The Tube-Sealing Test: A novel behavioral assay to quantify offspring-centered defensive behavior in postpartum mice

Perinatal mental health is a major public health concern, and postpartum women are particularly vulnerable to conditions such as postpartum obsessive-compulsive disorder and postpartum depression. Many postpartum symptoms involve heightened concern for infant safety, suggesting the presence of offspring-centered defensive processes that are distinct from general anxiety focused on self-safety. 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 sealing spontaneously, sealing-like behavior can be experimentally induced by chemogenetic activation of hypothalamic perifornical Urocortin-3 neurons, indicating that mice retain a latent motor pattern for this response. However, no method has existed for reliably quantifying sealing behavior under controlled laboratory conditions. To address this gap, we developed the Tube-Sealing Test (TST), an assay that measures bedding packed into tube-like openings attached to the home cage and enables repeated, non-invasive quantification of sealing behavior. Using this approach, we found that tube-sealing occurred rarely in males, intermittently in virgin females, and with markedly greater intensity in postpartum females. Although virgin and postpartum females showed a similar likelihood of performing sealing, postpartum females inserted substantially more bedding than either group. In postpartum females, exposure to a male intruder increased sealing specifically at the tube through which the intruder was introduced, indicating that sealing intensity is modulated by direct threat experience. Together, these findings establish the TST as a simple and reproducible method for quantifying sealing behavior and identify tube-sealing as a measurable component of offspring-centered defensive behavior.