The tube-plugging test: a simple assay that reveals offspring-centered defensive behavior in postpartum mice

Perinatal mental health is a major public health concern. Epidemiological studies indicate that approximately 10-20% of women in the postpartum period experience clinically significant depressive or obsessive-compulsive symptoms. Many postpartum symptoms involve heightened vigilance and behaviors aimed at protecting the infant from possible harm, suggesting the presence of offspring-centered defensive processes. One offspring-centered defensive behavior described in wild rats is entrance-sealing, in which lactating females plug the entrance of their burrow to limit access by potential intruders. Although laboratory mice rarely exhibit such behavior spontaneously, similar bedding-plugging behavior has been occasionally observed, suggesting that mice retain the capacity for this response. However, no method has existed for reliably quantifying such behavior under controlled laboratory conditions. To address this gap, we developed the Tube-Plugging Test (TPT), an assay that measures bedding accumulated at tube-like openings attached to the home cage and enables repeated, non-invasive quantification of plugging behavior. Using this approach, we found that tube plugging occurred rarely in males, intermittently in virgin females, and most robustly in postpartum females. Notably, plugging behavior exhibited substantial inter-individual variability, but was more temporally stable across days in postpartum females than in virgin females. In postpartum females, exposure to a male intruder altered plugging behavior at the tube through which the intruder was introduced, suggesting that plugging is modulated by direct social context. Together, these findings establish the TPT as a simple and reproducible method for quantifying plugging behavior and identify tube plugging as a measurable component of offspring-centered defensive behavior.