Assessing Spatial Learning and Memory in Small Squamate Reptiles

The overall goal of this procedure is to assess spatial memory and learning in small squamate reptiles using a modified Barnes maze. This method can help answer key questions concerning the spatial cognitive processes of non-model species, particularly in small squamate reptiles. The main advantage of this technique is that we have modified a traditional rodent paradigm, the Barnes maze, to allow for testing of spatial learning and memory in small lizards.

It’s possible that the maze could be similarly altered to be applicable to other species as well. For this protocol, it is necessary to purchase or construct the Barnes maze. Make certain that the goal holes are appropriately sized for the species of interest.

For this demonstration, fence lizards are in use, so the holes are sized to match. Position the maze in a quiet room with bright overhead lighting and spatial cues around the maze, such as doors and cabinets, to help the animals navigate. Above the maze, mount an auto-focus wide-angle lens webcam or other suitable camera.

The key is for the field of view to contain the entire maze. At least one meter to the side of the maze, position the computer used for animal tracking. Once positioned, mark the legs of the maze on the floor with a permanent marker or tape, so it can be adjusted back into its original position with ease.

Regarding the maze, randomly assign one of the escape holes as number one, and number the other holes consecutively up to number 10. Mark the numbers along the outer wall of the maze, so they do not serve as visible cues for the animals. Under the maze, if needed, use a small space heater or attach heat tape to increase the maze surface temperature so that the animals will be at their optimum body temperature for metabolic activity.

To begin, randomly assign goal holes to each animal. If the animals are housed outside of the testing room, bring their home cages into the room at least 30 minutes before testing. Be certain to provide them heat, if needed, so that they are at their ideal body temperature when tested.

On the computer, open the custom MATLAB tracking software and webcam application to get the video feed. Then, evaluate the field of view again and adjust the maze’s position as needed. Next, open the worksheet to record the basic experimental parameters, which should include the date, start and end times, animal IDs, assigned goal holes, number of training trial runs, notes, and observer’s initials.

Then, randomize the animals’testing order and start testing them in a systematic fashion. First, remove an animal from its home cage and gently place it in the middle of the maze. Then, cover it with an opaque plastic tub and allow the animal to acclimate for 30 seconds to the tub.

Meanwhile, mount the animal’s home cage under the maze directly below the assigned escape hole. Slide the top of the enclosure into the peg mounts, and put the basking rock in the cage, directly under the hole. Now, gently remove the plastic tub from the top of the maze and start recording the trial on the computer.

Allow the animal to explore the maze for ten minutes, and if anything atypical occurs, note it in the worksheet. If the animal falls or runs off the table, gently return it to the middle of the maze, provided it has not been out of the maze for more than 30 seconds. Otherwise, abort the trial and record this result in the notes section.

If the subject descends into the escape hole unassisted, the trial ends at that time. If the subject does not escape into the hole within 10 minutes, end the trial and gently guide the animal to its escape hole by hand, moving it head first. Once in its home cage under the maze, allow the animal to rest for at least two minutes.

Meanwhile, clean the top of the maze with a diluted soap mixture and paper towels. Some species, particularly those who make heavy use of chemosensory information, necessitate additional rinsing of the maze with hot water to eliminate all scent cues. Once the maze is cleaned, remove the enclosure from under the maze and continue testing the animals.

Each can run the maze up to five times per day, with a half-hour interval between trials. Repeat this training procedure until the subject has descended into its goal hole unassisted on three different trials. These success do not need to occur consecutively, and there are no limits on the number of trials needed.

The number of training trials to reach criterion can be burdensome. Alteration of the maze in an ecologically relevant manner, taking into consideration a species’natural history and behavior may help to alleviate this. Then, proceed with the probe trial on the following day to further test that animal.

Once the animals have learned their escape hole, use the probe trial to determine if it is navigating using a spatial strategy or some other navigational strategy. For this trial, rotate the maze 180 degrees, so the local cues within the maze will be in direct conflict with the spatial cues outside the maze. Then, test the animal using the same procedure as a training trial.

However, no cage is mounted under the escape hole. Thus, no olfactory cue from the cage can help guide the animal to its escape. If the animal fails to find the escape hole, just remove it from the maze and return it to its home cage.

Do not teach it where the hole is located. As before, always clean the maze before testing the next animal. Seven adult side-blotched lizards were tested as described.

Individuals took between 17 and 81 trials to advance to the probe trial, with the average near 50. It did take significantly less time for the group to find the escape hole in the fourth quartile of their trials than the first, which suggests learning. During the probe trials, individuals went to the spatially correct goal hole at a rate well better than chance and spent a majority of their time in this quadrant of the maze.

Thus, spatial cues in the room were being prioritize over local cues in the maze. After watching this video, you should have a good understanding of how to modify a Barnes maze to accommodate testing of a small squamate reptile, as well as how to run animals in training and probe trials. While attempting this procedure, it’s important to remember to take into account the natural history and behavior of the species of interest when designing a task to probe for spatial memory and learning.

Following this procedure, more detailed applications would be appropriate, such as decreasing reliability of spatial cues or making local cues more obvious. Doing so may reveal how the stability of different cues can modulate cue prioritization and memory.