Light/Dark preference test (zebrafish)

The preference for either dark or light environments is a behavioural trait seen across many species.

This protocol is a behavioural model for fish which has been validated as an assay to assess the anti-anxiety effects of pharmacological agents and the behavioural effects of toxic substances, and to investigate the (epi)genetic bases of anxiety-related behaviour.


Experimental setup

The Zantiks AD unit is used for the light/dark preference assay in adult zebrafish. The dividing insert and the black lid insert are used to create the experimental environment. The dividing insert splits the experimental tank to separate the 'dark' and 'light' environments and leave a shallow gap at the bottom for the fish to swim between the two.

When only tracking one fish in the tank the dividing insert is used. If you want to track more than one fish (i.e., up to four fish) in different arenas, length-ways inserts can create different arenas for each individual fish.

There are a few options available for setting up the light stimulus.

  1. Use the overhead light and the black lid insert will block the light creating the 'dark' environment on one side of the tank.
  2. Alternatively, the tank can be illuminated from below via the built-in screen, which is visible through the tank’s clear bottom. A square white light can be programmed to appear in one half of the tank.
Light Dark Box For Ad Unit
Inserts used in the Zantiks AD unit to create a light/dark experimental environment.

Experimental procedure


Results / Data output



Araujo, J., Maximino, C., Marques de Brito, T., Waneza Batista da Silva, A., Renata Matos Oliveira, K., de Jesus Oliveira Batista, E., Morato, S., Manoel Herculano, A., & Gouveia Jr., A. (2012) Behavioral and Pharmacological Aspects of Anxiety in the Light/Dark Preference Test. In: Kalueff A., Stewart A. (eds) Zebrafish Protocols for Neurobehavioral Research. Neuromethods, vol 66. Humana Press, Totowa, NJ.
Blaser, R.E., Chadwick, L., & McGinnis, G.C. (2010). Behavioral measures of anxiety in zebrafish (Danio rerio), Behavioural Brain Research, 208, 56-62. doi: 10.1016/j.bbr.2009.11.009.
Blaser, R.E., & Peñalosa, Y.M. (2011). Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test, Physiology & Behavior, 104(5), 831-837. doi: 10.1016/j.physbeh.2011.07.029.
Facciol, A., Tran, S., & Gerlai, R. (2017). Re-examining the factors affecting choice in the light–dark preference test in zebrafish, Behavioural Brain Research, 327, 21-28. doi:
Serra, E.L., Medalha, C.C., & Mattioli, R. (1999). Natural preference of zebrafish (Danio rerio) for a dark environment. Brazilian Journal of Medical and Biological Research, 32(12), 1551-1553. doi: 10.1590/S0100-879X1999001200016.