Features

Documentation

Introduction

The Zebrafish Automated T-maze is used to study spatial learning and memory along with conditioned behaviors in zebrafish. It consists of a chamber with test areas and returning pathways. Doors are placed within the test areas, which form a T-shape maze. The returning pathways allow the subject to swimming back to the starting area after reaching the goal area. This allows another trial to be conducted easily and quickly since the subject does not have to be removed and placed back in the maze by the experimenter. 

The Zebrafish Automated T-maze contains multiple sensors that control the opening and closing of the doors within the maze. The sensors also control the delivery of the food reward in the goal arm. The maze is available with software that configures and runs the selected protocol. The protocols include “left force choice” and “right force choice”, which force the subject to enter only the selected goal arm. “Left free choice” and “right free choice” protocols allow the subject to freely enter any goal arm, but the reward is only given in one arm. A “free choice” protocol is also present where the reward is given in both goal arms to observe preference behaviors in the subject. Additionally, the free choice protocol is conducted as a test trial to observe if the subject displays memory retention of the rewarded and punished arm. The sensors are configured according to the protocol selected; therefore, the experimenter has the ease of not manually controlling the doors or administering the rewards, which helps save time. 

The Zebrafish Automated T-maze also includes a water stirrer in the goal arms, which serves as positive punishment if the subject extends its maximum duration in the goal arm. Furthermore, the colored inserts placed outside both maze arms can be used to observe color preferences in zebrafish. 

Apparatus and Equipment

The Zebrafish Automated T-Maze consists of a test chamber with T-maze test areas and returning pathways. It includes seven auto doors, seven sensor arrays, two reward dispensers, two auto water stirrers, a control box, and other accessories. 

The maze measures 51 cm in width and 38 cm in length. The interior width of the maze arms measure 9 cm, and the interior wall height is 10 cm. The maze walls are clear with ½ inch thick acrylic. The maze can be filled to a water height of 10 cm. Two red and two green removable wall inserts are provided with the maze that can be inserted outside the goal arms. 

The sensors detect fish movement into the areas. Each door requires a pair of custom-designed sensor arrays. The length of each sensor array is 8 cm. The pellet dispensers are placed on the outside of the goal arms. Each dispenser has an LED light and an adjustable slot for food delivery. Each goal arm contains a removable water stirrer with adjustable speed to be used as positive punishment. 

The automated T-maze also comes with software to configure and run protocols. The maze doors are kept open or closed according to the experimental protocol to allow free choice of forced-choice behaviors.

Training Protocol

The Zebrafish Automated T-maze software configures and runs the following protocols:

Left forced choice – the doors leading to the left goal arm are kept open, and the one leading to the right goal arm is kept closed. The food reward is delivered at the end of the left goal arm.

Right forced choice – the doors leading to the right goal arm are kept open, and the one leading to the left goal arm is kept closed. The food reward is delivered at the end of the right goal arm. 

Left free choice – the doors leading to the right goal arm and left goal arm are kept open. However, the food reward is delivered at the end of the left goal arm only. 

Right free choice – the doors leading to the right goal arm and left goal arm is kept open. However, the food reward is delivered at the end of the right goal arm only.

Free choice – the doors leading to both goal arms are kept open, and the food reward is delivered at whichever arm the subject enters. 

Depending on the experimental protocol, the different sensors in the maze are automatically turned off or kept on, which allows the doors to remain open or closed or causes the food reward to be delivered. 

Additionally, the water stirrer can be utilized as positive punishment during the task to make the subject leave the goal area if it extends the maximum duration. 

Literature Review

Evaluation of Learning and Memory of Zebrafish 

Hieu et al. (2020) evaluated the learning and memory of zebrafish using a conditioned place preference test on a T-maze. A green cue card was placed outside the right goal arm, which produced an unfavored stimulus in the zebrafish and caused them to swim to the left goal arm. However, when the subject entered the left goal arm, a mild electric shock was administered for spatial conditioning training or the development of passive avoidance behavior. The subjects’ memory retention was evaluated during testing by analyzing the time they spent in the punished arm and the latency to enter the left goal arm. The results of control fish were compared with ZnCl2-treated fish and genetic mutant zebrafish with a leptin a (lepa) gene deficiency. The results indicated that ZnCl2 exposure did not affect learning behaviors since the number of electric shocks administered was similar to the control group. However, ZnCl2-treated fish spent more time in the punished arm after training and had a shorter latency of swimming to the punished arm. This shows that ZnCl2 exposure affects memory retention in zebrafish. The results from the leptin a fish revealed that learning and memory performance were similar to control fish. However, a greater number of freezing behaviors were observed during training. 

Evaluation of Directional and Color Preference in Zebrafish 

Bault, Peterzon, and Freeman (2015) evaluated directional and color preference in zebrafish using a T-maze. During the directional place preference test, the maze was placed in different directions to result in the fish swimming north/south, northeast/southwest, or east/west. The colored preference test utilized a combination of orange, yellow, green, blue, and purple. One colored sleeve was placed outside one goal arm, and another colored sleeve was placed outside the other goal arm during the color preference test. The maze was placed in a direction that was not preferred based on the directional place preference test. The results indicated that rotating the arms pointing west and east reduced the directional preference reported in males. Furthermore, when the directional preference was removed, both male and female zebrafish preferred colors with shorter wavelengths.

Data Analysis

The following parameters can be observed using the Zebrafish Automated T-Maze:

• Number of times the subject chose the right goal arm
• Number of times the subject chose the left goal arm
• Latency to visit the right arm
• Latency to visit the left arm 
• Number of times the subject displayed freezing behaviors
• Latency of freezing behaviors 
• Time taken to leave the goal arm 
• Number of times the correct arm was chosen 
• Number of times the incorrect arm was chosen

Strengths

The Zebrafish Automated T Maze includes sensors and software used to configure and run the protocol, which allows the experiment to be easily performed within a short time. The maze chamber also includes pathways that allow the subject to swim back into the starting area once a trial finishes, which reduces the hassle of handling the subject. The maze can be used for various protocols, including conditioned place preference testing, color discrimination, and learning and memory. 

Summary

• The Zebrafish Automated T-maze is used to study spatial learning and memory and conditioned behaviors in zebrafish.

• It consists of a chamber with test areas, returning pathways, doors, sensors, water stirrers, a colored wall insert, and pellet dispensers in each goal arm. 

• The doors placed within the maze testing area form the shape of a “T”.

• The returning pathways are present between the goal areas and the starting area and allow the subject to swim to the starting area after completing a trial. 
• The maze is available with software that configures and runs the protocol. 

References

Ngoc Hieu, B. T., Ngoc Anh, N. T., Audira, G., Juniardi, S., Liman, R., Villaflores, O. B., Lai, Y. H., Chen, J. R., Liang, S. T., Huang, J. C., & Hsiao, C. D. (2020). Development of a Modified Three-Day T-maze Protocol for Evaluating Learning and Memory Capacity of Adult Zebrafish. International journal of molecular sciences, 21(4), 1464. https://doi.org/10.3390/ijms21041464

Bault, Z. A., Peterson, S. M., & Freeman, J. L. (2015). Directional and color preference in adult zebrafish: Implications in behavioral and learning assays in neurotoxicology studies. Journal of applied toxicology: JAT, 35(12), 1502–1510. https://doi.org/10.1002/jat.3169