Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The overall goal of this experiment is to measure anxiety in rats, using the appropriate elevated plus maze. The main finding of this study is that the detection sensitivity of the anxiolytic drug effect can change depending on the features of the elevated cross maze apparatus. This study can help researchers set up the maze apparatus in the laboratory, appropriate for their experimental objective, and ensure a great recording of data.

Demonstrating the procedure will be a guest researcher from my laboratory. To begin set up the elevated plus maze apparatus in a customized closet container. The plus shaped metal framework of the maze should be elevated about half a meter from the floor.

Maintain a consistent brightness throughout the maze at the required intensity. At the overhead center point mount a digital video camera to record and monitor the tests. Now fix the maze arms onto the framework.

There are two types of open arms, either with or without 5MM ledges along the sides. There are also two types of closed arms. The walls can be translucent or opaque.

Attach the square central platform between the arms. The central platform and maze arm runway surfaces are colored black for better animal tracking by the automated behavioral analysis. Now with arms in place adjust the lighting intensity, so there is approximately 100 lux at the surface of the distal end part of each arm, and at the central platform.

Before each test clean the entire apparatus with 70%ethanol solution, followed by wiping it down with distilled water. Click the record button on the hard disk recorder, to start collecting images from the digital video camera. If the equipment is working then conduct the test.

Start the test by placing the rat on the central platform, so that it is facing an open arm, and close the closet doors. Then allow free exploration on the apparatus for five minutes. The experimental outcome may vary, depending on whether the rat is appraised facing an open or crossed arm.

It is better to always place rats, resting on open arm to prevent freezing, on first entry to the closed arm. After five minutes, stop the recording to end the test. Then return the rat to its cage, and count the number of feces and urination's in the maze before cleaning it.

Always perform the behavioral analysis using both manual observation, and automated analysis software. From the video manually record the number of entries the rat makes into the arms of the maze. An entry is defined as any instance when all four paws are on the arm.

Also record the time spent in each arm, and the number of stretched attending postures made while the rat is on the central platform facing towards an open arm, and backing into a closed arm. Perform these measurements using a stopwatch and a counter during the five minute test. To perform an automated analysis, open the video recording in a commercial software program.

The software tracks the animal on the maze, using the contrast between the animal and the dark floor. First assign the animal to a contrast blob of a specific size and then assign the center of the blob as the tracking point. Next for the full five minute test, have the software record the distance traveled, the time spent in different positions on open arms, and the total time spent on open and closed arms.

Sometimes the automated analysis cannot distinguish between entries and non entries into arms. Therefore, we recommend that manual observation is always carried out in addition to automated analysis. The results of rat exploratory behavior on the elevated plus maze with different open and closed arm designs, show that rats spend more time on open arms with ledges, than open arms without ledges.

They also made more entries onto those arms, thus ledges likely increase exploratory behavior on open arms. Rats showed significantly less stretched-attending posture on closed arms with transparent walls, than on closed arms with opaque walls. Suggesting that transparent closed arm walls decreased anxiety like behavior.

Rats also spent more time on the distal segments of the open arms when the closed arms had transparent walls, than when the closed arms had opaque walls. Furthermore, distal open arm exploration was significantly lower in mazes with no ledges and opaque arm combinations, than in ledges and transparent arm combinations. Therefore detection sensitivity maybe altered, depending on the combination of arm designs used.

To verify differences in detection sensitivity between different types of arm design, a further experiment was performed using the anxiolytic drug Diazepam. I brief, the effect of high dose Diazepam treatment was detected in the opaque wall apparatus without ledges, but not in the transparent wall apparatus with ledges. While attempting this procedure, it's important to remember to treat all animals under the same experimental conditions, and to make sure that all animals are placed facing the same open or closed arm at the start of each test.

After watching this video you should understand how to set up the elevated cross maze apparatus. It is important to remember that experimental outcomes can vary depending on arm design. So we recommend using appropriate arms for your experimental objectives.