Establishing a social behavior paradigm for female mice.

Introduction: Social behavior assessment in female mice has been historically challenged by inconsistent results from the classic three-chamber test, which reliably detects social preferences in males but fails to capture female specific social dynamics. Methods: We developed a modified three-chamber paradigm by replacing standard social stimuli with familiar cagemates (co-housed for 2 weeks, 1 week or 24 hours) to better assess sociability and novelty preference in female mice. Results: In the sociability phase, female mice showed a significant preference for interacting with cagemates compared to empty chambers. Crucially, during the social preference phase, test females demonstrated robust novelty seeking behavior, spending significantly more time exploring novel conspecifics compared to 2-week cagemates or 1-week cagemates. This preference trended similarly, though non significantly, with 24-hour cagemates. Notably, our paradigm enhanced social preference indices without altering total interaction time, confirming its specificity for detecting novelty driven exploration. Discussion: These findings overcome the limitations of traditional paradigms and establish a validated framework for studying female social behavior, with critical implications for modeling neurodevelopmental disorders like autism spectrum disorder (ASD) in female preclinical research.