The Light–Dark Preference Test for Larval Zebrafish

  • Peter J. Steenbergen
  • Michael K. Richardson
  • Danielle L. Champagne
Part of the Neuromethods book series (NM, volume 66)


The light–dark preference test is one of the most commonly used anxiety models in animal preclinical studies. This test assesses spontaneous/natural tendency of an animal to explore/avoid certain zones of a given environment, for instance a half black-half white box. This test has been validated for use in adult but not larval zebrafish. Here we provide the first method for assessing anxiety-like behaviors in the light–dark preference test in zebrafish larvae. We also provide a pharmacological validation of this assay as well as a simple method to render it more versatile for screening of both anxiolytic and anxiogenic drugs. This is important since the ability to characterize and document the behavior of larval zebrafish is crucial in the current research era, where zebrafish has been rapidly propelled at the forefront of high-throughput technology. Therefore, this 5-min protocol will not only contributes to enriching the repertoire of assays customized for larval zebrafish but also provides a method to assess complex phenotypes of brain function while being eventually amenable to high-throughput screening in the near future.

Key words

Anxiety Automation Avoidance behavior Development Drug screening Exploration Stress Hypothalamic-pituitary-interrenal axis 



The authors gratefully acknowledge the support of the Smart Mix Program of The Netherlands Ministry of Economic Affairs and The Netherlands Ministry of Education, Culture and Science.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Peter J. Steenbergen
    • 1
  • Michael K. Richardson
    • 1
  • Danielle L. Champagne
    • 1
    • 2
  1. 1.Department of Integrative Zoology, Institute of BiologyLeiden UniversityLeidenThe Netherlands
  2. 2.Department of Organismal Animal Physiology, Institute for Water and Wetland ResearchNijmegen UniversityNijmegenThe Netherlands

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