Protocol for Elevated Plus Maze

(Final)

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.

Specific Testing Procedures

1. Testing is to be done in pairs with the dual mazes. Mice are set up and transported from the holding area in home cage pairs. Walk the cage to the testing room and place it on the table adjacent to the plus maze. Testing begins as soon as possible. The time from removal from the home cage to the start of testing would be less than 10 sec if camera/VCR and identifying card are ready for each mouse prior to bringing it into the room.

2. The camera/VCR should be started and recording at this point. Stopping and starting the VCR between tests of individual mice is recommended to save time on the tapes. Start with the +-maze furthest from the control/monitor booth. Place the 3x5 card under the camera view briefly to identify the mouse to be tested. Make certain that the card is placed at a level where the camera is focused.

3. Gently remove the cage lid and place it on the towel on the table and pick the mouse up. The experimenter is standing off the end of one open arm. The mouse is placed quickly (but not rushed or with a jerking motion) onto the center square between the arms, and facing the opposite open arm. Thus the mouse is facing away from the experimenter upon placement.

If the mouse is lowered from a height of approximately 18" at a smooth rate, it will momentarily freeze upon touching the floor of the plus maze. If the rate is too slow, the mouse will grab the edge of a closed arm and the placement will be disrupted. If the mouse is moved too rapidly, it will likely bolt upon release and immediately move out into an arm, and perhaps jump off the maze. The key is smooth continuous movements, from holding cage to maze. A right-handed experimenter should be holding the cage lid with the left hand and handling the mouse with the right hand. It is possible to replace the cage lid on the cage as one is removing the mouse so that it will not be rattled as the mouse is placed on the maze. In Portland, the lid of the cage was placed on towel. Experimenter moved to end of open arm with cage and mouse. Then picked up mouse, etc. Experimenter was holding cage in left hand, mouse in right,

rather than lid.

4. Quickly place the empty cage back on the table and move to the VCR/monitor behind the partition. Immediately after placement, a timer separate from the VCR timer is started. Quiet stopwatches or constantly running visible clocks work. If a mouse falls off an arm to the floor, quickly but smoothly move to the maze, and replace the mouse onto the center square facing an open arm as at the beginning of the test. Extend the total trial time by the duration of the time off the maze. Make a note of this event.

5. At the end of the five min test for the first mouse leave that mouse on the maze for the few additional seconds until the second mouse's five min is finished. The experimenter would move away from the mazes to the area where the television monitors are in order to watch the mice in an area not visible to the mouse.

6. At the end of the five minute test for the second mouse, remove the wall for the closed arm closest to the camera despite where the mouse is located on the maze. If the mouse is on a closed arm, persuade it to walk onto one of the open arms or the closed arm with wall removed. Remove the mouse with your fingers.

7. Counts of fecal boli on closed and open arms and the center square are recorded prior to cleaning the maze.

8. After mice are placed back in the home cages at the end of the test, the VCR stopped, the mazes are cleaned and the index cards for the next pair are prepared. When cleaning, remove the other closed arm wall for better access to the maze surface but leave the low walls on the open arms in place.

9. Mice are then returned to the holding area.

General Scoring Procedure

To be elaborated later. Simple measures of time in the arms/center, entries into each arm, and

forays to the end of each arm are what is planned. Only fecal boli and notes about aberrant reactions to handling or falling off of the maze need be recorded at the time of testing.