Light/Dark Plus Maze (zebrafish, xenopus, other)

Preference for light or dark areas and exploration of novel environments are some of the most widely used behaviour assays to measure anxiety. Here we present an adaption of the elevated plus maze and the light/dark box as a robust and versatile measure of anxiety and exploratory behaviours in aquatic and terrestrial organisms. The Zantiks light/dark plus maze is designed to fit within the LT system.

In the following zebrafish version of this task, fish are allowed to explore freely in a plus maze which alternates between all 4 arms dark, and 2 bright arms and 2 dark arms.


Experimental setup

The light/dark plus maze has been designed for use in the Zantiks LT system. The experimental setup is outlined in the video below. One animal can be placed in the plus maze at a time. A clear acrylic sheet is then placed over the top followed by a black acrylic sheet with a cut out for two opposing arms of the plus maze and the central zone. The black top sheet can be rotated to determine which arms are dark and which are bright, this can be alternated/randomised between trials

Set up of the light dark plus maze in the LT


Experimental procedure

In this assay turning the overhead lights on within the Zantiks system will create two bright arms and two dark arms. The model organisms can be allowed to roam freely between these arms for the duration of the assay. Alternatively, the lights can be turned off and on within the system at an interval of your choice. This creates an environment which alternates between all 4 arms remaining dark when the overhead lights are off, and a choice between the 2 bright arms and 2 dark arms when the overhead lights are on within the system.

In the example video below we show an adult zebrafish exploring the light/dark plus maze whilst alternating the overhead lights between on and off.

Adult zebrafish exploring the light/dark plus maze under alternating light and dark cycles


Results/data output

From the example experiment outlined above, zone use in terms of distance travelled in zone (mm), entries into zone and time spent in zone (sec) were exported for each of the four arms and for the central square zone (see plus maze asset for details). Example data for time spent in zone between a single lights off and lights on cycle is shown below.

Please note: this is example data output from a single fish and is not a full experiment.

Time In Zone Plusmaze
Example data from Zebrafish light/dark plus maze

Protocol scripts downloads

There is one example script for the light/dark plus maze assay for use in the LT system. This script can be adapted to change the number and duration of overhead lights on/off cycles.

There are two assets that you will use. The arena asset includes the entire plus maze. The zones asset will 'divide' the plus maze into the four arms plus a square central zone.

  1. LT_plus_maze.zs


You will need to upload the asset into the Asset directory on your Zantiks Control Console and ensure the correct asset name is in the LOAD(ZONES,"name_of_asset") and the LOAD(ARENAS,"name_of_asset") command in the script.

See the Calibrating your Zantiks unit page and Asset building in the LT unit page for details on how to create assets customised to your system.



Fossat, P., Bacqué-Cazenave, J., Deurwaerdère, P. De, Delbecque, J.-P., & Cattaert, D. (2014). Anxiety-like behavior in crayfish is controlled by serotonin. Science, 344(6189), 1293–1297.