Barnes Maze: A Method For Assessing Spacial Learning and Memory in Lab Animals

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Basic Housing of Mice

1. House the mice in plastic cages with solid bottoms and sides, and a layer of soft bedding and nesting material such as paper shredding. Use bedding that is composed of shredded corn cobs or wood shavings, and ensure that the bedding is changed regularly for sanitation.
2. Provide access to food and water ad libitum. The top of the plastic cage houses a food hopper. Dispense properly formulated mouse food pellets from the food hopper and provide a water dispenser. Ensure that the cage top features an air filter to protect mice from outside contaminants.
3. Maintain the natural circadian rhythms of the mice by following a 12-hour light-dark cycle in the housing facility. Conduct behavioral testing at the same time of day, ideally during the animal's dark cycle, such as during the evening hours.
   NOTE: Care should be taken to distinguish mice in the housing. Various methods for distinguishing mice are available, such as ear punches, ear tags, and tail markings.

2. Barnes Maze Testing: Construction

1. Obtain a circular wooden board that is 120 cm in diameter.
2. Cut 20 circular holes (4.5 cm in diameter) around the perimeter of the circle. Position each hole to be 2.5 cm away from the maze edge and 13 cm apart from neighboring holes.
3. Smooth the surfaces and paint them glossy white (a bright color is recommended).
4. Designate one side of the maze board for insertion of small cup hooks roughly 2–3 cm away from each of the 20 holes and place two hooks for each hole (one on either side of each hole).
5. Use shallow black (plastic) disks for covering holes on side of maze with hooks. Use hooks to secure thick rubber bands in order to hold the disks onto the bottom of the maze.
6. Securely mount the Barnes maze 120 cm above the ground and away from other similarly tall objects like tables or chairs. A box or stool can be used to support the middle of the maze.
7. Place large white posters with one shape on each wall (triangle, circle, and cross) as extra-maze cues on 3 sides of the maze. Maintain the posters on the walls around the maze for each trial.
8. On last side of maze, set up a solid black curtain to hide observers so that data can be recorded accurately without the researchers being visible to mice on the maze.
9. Suspend a video camera over the arena with a bird's-eye view of the entire maze surface.
10. Clean every surface of the maze (black disks and target box included) with water then a 70% ethanol solution before and after each mouse trial.
11. Place a 100 W light source 25 cm over center of maze (turn on/off at the start/finish of trial) and be sure to have the disks and hooks facing the floor.
    NOTE: All other overhead lights in the room should be turned off during testing. The addition of an ultrasonic noise maker hung next to 100 W light is recommended. Turn it on/off at the start/finish of each trial.

3. Barnes Maze Testing: Procedure

NOTE: Ensure that all the maze components are cleaned with water and 70% ethanol solution before and after each trial, allowing for time to dry completely before testing resumes. Be sure to have cleaning supplies ready as well as timers for the trials.

1. House all the mice in groups while using a reliable identification method. Keep mice outside of the testing room when they are not actively running on the maze. This is to ensure they are not subjected to the ultrasonic noisemaker prematurely.
2. Handle mice by gently picking up and holding them from the base of the tail, with the paws kept on one's hand.
3. For training, have the mice run on the maze daily once per day for 7 days in a row to assess learning/acquisition.
   NOTE: A single long-term trial can be done on a later date to assess memory/retention. The current protocol trained adolescent mice beginning at postnatal day 32 and a long-term trial was performed in adults at 4 months of age.
4. Randomly assign each mouse to a target hole to use throughout the testing period. The holes can be labeled 1–20 on the bottom of the maze or on the outside of the maze perimeter, where they are not visible to the mouse in the maze.
5. Replace the assigned hole disk with a small black box (23 x 11 cm). Firmly attach it to the Barnes maze using rubber bands connected to the nearest hooks.
   NOTE: The target box is shallow enough so that the mouse can easily step down into it or contains a step to ensure that the mouse does not have to jump down into it.
6. Place the mouse onto the center of the maze beneath a cup to acclimate for 30 seconds until the test begins. Begin video recording.
7. Turn on the light and ultrasonic noisemaker. Lift the cup via a string mechanism to avoid biasing the initial heading of the animal. Start a timer and sit behind the curtain to observe.
8. After the mouse enters the target hole, cover the hole with the same opaque cup used at the start of the trial or a binder and turn off the ultrasonic noisemaker. If the mouse has not entered the target after 5 minutes have elapsed, corral the mouse into the target hole.
   1. Ensure the mouse enters the hole. When mouse is inside the target box, cover the hole and turn off the noisemaker. Allow the mouse to remain in the target box for 1 minute undisturbed.
9. Although the target hole will remain constant for each mouse throughout the training period, run mice in a random order each day to ensure that they are not following any cues/scents left by the previous mouse.