The three-chamber paradigm is a widely used task to evaluate spatial and non-spatial learning as well as memory in zebrafish. The watertight apparatus from MazeEngineers is outlined by a dark panel; covering one side of each compartment as a visual cue to provide an axis of orientation for right/left discrimination. The apparatus is primarily used for learning and memory but can also be used for various toxicity experiments.


Price & Dimensions


$ 1990

Per Month
  • Total Width: 22cm
  • Total Length: 44cm
  • Total Height: 30cm
  • Middle chamber Width: 22cm
  • Middle chamber Length: 18cm
  • Door: Height: 12cm. Width: 10cm.
  • Choice Area: Width: 22cm. Length: 13cm



Environmental enrichment of captive animals can have a significant impact on their behavioral flexibility and mental cognition. The effects of environmental enrichment have been studied in terrestrial animals (see more here). However, this effect has yet to be thoroughly studied in fishes. Captive rearing for population rehabilitation can immensely gain from the understanding of how enriching the environments of the captive fishes can fine-tune and shape their behaviors. It is particularly important because fishes raised in standard hatcheries tend to have lower survivability hierarchy than their wild counterparts, thus not efficiently, nor effectively aiding the rehabilitation endeavors.

The behavioral flexibility of the fishes can be tested using a flexibility test. The test involves a tank with a partition that divides it into two chambers of which one holds a feeder. The partition is equipped with a single exit, to the feeder chamber, that is either a simple hole in the divider or a more complex tube route to the exit.

Apparatus and Equipment

Tanks of dimension 90 x 45 x 30 cm are used. Environmental enrichment can be done using objects such as fine grain gravel, upturned, black plastic pots (10 cm in diameter) to create areas of shelter, plastic plants (12–30 cm tall) and novel objects such as plastic plants of different colors, grey PVC-pipes (3 cm in diameter).

For assessing flexibility, two types of partitions are used: divider with exit hole (5 cm wide) in it and divider with tube access to the exit hole.

Training Protocol

The subjects should be housed in tanks that are maintained at constant temperature and pH value. It is advisable that these conditions be maintained throughout the sessions to avoid unnecessary harm or stress to the fish. It is also important to keep the water aerated and the quality of water maintained at an acceptable level.

Environment Enrichment

Subjects are grouped into 4 groups. For a rearing phase of 12 weeks, provide the following set-ups for each group,

  • Group I (Full): Full 12 weeks of the enriched environment.
  • Group II (Early): First 5 weeks in an enriched environment then returned to the regular holding tank.
  • Group III (Late): Last 5 weeks in an enriched environment.
  • Group IV (Control): No enriched environment for the entire rearing phase.

Flexibility Test

The testing is done over the course of 4 consecutive days. Position the divider wall with exit hole and the opaque partition wall blocking the exit hole and the feeder chamber. Transfer individual fish into the test tank and allow 30 minutes of settling period. After the expiration of 30 minutes, remove the opaque partition wall.

For each testing day, move the exit to a different position. For days 1 & 2, use the divider wall with exit hole either on left or right and allow 15 minutes for the fish to find it. For days 3 & 4, use the divider wall with access tube to exit hole. One of the tube set-ups requires the fish to swim down and up into the tube, and the other set-up requires the fish to swim down into the tube. For these trials, the cut-off time is 20 minutes to complete the trial.

Evaluation of recency and duration of environmental enrichment on behavioral flexibility

Bergendahl et al. evaluated the effects of environmental enrichment on juvenile rainbow trout. Fishes were divided into 4 groups that received different levels of enrichment or no enrichment at all for 12 weeks. On completion of the rearing phase, the fishes were individually tested in the flexibility test. It was observed that the Full treatment group performed consistently better than the other groups in the flexibility test.


The original version of the maze was rectangular in shape. (D. Arthur, E.D. Levin, 2001)

The most recent version of the three-chamber maze was developed by Eddins 2009 for investigating the effects of nicotine on learning in zebrafish.

The testing apparatus consists of a semi- cylindrical pipe unit divided into three chambers. The chambers are named as the start chamber, right choice chamber, and the left choice chamber. Plexiglas rods are extended through both sides of the apparatus.

The inner side of the rod is provided with rotatable plastic partitions that permit the entry of the subjects.

The partitions are circular in shape and vertical in position. The partitions are attached to approx. 13 cm long rails. The rails can move freely from the central chamber to the end wall of the apparatus.

In the original version, a fishnet was waved, but in this modified version, a plastic beaker is dropped just above the start chamber as a threatening stimulus in order to evoke a response.

Sample Data

The following data are observed in the flexibility test

  • Number of fish that found the route to feeder chamber
  • Swim velocity
  • Time spent immobile
  • Time taken to find the exit

Strengths & Limitations


Environment enrichment plays an important role in the survivability success of hatchery-raised fishes as rehabilitation population. It has been observed that the duration of exposure to the enriched environment and the recency of exposure both affect the behavioral flexibility and cognitive abilities in fish. The flexibility test is a simple test to assess these abilities. This simple apparatus is an efficient and valuable tool for determining the effects of environmental enrichment.


Handling and rearing environments can impact the fish performance. Undue stress and anxiety can result in incorrect results and observations. Presences of cues or other disturbances can also affect the performances. It is imperative to maintain constant tank temperature and other environmental factors during the test to avoid unnecessary influences on the performances.

Summary and Key Points

  • Environmental enrichment impacts behavioral flexibility and mental cognition of captive animals.
  • Enriching the environments of the captive fishes can fine-tune and shape their behaviors thus increasing the success of rehabilitation efforts.
  • The duration and recency of exposure to structural, environmental enrichment affect the behavioral development.
  • Flexibility test allows assessment of flexibility in habitat use in fishes.
  • Two types of partitions are used in flexibility test; one with an exit hole and the other with tube access to the exit hole.


Bergendahl I.A, Salvanes A.G.V, Victoria A. Braithwaitec V.A (2015) Determining the effects of duration and recency of exposure to environmental enrichment. Appl. Anim. Behav. Sci. DOI: