Description

The Zebrafish associative learning chamber is a simple test for evaluating visual discrimination with an associative learning task. The MazeEngineers test allows for simple insertion of color cues for zebrafish to navigate between the Start chamber to the target chamber, and includes the entire package including insertable doors and color cues for your experiment.

• Acrylic Chamber size:
  • Length: 60cm
  • Width: 47cm
  • Height: 25cm
• Start Chambers:
  • Length: 15cm
  • Width: 10.5cm
  • Height: 25cm
• Target Chambers:
  • Length: 15cm
  • Width: 10.5cm
  • Height: 25cm
  • One removable colored cue card (yellow, blue, red, or green)

• Open Compartment:
  • Length: 60cm
  • Width: 25cm
  • Height: 25cm
• Transition:
  • 9 cm opening between target chamber & open compartment, which can be closed with an sliding transparent door

Documentation

Introduction

Zebrafish’s associative learning multi-chamber tank is a novel task developed by Fernandes et al., 2016. It is a visual discrimination task that assesses and analyzes the visual discrimination in adult zebrafish. It is an approach in assessing the cognitive and mnemonic characteristics of the zebrafish. Studying the learning abilities of the zebrafish is a significant tool in identifying the genes that govern vertebrate neural plasticity (Gerlai, 2011).

The novel zebrafish associative learning multi-chamber tank is an aquarium retrofitted with acrylic inserts. The tank serves as the maze in which zebrafish are supposed to associate a visual stimulus with a reward. The reward to be used in this apparatus is a stimulus shoal, as it has been recently shown that associative learning in zebrafish could be reinforced at the sight of a group of conspecifics (Al-Imari and Gerlai, 2008). The relationship between the time the fish spent in each chamber and the frequency of visits in the target chamber could be observed and regarded as the acquisition of CS-US association. This new learning apparatus is a significant tool in demonstrating that complex behaviors such as learning, memory, and social skills are detected later in the development of the zebrafish (Liu et al., 2016).

Apparatus and Equipment

The zebrafish associative learning multi-chamber tank (60 cm x 47 cm x 25 cm) is a rectangular acrylic device. Four chambers are constructed along the length of the maze on either side. Each chamber is  15 cm x 10.5 cm x 25 in dimensions with a colored removable cue card: yellow, blue, red, or green. The chambers are separated by an open compartment (60 cm x 25 cm x 25 cm). A 9cm tube connects the target chambers and the open area. A transparent door guards the opening between the target chamber and the tube. This transparent door allows the experimental fish to see the stimulus fish (reward) and the cue and at the same time. It also prevents the stimulus fish from escaping the target chamber. A slot for a second door is situated at the tube entrance, which is connected to the open compartment. This second door is placed at the entrance as soon as the experimental subject enters the tube.

The overall construction of the chamber is simple, and the acrylic inserts do not have to be glued in confusing angles and shapes.

Training Protocol

Three phases comprise the experimental procedure: habituation, training, and the probe.

Habituation

Experimental fish are to be put in the maze per groups. The size of the groups gradually decreases as the days go by, with 16 fish per group on the first day (one 20 min long exploration session), 8 on the second day (two separate 10 min exploration sessions), 4 on the third day (two separate 10 min exploration sessions), to 2 on the fourth day (four exploration sessions of 5 min each), and finally to 1 on the fifth day (four 5 min long exploration sessions). The subject/s is/are to be placed in a different start chamber for each habituation trial. Only the start chamber from which the fish are released should be maintained open, and the other three chambers are to remain closed. Meanwhile, all the reward chambers should be left open.

Training Phase

After the habituation, the experimental zebrafish would now proceed to the training phase. Subjects should be placed in the behavioral testing room. The view of the maze from the holding tanks is blocked by a black metal divider (120 cm x 180 cm). Fish are then transported from the holding tank to the experimental tank. Each experimental fish is placed into the start chamber. All target chambers are left open so the zebrafish could freely choose which target chamber it would go to. One of the target chambers contained the reward, a stimulus shoal. The other three target chambers are non-rewarded. During each training day, each experimental fish undergo four training sessions. The trial ends 5 minutes after the opening of the start chamber.

Probe

During the probe, target chambers are open with their color cue cards in the absence of the reward fish. The probe starts when the door of the start chamber is raised and ends 5 minutes later. Each experimental zebrafish is to receive a single probe trial. For automated recording, an overhead Noldus Ethovision XT. Measure the time it will take for the experimental fish to go to the target chamber. Record the results and compare for the course of 5 days, as seen in the sample data, Fig. 1.

Evaluation of zebrafish latency to the reward

With this multi-chamber tank, evaluation of the time it would take for the experimental zebrafish to go to the reward chamber that contains the stimulus fish could be done, as demonstrated by Fernandes et al. (2016).

Validation of color preference

This apparatus could be used to validate the color preference of zebrafish for learning and memory, which was first published by Avdesh et al. in 2012. The removable color cues of the chamber (yellow, blue, red, and green) serve for this purpose.  The number of entries to the particular colored chamber could be recorded for evaluation.

Evaluation of memory acquisition of color and reward association

The acquisition of color and reward association could also be evaluated using this multi-chamber apparatus, as demonstrated by Fernandes et al. (2016). It can be observed during the probe trial when there is no reward in any of the colored chambers. Observe whether the experimental zebrafish would still spend a high percentage of time in the colored chamber that once contained the reward.

Sample Data

Figure 2. A)  Percent of time experimental fish spent in the chamber marked by the correct color cue. B) The number of entries to each chamber.

Strengths & Limitations

This multi-chamber tank design for assessing zebrafish associative learning is more advantageous as compared to currently existing paradigms. Compared to the T-maze, the multi-chamber apparatus allows the investigator to observe the zebrafish’s choices when encountered with more than two options. Compared to the plus or radial mazes, the multi-chamber tank is easier to assemble and has a smaller physical footprint. The design of the physical barriers allows the zebrafish to make clear choices that could easily be monitored and quantified both from the side and overhead. Observing the experimental fish from the side enables the evaluation of a number of behavioral responses such as fin erection display which is a manifestation of aggression, an erratic movement which manifests fear, and other motor patterns previously described in the zebrafish. Also, since the apparatus is small, multiple tank studies could be done facilitating higher throughput.

Summary and Key Points

• This multi-chamber tank for zebrafish associative learning is a simple, easy to assemble apparatus.
• This apparatus enables the investigator to observe the behavior of the zebrafish when encountered with more than two options.
• The design of the tank allows for easy monitoring and quantification.
• Multiple setups of this apparatus could be utilized for a high-throughput study.
• Significant applications of this simple and versatile multi-chamber tank apparatus include behavioral studies that aim to understand the complex mechanisms of memory and learning in zebrafish, and in vertebrates in general.

References

Al-Imari, L., & Gerlai, R. (2008). Sight of conspecifics as reward in associative learning in zebrafish (Danio rerio). Behav Brain Res, 189(1): 216-19. doi: 10.1016/j.bbr.2007.12.007.

Avdesh, A., Martin-Iverson, M.T., Mondal, A., Chen, M., Askraba, S., Morgan, N., Lardelli, M., Groth, D.M., Verdile, G., Martins, R.N. (2012). Evaluation of color preference in zebrafish for learning and memory. J Alzheimers Dis. 28(2):459-69. doi: 10.3233/JAD-2011-110704.

Fernandes, Y.M., Rampersad, M., Luchiari, A.C., & Gerlai, R. (2016). Associative learning in the multichamber tank: A new learning paradigm for zebrafish. Behav Brain Res, 312, 279-84. doi: 10.1016/j.bbr.2016.06.038.

Gerlai, L. 2011. Associative learning in zebrafish (Danio rerio). Methods Cell Biol, 101: 249-70. doi: 10.1016/B978-0-12-387036-0.00012-8.

Xiuyun, L., Yinglan, Z., Jia, L., Qiaoxi, X., Ning, G., & Qiang, L. (2016). Social preference deficits in juvenile zebrafish induced by early chronic exposure to sodium valproate. Front Behav Neurosci, 10: 201. doi:  10.3389/fnbeh.2016.00201