Phenotyping spontaneous locomotor activity in inbred and outbred mouse strains by using Digital Ventilated Cages

Mouse strains differ markedly in all behaviors, independently of their genetic background. We undertook this study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in the study design to ensure experimental reproducibility and comply with essential animal welfare principles.