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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.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Journal peer review information: Nature Protocols thanks Christian Broberger, Carmen Sandi and other (anonymous) reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Wang, F. et al. Science 334, 693–697 (2011):

Zhou, T. et al. Science 357, 162–168 (2017):

Change history

  • 11 April 2019

    In the version of this paper originally published, an affiliation for Zhengxiao Fan was omitted. In addition, the Reporting Summary incorrectly indicated that human research participants had been used in the study, instead of animal subjects. These errors have been corrected in the PDF and HTML versions of the protocol.


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We thank the Hu laboratory members for valuable discussions and advice. This study was supported by grants from the National Natural Science Foundation of China (31830032, 81527901, 91432108 and 31225010), the National Key R&D Program of China (2016YFA0501000) to H.H., the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2017PT31038 and 2018PT31041) and the 111 Project (B13026) to H.H.

Author information

T.Z., H.Z. and H.H. designed the experimental strategy. Z.F., T.Z. and H.Z. optimized experimental procedures. Z.F., S.W., Y.W. and H.H. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Correspondence to Hailan Hu.

Supplementary information

Reporting Summary

Supplementary Video 1

Training stage of the tube test.

Supplementary Video 2

Testing stage of the tube test.

Supplementary Video 3

Annotation of different behavior epochs during a tube test trial. Adapted with permission from Zhou et al.26.

Supplementary Video 4

Optogenetics in tube test. A tube with a 15-mm slit is used for optogenetics. After a stable tube test rank is determined, acute dmPFC photostimulation of a previously rank 3 mouse induces winning against a rank 1 mouse. Adapted with permission from Zhou et al.26.

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Fig. 1: Tubes.
Fig. 2: An example showing the stability of tube test rank and time spent in the tube for different ranked pairings.
Fig. 3: Example of daily tube test ranking of one cage of four mice injected with AAV-CAG-ChR2(H134R) virus before and after acute dmPFC photostimulation of the rank-3 mouse at day 0.
Fig. 4: Comparison of the behavioral performances of photostimulated mice during light-off and light-on trials.


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