Neurophenotyping of Adult Zebrafish Using the Light/Dark Box Paradigm

Part of the Neuromethods book series (NM, volume 51)


The light/dark box test, traditionally used to quantify rodent anxiety-like behavior, has recently been applied to the adult zebrafish (Danio rerio). Utilizing the fish’s scototaxis (aversion to bright areas and natural preference for the dark), this paradigm can be used to assess levels of anxiety in adult zebrafish. The light/dark box is a simple and time-efficient one-trial test that does not require pre-training the animals. Importantly, this novelty-based paradigm may also represent a useful tool for studying the pharmacological modulation of zebrafish behavior. Summarizing the experience with this model in several laboratories, here we outline a protocol for the neurophenoptyping of zebrafish anxiety-like behavior using the light/dark paradigm.

Key words

Zebrafish Light/Dark box Scototaxis Anxiety Novelty-based paradigm 



The study was supported by Tulane University Intramural funds, Provost’s Scholarly Enrichment Fund, Newcomb Fellows Grant, LA Board of Regents Pfund and NARSAD YI awards, Zebrafish Neuroscience Research Consortium (ZNRC), as well as CAPES/Brazil.


  1. 1.
    Egan, R.J. et al. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav. Brain Res. 205(1), 38–44 (2009).PubMedCrossRefGoogle Scholar
  2. 2.
    Stewart, A. et al. The developing utility of zebrafish in modeling neurobehavioral disorders. Int. J. Comp. Psychol. 23(1), 104–121 (2010).Google Scholar
  3. 3.
    Levin, E.D., Bencan, Z., & Cerutti, D.T. Anxiolytic effects of nicotine in zebrafish. Physiol. Behav. 90(1), 54–58 (2007).PubMedCrossRefGoogle Scholar
  4. 4.
    Gerlai, R. et al. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacol. Biochem. Behav. 67(4), 773–782 (2000).PubMedCrossRefGoogle Scholar
  5. 5.
    Wong, K. et al. Analyzing habituation responses to novelty in zebrafish (Danio rerio). Behav. Brain Res. 208(2), 450–457 (2010).PubMedCrossRefGoogle Scholar
  6. 6.
    Sackerman, J. et al. Zebrafish behavior in novel environments: effects of acute exposure to anxiolytic compounds and choice of Danio rerio line. Int. J. Comp. Psychol. 23, 43–61 (2010).PubMedGoogle Scholar
  7. 7.
    Blaser, R. & Gerlai, R. Behavioral phenotyping in zebrafish: comparison of three behavioral quantification methods. Behav. Res. Methods. 38(3), 456–469 (2006).PubMedCrossRefGoogle Scholar
  8. 8.
    Bourin, M. & Hascoet, M. The mouse light/dark box test. Eur. J. Pharmacol. 463(1–3), 55–65 (2003).PubMedCrossRefGoogle Scholar
  9. 9.
    Hascoet, M., Bourin, M., & Dhonnchadha, B.A. The mouse light-dark paradigm: a review. Prog. Neuropsychopharmacol. Biol. Psychiatry. 25(1), 141–166 (2001).PubMedCrossRefGoogle Scholar
  10. 10.
    Fraser, L.M. et al. Measuring anxiety- and locomotion-related behaviours in mice: a new way of using old tests. Psychopharmacology (Berlin), 211(1), 99–112 (2010).Google Scholar
  11. 11.
    Shimada, T. et al. The modified light/dark transition test in mice: evaluation of classic and putative anxiolytic and anxiogenic drugs. Gen. Pharmacol. 26(1), 205–210 (1995).PubMedCrossRefGoogle Scholar
  12. 12.
    Blaser, R.E., Chadwick, L., & McGinnis, G.C. Behavioral measures of anxiety in zebrafish (Danio rerio). Behav. Brain Res. 208(1), 56–62 (2010).PubMedCrossRefGoogle Scholar
  13. 13.
    Maximino, C. et al. A comparative analysis of the preference for dark environments in five teleosts. Int. J. Comp. Psychol. 20, 351–367 (2007).Google Scholar
  14. 14.
    Grossman, L. et al. Characterization of behavioral and endocrine effects of LSD on zebrafish. Behav. Brain Res. 214(2), 277–284 (2010).Google Scholar
  15. 15.
    Maximino, C. et al. Scototaxis as anxiety-like behavior in fish. Nat. Protoc. 5(2), 209–216 (2010).PubMedCrossRefGoogle Scholar
  16. 16.
    Maximino, C. et al. Parametric analyses of anxiety in zebrafish scototaxis. Behav. Brain Res. 210(1), 1–7 (2010).PubMedCrossRefGoogle Scholar
  17. 17.
    Serra, E.L., Medalha, C.C., & Mattioli, R. Natural preference of zebrafish (Danio rerio) for a dark environment. Braz. J. Med. Biol. Res. 32(12), 1551–1553 (1999).PubMedCrossRefGoogle Scholar
  18. 18.
    Malmberg-Aiello, P. et al. Mouse light/dark box test reveals anxiogenic-like effects by activation of histamine H1 receptors. Pharmacol. Biochem. Behav. 71(1–2), 313–318 (2002).PubMedCrossRefGoogle Scholar
  19. 19.
    Shin, J.T. & Fishman, M.C. From Zebrafish to human: modular medical models. Annu. Rev. Genomics Hum. Genet. 3, 311–340 (2002).PubMedCrossRefGoogle Scholar
  20. 20.
    Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th ed. (University of Oregon Press, Eugene, 2007).Google Scholar
  21. 21.
    Takao, K. & Miyakawa, T. Light/dark transition test for mice. J Vis Exp. 1, 104 (2006).PubMedGoogle Scholar
  22. 22.
    Prior, H. & Sachser, N. Effect of enriched housing environment on the behaviour of young male and female mice in four exploratory tasks. J. Exp. Amin. Sci. 37, 57–68 (1994).Google Scholar
  23. 23.
    Chapillon, P. et al. Rearing environmental enrichment in two inbred strains of mice: 1. Effects on emotional reactivity. Behav. Genet. 29(1), 41–46 (1999).PubMedCrossRefGoogle Scholar
  24. 24.
    Roy, V. et al. Environmental enrichment in BALB/c mice: effects in classical tests of anxiety and exposure to a predatory odor. Physiol. Behav. 74(3), 313–320 (2001).PubMedCrossRefGoogle Scholar
  25. 25.
    Neuhauss, S.C. Behavioral genetic approaches to visual system development and function in zebrafish. J. Neurobiol. 54(1), 148–160 (2003).PubMedCrossRefGoogle Scholar
  26. 26.
    Mueller, K.P. & Neuhauss, S.C. Behavioral neurobiology: how larval fish orient towards the light. Curr. Biol. 20(4), R159–R161 (2010).PubMedCrossRefGoogle Scholar
  27. 27.
    MacPhail, R.C. et al. Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Neurotoxicology 30(1), 52–58 (2009).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Pharmacology and Neuroscience Program, Zebrafish Neuroscience Research Consortium (ZNRC)Tulane University Medical SchoolNew OrleansUSA
  2. 2.Institute of Biological Sciences, Universidade Federal do ParáBelémBrazil
  3. 3.Department of Psychology and EducationUniversidade de São PauloRibeirão PretoBrazil
  4. 4.Behaviour Theory and Research NucleusUniversidade Federal do ParáBelemBrazil
  5. 5.Department of Pharmacology and Neuroscience ProgramTulane University Medical SchoolNew OrleansUSA
  6. 6.Department of Pharmacology and Neuroscience ProgramTulane University Medical SchoolNew OrleansUSA

Personalised recommendations