Using the tube test to measure social hierarchy in mice

Investigation of the neural mechanisms underlying social hierarchy requires a reliable and effective behavioral test. The tube test is a simple and robust behavioral assay that we recently validated as a reliable measure of social hierarchy in mice. The test was demonstrated to produce results largely consistent with the results seen when using other dominance measures, including the warm spot test, territory urine marking or the courtship ultrasound vocalization test. Here, we describe a step-by-step procedure to use the tube test to measure dominance within a cage of four male C57/BL6 mice as an example application. The procedure comprises three stages: habituation, training to pass through the tube, and the tube test itself. The social rank of each mouse is determined by the number of wins it gains when competing against the other three cagemates. A stable rank is derived when all mice maintain the same ranking for 4 consecutive days. The time required to acquire a stable rank usually varies from 4 to 14 d. An additional 5 d is required for habituation and training. In this protocol, two mice meet in a tube and the more dominant one pushes out the other one. The social hierarchy of a group of mice can thus be measured.