We show that a three-chamber choice task developed for rodents can be adapted to assess the acquisition and reversal of visual discrimination learning in zebrafish. Our behavioral chamber is designed to assess cognitive performance. Once acquired, zebrafish remember the task eight weeks later, making it suitable for studies with zebrafish mutants and/or following drug exposure.
Demonstrating the procedure will be Dr.Cassie Rowe, a formal doctoral student from the Connaughton Laboratory. To prepare a three-chamber choice testing chamber, fix an aluminum U-shaped channel to the interior glass walls of a 40 liter 50 by 30 by 30 cubic centimeter aquarium to separate the aquarium into three chambers with the central chamber measuring 10 by 30 by 30 cubic central centimeters. Then fit 10 centimeter high opaque dividers constructed out of gray PVC sheets into both sides of the aluminum channels.
For each discrimination task, use hook and loop tape to place individual beige, black, or white felt pieces onto the outer back, side, and bottom of the choice chambers. As a reward, place four adult zebrafish that otherwise will not be used in the study in a small clear tank in the far back corner of each choice chamber to create a group of conspecifics. For group acclimation for each of the three days, attach the beige background to the outside of both choice compartments and submerge a live shoal tank in each of the compartments, then add five to six zebrafish to the central starting chamber with both sliding doors open and allow the fish to roam freely for 30 minutes.
The experimental zebrafish should be able to interact and socialize with the shoal fish through the tank as a reward after crossing into either choice compartment during acclamation. A fish is considered to have entered one of the side chambers when its entire body enters the chamber. For individual acclimation, after setting up the chamber in the same manner, place an individual zebrafish in the center starting chamber with the sliding doors closed.
After two minutes, open both doors simultaneously and make sure that the first swims from the central chamber through either door a total of 10 times in 30 minutes. Reward the fish each time it enters one of the side chambers. After completing the acclimation, place a white felt piece to the outside of one choice compartment and a black felt piece to the outside of the other choice compartment and place a shoal reward only in one of the choice compartments.
This becomes the rewarded side. To begin the acquisition, place a single experimental fish in the starting chamber with the choice compartments closed off. After a two-minute acclimation, simultaneously open both doors and start a stopwatch to assess the choice latency.
To denote the choice response, when the fish makes a choice by entering one of the side compartments, stop the timer. If the fish correctly selects the preferred side, immediately close the door between the central chamber in that side to restrict the fish to the preferred side for one minute to allow the first to be rewarded by interacting with the shoal tank. Score this trial as C for correct.
If the fish swims through the incorrect door, use a net to gently transfer the fish back to the central chamber, close both doors and score the trial as I for incorrect. If the fish does not make a decision within two minutes of the doors being opened, move the fish to the correct side for one minute and score the trial as M for marked. Once the fish has been returned to the central chamber, wait one minute before performing the task again.
After each fish has completed eight acquisition trials, categorize fish that have successfully selected the correct side of the tank at least six out of eight times as high performing and the fish that do not meet this criterion as low performing. Once identified, house the high and low-performing fish separately. When the fish have demonstrated the ability to solve a simple discrimination task, attach the black and white felt to the same sides of the choice chambers as for the acquisition task and submerge the shoal tank in the far back corner of the side opposite to that of the previously rewarded choice chamber.
Test the fish individually as demonstrated. As the rewarded side is reversed compared to the acquisition task, the reversal task assesses whether the fish can learn where the reward is located irrespective of the color of the background. When a total of eight trials have been completed for the three consecutive treatment days, report the results for the experiments as group averages for each two-trial block on each of three reversal days, keeping the data for high and low-performing fish separate to determine whether the two groups display the same level of performance during reversal as they did during acquisition.
After three days of training, experimental animals conditioned using a shoal-based reward take an average of 125 seconds to reach their first decision and an average of 725 seconds to complete the entire individual acclimation task. No significant side preference during acclimation is observed and the number of excluded fish is minimal compared to other previously tested reward types. During the acquisition phase, the number of high-performing animals increases daily with more high-performing fish observed on acquisition day three compared to day one.
The initial choice latency for all of the fish decreases across the three acquisition days indicating an improved performance with each day of acquisition. The same trend is observed when only the high-performing fish group are considered. As shown in this discrimination ratio analysis, all of the fish perform above chance by the end of acquisition, indicating that the fish have learned the discrimination task.
Following the acquisition of discrimination learning, the fish demonstrate strong reversal behavior and an increased discrimination over the three-day learning period. The three-chamber choice paradigm can also be applied to the examination of disease complications. For example, in this analysis, hyperglycemic zebrafish acclimation and acquisition were evaluated.
This task provides a robust assay that can be applied to various studies examining behaviorally-linked diseases such as hyperglycemic complications of diabetes, Alzheimer's and dementia.
