Substrain-specific behavioral variation in female C57BL/6 and C57BL/10 mice

Introduction: Inbred mouse strains are essential to biomedical research, yet accumulating mutations and substrain divergence introduce phenotypic variability that can confound experimental outcomes. This study investigates behavioral differences among 13 inbred mouse substrains: eight C57BL/6 (B6) and five C57BL/10 (B10), bred in-house to control for environmental effects. Methods: Female F1 offspring underwent a standardized battery of behavioral assays-open field test (OFT), locomotor response to cocaine (LOCO), fear conditioning (FC), prepulse inhibition (PPI), and the forced swim test (FST)-chosen for their relevance to models of psychiatric and substance use disorders. Results: Significant substrain-specific differences were observed across all behaviors. In the OFT, B6 substrains such as C57BL/6J showed higher activity than others, while B10 substrains exhibited distinct edge-zone preference patterns. Cocaine-induced locomotor stimulation varied significantly among B6 substrains but not among B10. In FC, substrain differences emerged in pre-training, contextual, and cued freezing behavior, particularly among B10 substrains. PPI testing revealed substrain-dependent variation in acoustic startle responses, with C57BL/10J displaying consistently lower startle amplitudes. In the FST, substrain-specific differences in swimming velocity and high mobility duration and frequency were found within the B6 group, while swimming distance showed substrain variation within the B10 group. Discussion: These findings demonstrate substantial phenotypic variability among closely related substrains, underscoring the critical importance of substrain selection in behavioral research. By focusing on female mice (a group underrepresented in prior work), this study addresses an essential gap and provides insights for researchers designing preclinical models of psychiatric disorders. The results provide the basis for studies in reduced complexity crosses to identify causal genetic variants underlying behavioral traits.