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The zebrafish associative learning multi-chamber tank was innovated by Fernandes et al. in 2016 as a novel tool for assessing visual discrimination in adult zebrafish. This task aims to evaluate the cognitive and mnemonic characteristics of these fish, which are pivotal for understanding vertebrate neural plasticity (Gerlai, 2011).
This unique aquarium is equipped with acrylic inserts that transform it into a maze where zebrafish learn to associate visual stimuli with rewards. In this setup, the reward is represented by a shoal stimulus, following recent findings that zebrafish can reinforce associative learning upon encountering groups of conspecifics (Al-Imari and Gerlai, 2008). The relationship between the time spent in each chamber and the frequency of visits to the target chamber allows observation of the acquisition of CS-US associations.
This innovative learning apparatus serves as a crucial tool in demonstrating that complex behaviors such as learning, memory, and social skills emerge later in the development of zebrafish (Liu et al., 2016).
The zebrafish associative learning multi-chamber tank (dimensions 60 cm x 47 cm x 25 cm) is a rectangular acrylic apparatus designed for cognitive testing. Along its length, four chambers are evenly spaced on each side of the maze. Each chamber measures 15 cm x 10.5 cm x 25 cm and features a removable colored cue card: yellow, blue, red, or green. These chambers are separated by an open compartment (60 cm x 25 cm x 25 cm). A 9 cm tube connects the target chambers to the open area, with a transparent door regulating access between the target chamber and the tube.
This transparent door allows experimental fish to view both the stimulus fish (reward) and the cue simultaneously while preventing the stimulus fish from leaving the target chamber. A slot for a second door is positioned at the entrance of the tube leading to the open compartment. This second door automatically closes once the experimental subject enters the tube.
The chamber’s design is straightforward, with acrylic inserts that fit seamlessly without requiring complex angles or shapes to be glued together.
Three phases comprise the experimental procedure: habituation, training, and the probe.
Experimental fish are grouped and introduced into the maze over several days, with group sizes decreasing gradually. On the first day, each group consists of 16 fish undergoing a single 20-minute exploration session. This is followed by groups of 8 fish on the second day, with two separate 10-minute exploration sessions. On the third day, groups of 4 fish engage in two separate 10-minute sessions. The fourth day involves groups of 2 fish participating in four exploration sessions, each lasting 5 minutes. Finally, on the fifth day, individual fish undergo four 5-minute exploration sessions.
For each habituation trial, the subject(s) are placed in a different start chamber. Only the start chamber from which the fish are released remains open, while the other three chambers are closed. Meanwhile, all reward chambers are kept open throughout the trials.
Following habituation, the experimental zebrafish progress to the training phase. Subjects are transferred to the behavioral testing room where the view of the maze from holding tanks is obscured by a black metal divider measuring 120 cm x 180 cm. Each fish is then transported from the holding tank to the experimental tank and placed into the start chamber. All target chambers remain open to allow zebrafish to freely choose their destination. One of the target chambers contains the reward—a stimulus shoal—while the other three are non-rewarded.
During each training day, every experimental fish undergoes four training sessions. Each session concludes 5 minutes after the start chamber is opened.
In the probe phase, the target chambers are accessible with their respective color cue cards, but without the presence of the reward fish. The probe begins upon raising the door of the start chamber and concludes after 5 minutes. Each experimental zebrafish undergoes a single probe trial. For automated data collection, an overhead Noldus Ethovision XT system records the time taken by each experimental fish to reach the target chamber. Results are recorded and compared over a period of 5 days, as illustrated in the sample data (see Fig. 1).
Using this multi-chamber tank setup, it is possible to assess the time it takes for experimental zebrafish to reach the reward chamber containing the stimulus fish, as demonstrated by Fernandes et al. (2016).
This device enables validation of zebrafish color preference in learning and memory tasks, initially described by Avdesh et al. in 2012. The removable color cues within the chambers (yellow, blue, red, and green) facilitate this assessment. The frequency of entries into each colored chamber can be recorded for evaluation purposes.
This multi-chamber apparatus allows for evaluating the acquisition of color and reward association, as demonstrated by Fernandes et al. (2016). This assessment can be conducted during the probe trial, where no reward is present in any of the colored chambers. It examines whether the experimental zebrafish continues to spend a significant amount of time in the colored chamber previously associated with the reward.
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.
This multi-chamber tank design for assessing zebrafish associative learning offers several advantages over existing paradigms. Unlike the T-maze, it allows researchers to observe zebrafish behavior when presented with more than two options. Compared to plus or radial mazes, the multi-chamber tank is easier to assemble and occupies less space. The design features physical barriers that enable zebrafish to make distinct choices, easily monitored and quantified from both side views and overhead perspectives.
Observing experimental fish from the side enables assessment of various behavioral responses such as fin erection (indicative of aggression), erratic movements (indicative of fear), and other motor patterns specific to zebrafish. Additionally, its compact size facilitates concurrent studies across multiple tanks, enhancing experimental throughput.
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
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