Effect of Sex and Environmental Enrichment on Predator Odor Contextual Fear Conditioning and Extinction in Laboratory Mice

This study investigated the effects of sex and environmental enrichment (EE) on predator odor, contextual fear conditioning, and extinction in BALB/c mice. Forty adult mice (20 males, 20 females) were randomly assigned to four groups (n = 10 each): male enrichment, female enrichment, male non-enrichment, and female non-enrichment. Enriched groups were housed in larger cages (60×40×20 cm) with tunnels and nesting materials, while non-enriched groups were housed in standard laboratory cages (30×20×15 cm). The experiment included a multi-day predator odor contextual fear conditioning paradigm. Days 1-3 consisted of a 10-minute familiarization session, a 10-minute predator odor exposure session (using cat feces), and a 10-minute conditioned context session. Extinction sessions (10 minutes each) were conducted on Days 4, 6, and 10. Behavioral responses (e.g., avoidance, freezing, locomotion, grooming) were assessed across all sessions. Results revealed that EE significantly reduced anxiety-related behaviors (e.g., hiding, freezing) and enhanced exploratory and grooming behaviors. Sex differences were also observed: male mice exhibited longer freezing and hiding durations, while females showed higher locomotor activity. Temporal patterns showed distinct dynamics in fear and exploratory behaviors, with EE facilitating adaptive coping mechanisms. These findings suggest that EE provides a protective effect against fear-related behaviors and that sex differences exist in the response to predator odor fear conditioning, highlighting the importance of considering both factors in preclinical anxiety research.