Learning in a colour-cued plus-maze (Adult fish)

The aim of this experiment is to measure learning in a plus maze. Zebrafish are trained to use different coloured cues (coloured pieces of card placed at the end of the plus maze) in order to find a food reward. Each day, the position of the coloured cues is changed, requiring the fish to deduce where the food reward can be found. The amount of time needed to find the food reward over successive days is then recorded and a learning curve plotted. The time spent in each arm is also recorded.

Experimental setup

The tank is made from glass and contains four arms of the same length. One of the arms leads to a large area where the food reward is given. Each arm of the tank is 30cm long and 10cm wide. The large arena measured 20cm by 20cm.

Colour cues are provided by taping a coloured plastic sheet (in this case, red, green or yellow) to the inside of the arm using sticky tape. The long sides of the arms and bottom of the tank are clear (i.e. uncovered glass). The tank is lit from above with white light and from below with infra-red light (provided by an infra-red floor). The back of the large arena (where the food reward will be given) is covered with red plastic and does not move during the duration of the experiment. Two of the other three arms are covered with a moveable colour cue; one arm has a yellow insert and a second arm has a green insert (the third arm, where the fish will be introduced into the maze is left uncovered and is therefore clear).

A food reward is given upon completion of the task – successfully navigating to the large arena. As a food reward we use “New Life Spectrum Small Fish Formula” from New Life International Inc. These are described as natural colour enhancing 0.5mm sinking pellets.

Experimental procedure

1) The fish to be measured should be starved for 2 to 3 days before the beginning of the experiment, and should not be fed in between the learning trials.

2) The fish to be tested in the learning protocol first need to be habituated to the setup. They are placed as a group into the plus maze and left overnight. The colour cues are in place during this habituation phase. The fish can also be habituated to the novel food – the Small Fish Formula sinking pellets – although we did not find this to be necessary for successful completion of the experiment.

3) The next morning (the first day of testing) fish are placed into separate holding tanks where they will remain for the duration of the experiment. The fish are not fed in between testing in the plus maze.

4) Before the start of the experiment, the position of the colour cues on the short arms (green and yellow cues) are moved and the positions recorded. The red colour in the holding tank is never altered during learning. The water in the plus maze is changed every day (this can be made easier by using a suction pump setup to remove the water). Ten food pellets are added to the large arena as a reward.

5) A single fish is released into the arm with no colour cue, and the time needed to find a food reward is recorded. The fish’s position is recorded by Videotracking (We use Zebralab from Viewpoint but other software may also be suitable). The time needed to find the food reward is recorded, as well as the time spent in each of the arms.

6) When the fish successfully enters the arena and finds the reward it is given 30 seconds to eat the food. It is then gently caught and replaced in its holding tank.

7) The food pellets are renewed in order to keep a stable number over time, and a second fish can then be measured.

8) The experiment can be repeated on the next day in a similar manner. The colour cues are first repositioned on the short arms, and the fish released into the clear third arm.

9) The experiment is repeated every day for 6 days. Finally, the behaviour of the fish is also re-measured one week later, on experimental day 13 in order to look at long-term memory.

10) The results can be manually copied into Microsoft Excel and analysed (or be analysed using an appropriate statistical package).