The hidden impact of cage position on behavioral phenotyping and reproducibility

Cage position within an animal-room rack is among the most routine housing decisions, yet it shapes both ambient illumination and husbandry contact. We asked whether this alone, in naive, untreated mice, alters adult behavior. Male and female ICR mice were housed from birth to adulthood on the uppermost or lowermost rack shelf. Lower-shelf mice left the dark compartment of the light-dark box faster, the only difference surviving correction, and showed less risk-assessment behavior in the elevated plus maze in both cohorts. Spatial memory, locomotor activity, nociception and ultrasound-measured optic nerve diameter showed no detectable difference, despite 70 versus 20 lx in-cage illumination. Lower-shelf mice showed greater novel-object preference, but not once cage clustering was modeled. Here, we show that cage position is associated with altered exploratory behavior in animals given no experimental manipulation, potentially identifying a hidden confounder that counterbalancing can remove.