Optimization and evaluation of a chronic variable stress model of depressive-like behaviors in mice

Chronic variable stress (CVS) paradigms are essential for modeling depression- and anxiety-related disorders. However, methodological heterogeneity in stressor selection, duration, and scheduling frequently compromises reproducibility and behavioral consistency across studies.

We systematically optimized a CVS protocol in C57BL/6 J mice to reliably elicit robust depressive- and anxiety-like phenotypes while minimizing somatic injury and mortality. The protocol employed quasi-randomized application of restraint, social defeat, tail suspension, and foot shock, administered twice daily with a 2-hour inter-stressor interval over 12 days. Brief, non-injurious stressor variants were utilized to reduce physical harm without compromising efficacy.

A 12-day, but not 6-day, regimen induced stable and reproducible behavioral deficits. The 2-hour inter-stressor interval maximized behavioral impact. Utilizing induction stressors as post-stress behavioral assays introduced habituation confounds. The protocol demonstrated predictive validity, as chronic fluoxetine reversed depressive-like behaviors. While female mice exhibited immediate deficits comparable to males, longitudinal assessment revealed sexual dimorphism in recovery trajectories.

Unlike conventional CVS protocols that vary widely in duration and stressor intensity, our optimized 12-day protocol achieves comparable behavioral outcomes within a shorter timeframe using brief, non-injurious stressor variants. Furthermore, we identified the confounding effect of stressor reuse in behavioral assessment, thereby enhancing interpretive clarity of stress-induced phenotypes.

This optimized CVS protocol provides a rapid, reproducible, and pharmacologically sensitive platform for investigating neural mechanisms underlying stress-related psychopathology, with applicability across both sexes and guidelines for avoiding common methodological pitfalls.