Mice and Timeline of Behavioral Testing

Fifteen male and female mdx (C57BL/10ScSn-mdx/J) and fifteen male and female wild-type (C57BL/10ScSnJ) mice were bred from homozygous mdx and wild-type lines, respectively at the animal facility of the Leiden University Medical Center (Leiden, The Netherlands). Mice were housed in individually ventilated cages (Green line, Sealsafe Plus GM500, Tecniplast, Italy) with 12 h light/dark cycles, and had ad libitum access to water and standard RM3 chow (SDS, Essex, United Kingdom) under SPF (FELASA 2014) conditions. At the age-range of 8–16 weeks, mice were transported as one cohort to the behavioral facility of the VU University (Amsterdam, The Netherlands). Upon arrival, mice were kept in individually ventilated cages (Green line, Sealsafe Plus GM500) on sawdust bedding and enriched with cardboard nesting material in the same social groups and from each cage a blood sample of 1 mouse was taken to confirm health status. Four weeks later, after confirmation of the SPF (FELASA 2014) health status, mice were moved to a different floor where behavioral experiments were carried out. Here, mice were housed in Makrolon type II conventional open cages (Type 2 short, model 1284 or 1264, Tecniplast, Italy) on sawdust bedding and enriched with cardboard nesting material. Over the course of the experiments, males were housed individually, while females were socially housed in groups of 2–3 mice per cage, except for when mice went into the PhenoTyper cages in which all mice are individually housed. At the facility of VU University, mice were housed with 12 h light/dark cycles and were ad libitum provided with water and regular chow (2018 Teklad, Harlan Laboratories, Horst, the Netherlands). The experiments were approved by the Animal Ethics Committee of VU University and performed according to Dutch regulation for animal experimentation, and in accordance with EU Directive 2010/63/EU.

To accommodate and standardize differences in age at behavioral testing, mice were assigned to 3 different batches, with the oldest mice in the first batch and youngest mice in the last batch. Mice were subjected to a series of experiments. First, at an average age of 18 weeks (youngest 16 weeks, oldest 21 weeks) mice were housed in PhenoTyper home-cages for seven days in which their spontaneous behavior was tracked with a camera-system for two and a half days, followed by a four-day discrimination and learning phase experiment to assess cognitive flexibility. Mice then had a resting period of 1–2 weeks. Thereafter, dark-light box and Y-maze tests were done on two consecutive days. Mice were sacrificed one to three days after the Y-maze test (Figure 1).

Overview of behavioral tests. Mice started at the age of ~18 weeks in the PhenoTyper home-cage in which spontaneous behavior was tracked for 2.5 days. After the spontaneous behavior assessment, mice were subjected to the DL/RL task for 4 days. After a resting period of 1–2 weeks, the DLB and the Y-maze tests were executed. SB, spontaneous behavior; DL, discrimination learning; RL, reversal learning; DLB, dark-light box.