Protocol for Elevated Plus Maze.

Basic Features

The test will be a five min test on the maze immediately following transport from the holding area in the home cage.

General Apparatus Setup and Room Configuration

1. Mazes are to be placed in an area where as much symmetry as possible is achieved from the room surroundings. What is most important is to balance the room with regard to open versus closed arms. For example, if a blank wall faces one closed arm then a similar blank wall should face an adjacent open arm. Then the other two arms should also have somewhat similar surroundings (Cabinet, wall, door, etc). The two mazes themselves should have very similar surroundings and be at the same orientation with respect to the camera, not as mirror images. This will greatly aid the later scoring from video tape. Without building special barriers, this is not easy to achieve, and I think we simply need to do the best we can without resorting to complete construction of false walls/curtains/etc. Place the maze arms at 45 degree angles with respect to walls. A small table adjacent to a wall will sit at about 4 ft from the maze if possible. This table would thus be between the angle of a pair of open and closed arms if the 45 degree arrangement is used. This table is for placing the home cage when mice are transported into the room.

2. Lighting level should be comparable to the water maze lighting. Tungsten lamps with 15 w light bulbs should be placed at the same level as for the water maze, making them approximately 1.5 m above the surface of the maze. (Ours are 1.37m above.) The lighting at the center of the maze should be approximately 7 fc (74.9lux). A tall partition is used to prevent glare from the lamp above the other maze.

3. Quiet is important. This includes surrounding rooms as well as test room conditions. Great care must be taken when opening cages for placement of mice. Rattling of cage lids should be avoided. The experimenter should wait quietly behind a barrier during a trial.

4. Camera setup is at the 45 degree angle between one open arm and one closed arm to aid the symmetry point #1 above. The VCR/monitor setup must be out of view of the maze behind an opaque barrier. Make certain to turn the sound down (completely) on any tv monitor. Monitor the mice on the maze by watching the tv monitor rather than the maze.

General Testing Procedures

1. Fecal boli are removed prior to isopropyl wipe. The plus-maze arm surfaces and closed sides are cleaned with 10% isopropyl. This is done prior to each mouse's test. Moisten a Kim-Wipe type of paper with the alcohol and wipe whole maze surface, concentrating also at the exposed edges of the closed arm walls. Mice tend to spend a lot of time sniffing there. A few seconds for evaporation and diffusion of odor is all that is required.

2. Mice are to be moved from the holding area to the testing room in their home cage with water bottle removed.

3. VCR settings would ideally include fast tape speed (2 hr/tape) for better resolution (especially if later scoring requires some slow motion playback), but this will require more tapes. Slower speeds will work ok, depending on the VCR quality. Videotaping integrity should be checked/doublechecked at regular and frequent intervals. In addition, elapsed tape time must be monitored closely so that tapes do not run out in the middle of a test.

Identification of the individual mouse will be done by placement of a 3x5 card under the camera view immediately prior to each mouse's test. This card should have an identifying subject number only so as to aid in blindness to genotype (as much as possible given coat color) at tape scoring.

4. Handle mice by hands (not forceps) for all plus maze testing. Experimenters will wear surgical type gloves for this handling. Hand handling can be more consistent and less likely to "upset" that mouse who gets a particularly hard pinch from forceps. Placement on the maze is done by hand to ensure consistency and orientation.

5. Mice for am or pm session (16 cages; 32 mice) are moved to the holding area just prior to the beginning of testing. Bottles/food available, with bottles removed only during transport into test room and testing.