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

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.


14) Positional reflexes
Assessed only in case animal is continuously lying on the ground -2 not present -1 reduced 0 no alterations

Analysis of fecal corticosterone metabolites
The collection of fecal samples was carried out in the morning (7 to 11 a.m.) directly after the Open field paradigm. Feces of the mice (n=200) were collected in the Open field arenas, where the animals were placed and tested individually. In order to collect a sufficient amount of sample material, animals were then placed individually into Makrolon open cages type II (Ehret GmbH & Co. KG, Emmendingen, Germany), supplemented with bedding material (Lignocel Select, J. Rettenmaier & Söhne GmbH & Co. KG, Rosenberg, Germany), and feces were collected from these cages after two hours. The samples were stored frozen at -20°C. For processing, feces were dried and homogenized, and aliquots of 0.05 g were extracted with 1 ml of 80% methanol 1 . The samples were analyzed using a 5-pregnane-3,11,21-triol-20-one enzyme immunoassay, which has been established and fully validated for the measurement of fecal corticosterone metabolites in mice 2,3 .

Analysis of nest building behavior
For the image-based analysis of nest building behavior we applied the following scoring scheme: Score 1: Nestlets are almost not manipulated (> 90% are intact); Score 2: Nestlets are slightly manipulated (50-90% are intact); Score 3: nestlets are largely manipulated (50-90% are torn); Score 4: Flat nest (> 90% are torn, shreds are placed in one quarter of the cage, < 50% of the walls are higher than the mouse); Score 5: Nearly perfect nest (> 90% are torn, > 50% of the walls are higher than the mouse); Score 6: Perfect nest (> 90% are torn, > 90% of the walls are higher than the mouse).

Supplementary Figures
Supplementary Figure S1. Burrowing performance. In the first burrowing test session during the light phase (a), a relatively low mean performance was observed among all age groups.
Age-and sex-related differences were not detected (interaction p=0.6497, age phase p=0.3715, sex p=0.8856). Two-way ANOVA, followed by Bonferroni multiple comparison tests. * p<0.05. Colored dots refer to the respective age group (red P25, orange P36, green P50, yellow P65, blue P120). Error bars indicate the standard error of the mean (SEM).