of the weekly experimental scheme. B burrowing.