Description
Specifications |
Acrylic Y-maze with walls and translucent base |
Length: 15cm |
Width: 7cm |
Height: 10cm |

Introduction
The Automated Avoidance Zebrafish Y-maze is an advanced operant conditioning assay designed to study zebrafish avoidance behaviors when exposed to aversive stimuli. It enhances the traditional Zebrafish Y-maze Avoidance setup, which relies solely on visual cues displayed via an LCD screen beneath the maze. The automated version incorporates additional stimuli, including auditory cues from speakers and aversive stimuli administered through electric shocks via steel mesh plates on the opposite walls of each maze arm. This system also includes video tracking software that manages the protocol, controlling the duration and timing of each stimulus, thereby reducing human error and saving time.
In this learning task, zebrafish must learn to associate a colored cue with an electric shock. All regions of the floor are colored red by the LCD screen except for one area designated as the “goal region,” which remains gray. The zebrafish must swim to the goal region within a set time limit (usually 15 seconds) to avoid an electric shock administered throughout the maze, except for the goal arm, until the trial ends. An auditory cue signals the start of each trial and can also serve as a conditioned stimulus alongside or instead of visual cues.
The Automated Avoidance Zebrafish Y-maze offers significant flexibility and can be easily adapted for various experimental protocols, eliminating the need for multiple behavioral apparatuses. For instance, it can be used for visual and auditory discrimination paradigms by placing different stimuli in each maze arm. This maze is valuable for studying zebrafish behaviors, which can be extrapolated to understand human behavioral phenotypes in behavioral neuroscience. Additionally, it aids in investigating the neural basis of behaviors, neuropsychiatric disorders, and the effects of drugs on the brain (Marcon, Benvenutti, Gallas-Lopes, Herrmann, & Piato, 2021).
Apparatus and Equipment
The Automated Avoidance Zebrafish Y-maze features an acrylic structure with three arms arranged in a Y-shape, complemented by video tracking software. Each arm of the maze measures 15 cm in length, 7 cm in width, and 10 cm in height. A camera positioned 60 cm above the maze captures the behavior of the subjects. The video tracking software is responsible for setting up the experimental protocol and managing the experiment.
The maze includes various conditioning stimuli. An attached speaker delivers auditory stimuli with frequency tones ranging from 100 to 20,000 Hz and a volume scale from 1 to 100. Visual stimuli are presented on an LCD screen placed beneath the maze, controlled by specialized software. Mesh grids cover all maze walls, and steel mesh plates on both sides of the maze arms can deliver electric shocks. These shocks have a DC setting of 5V (0.12 mA), 50-ms duration, 10 Hz, and an AC setting of 2.5V. The voltage, current, and duration can be adjusted as needed.
The video tracking software regulates all stimuli, including auditory cues, visual signals, and electric shocks, all of which are customizable. The setup includes a control box connected to a PC that oversees all operations.
Training Protocol
Separate the subjects by placing them individually in 1-liter tanks a day before the experiment.Ā
Literature Review
Evaluation of associative learning in zebrafish using an Automated Avoidance Y-MazeĀ
Aoki, Tsuboi, and Okamoto (2015) designed the Automated Avoidance Y-maze to investigate associative learning in zebrafish (Danio rerio) and enhance experimental efficiency. They used wild-type zebrafish aged 6-12 months. The maze was placed in a soundproof box atop a 24.1-inch LCD screen. The subjects’ behaviors were recorded with a video camera connected to a computer. The video software controlled auditory tones via a speaker and displayed visual cues on the screen. Electric shocks were administered through steel mesh plates in the maze arms. Additionally, small fans integrated into the screen maintained the water temperature between 29-30°C and dissipated heat from the screen.
The experiment began with twenty initial trials without shocks, followed by one hundred trials with shocks. During the training, the goal region was highlighted in red on the LCD screen, and the subject had to reach the goal within a set time limit (30 seconds for ‘no shock’ trials and 15 seconds for ‘shock’ trials). If the subject failed to reach the goal region during ‘shock’ trials, electric shocks were delivered throughout the maze except for the goal arm. Subjects exhibited active locomotion throughout all trials. Results showed that in the ‘no shock’ trials, subjects consistently reached the goal region within 15 seconds, though there was no initial preference for the goal arm. In the ‘shock’ trials, subjects achieved a 100% success rate in reaching the goal region by trials 41-43. However, between trials 44-61, there was a decline in success despite repeated shocks, with subjects only rushing to the goal region once the shock was administered. During trials 62-120, the success rate improved, with subjects swimming directly to the goal region. Overall, the findings indicated that the subjects demonstrated associative learning of electric shocks with red color cues and learned to exhibit avoidance behaviors in response to these cues.
Data Analysis
The following parameters can be observed using the Automated Avoidance Zebrafish Y-Maze:
- The arm the subject visited firstĀ
- Time taken to reach the goal regionĀ
- The number of times the subject reached the goal region within the required time limitĀ
- The number of times the subject did not reach the goal region within the required time limitĀ
- Trial durationĀ
Strengths and Limitations
Summary
- The Automated Avoidance Zebrafish Y-Maze is an operant conditioning assay used to study avoidance behaviors in zebrafish when presented with an aversive stimulus.
- It consists of a Y-Maze with an LCD screen placed below the maze that displays visual cues throughout all regions of the maze.Ā
- It also includes speakers for providing auditory cues and mesh plates equipped on opposites sides of each maze wall that administers electric shocks throughout the maze.Ā
- A video tracking software sets up the protocol and controls the experiment. It can control the duration of each stimulus and when it will be presented.Ā
- The Automated Avoidance Zebrafish Y-Maze eliminates the need to control multiple stimuli, which reduces human error and also helps save time
- The Automated Avoidance Zebrafish Y-Maze can be modified and adapted for various behavioral experimental protocols, such as visual discrimination and auditory discrimination paradigms.Ā
References
Aoki, R., Tsuboi, T., & Okamoto, H. (2015).Ā Y-maze avoidance: an automated and rapid associative learning paradigm in zebrafish.Ā Neuroscience research,Ā 91, 69ā72. https://doi.org/10.1016/j.neures.2014.10.012
Marcon, M., Benvenutti, R., Gallas-Lopes, M., Herrmann, A. P., & Piato, A. (2021). What do zebrafish prefer? Directional and color preferences in maze tasks.Ā bioRxiv.Ā https://doi.org/10.1101/2021.12.22.473814