Effects of Sex Differences on Conditioned Fear Extinction and Safety Learning in C57BL/6J Mice

Objectives: Females are often underrepresented in preclinical fear research due to concerns over estrous cycle related variability. This study examined whether there were differences between female and male C57BL/6J mice in terms of fear extinction and safety learning, aiming to verify the inclusion of both sexes in fear regulation research. Methods: Mice underwent a 5-day fear conditioning and extinction protocol, with recent (Day 6) and remote (Day 13) retrieval tests. A separate cohort received unpaired tone-shock safety conditioning over two days, followed by recent and remote retrieval. Freezing percentage and locomotor distance, among other measures, were quantified to compare behavioral responses between sexes. Results: During fear acquisition and extinction, females and males showed comparable conditioned fear and progressive extinction, with no sex differences in freezing percentage, bout counts, or locomotor distance. Freezing remained low during both recent and remote retrieval in both sexes. In the safety-conditioning task, the safety cue reduced freezing relative to contextual baseline, contextual freezing declined from recent to remote retrieval, and no sex differences were observed across measures. Conclusions: Female and male C57BL/6J mice exhibit equivalent performance in auditory fear conditioning, extinction, retrieval, and safety learning under matched conditions. These findings support equitable inclusion of both sexes in preclinical fear-regulation studies, enhancing translational relevance without added behavioral variability.