This method can help answer key questions in the behavioral neuroscience field about whether a genetic mutation or environmental variable can impact social behaviors or the acquisition and retention of spacial memories. The main advantages of these techniques are that both the studies can be simply constructed with minimal financial investment and that neither protocol requires the animals to experience extreme or stressful stimuli. The Damsel in Distress method can provide insight into mouse pro-social behaviors, but it can also be used as an empathy measure in other mammalian model organisms.
Before beginning the Barnes Maze Testing Procedure, clean all of the maze components with water and 70%ethanol. When the components are dry, assemble the maze and randomly assign each mouse to a target hole to use throughout the testing period. The holes can be labeled one to 20 on the bottom of the maze or on the outside of the maze perimeter where they are not visible from within the maze.
To start a training test, replace the assigned hole disc with a 23 by 11 centimeter black box and use rubber bands and hooks to firmly attach the box to the maze. Then, place a mouse onto the center of the Barnes Maze beneath a cup and allow the mouse to acclimate for 30 seconds. At the end of the acclimation period, begin the video recording and turn on the light and ultrasonic noise maker.
Lift the cup via a string mechanism to avoid biasing the initial heading of the animal and start a timer behind an observation curtain. Track the animal's movements on a piece of paper with a diagram of the maze and use the diagram to analyze the mouse's search strategies as well as to determine the number of holes that were explored in the quadrant opposite the target. When the mouse enters the target hole, cover the hole with a heavy opaque binder and turn off the ultrasonic noise maker.
If the mouse has not entered the target after five minutes, corral the animal into the target hole. Then, allow the mouse to remain in the target box for one minute undisturbed, making sure to turn off the ultrasonic noise. Then, return the animal to its cage.
When all of the mice have been tested, manually review their recording to track the overall movement of the mouse to determine the number of incorrect holes explored before and after the target is found, as well as the number of holes from the target hole to the first hole explored and any notable grooming behavior. For exploratory behavior Damsel in Distress Testing, place a closed and empty stress chamber into the center of an empathy arena and begin recording with the suspended camera. Gently place a male mouse beneath a cup at the lower left corner in the arena for 30 seconds.
Then, remove the cup via a string mechanism and allow the mouse to explore for 10 minutes taking care to stay out of the field of view during the recording. At the end of the exploration period, move the mouse from the arena into a holding cage for five minutes and stop and save the recording. For social responsiveness Damsel in Distress Testing, gently manually restrain a female mouse and lower the mouse into a restraint chamber.
Close the open end of the chamber behind the mouse and confirm that the mouse is unable to turn around. Begin recording with the suspended camera and place the restraint chamber inside in the center of the arena. Allow the animal to acclimate for 10 minutes taking care to remain out of the field of view.
Then, transfer the male mouse from the holding area back into the empathy arena for 10 seconds and allow the male mouse an additional five minutes to explore the arena taking care to remain out of the field of view. At the end of the exploration period, stop and save the video file and return both mice to their cages. Then, replace the corn bedding and sanitize both the arena and the stress chamber with 70%ethanol.
To analyze the data, open the video file in the appropriate tracking software and place a transparent sheet over the screen. Outline the square of the arena with a marker and divide the arena square into nine equal compartments. Review the video data for the first five minutes of the initial exploratory measure recording the number of compartments crossed, the time spent in the center square of the arena, the number of digging and grooming episodes, and the number of times the mouse touched the empty center restraint chamber.
Then, review the video data for the five minutes of the social responsiveness measure immediately after the male is reintroduced into the arena with the trapped female recording the same data as for the initial exploratory measure, but adding tracking of the number of times the male mouse touched noses with the trapped female mouse. When Barnes Maze trained neonates are run as adults in a single long term trial, the ethanol injected mice have a harder time remembering where the target hole was located than saline injected control animals. Ethanol treated mice also explore significantly more holes in the opposite quadrant from the target, indicating a failure to remember the correct general spacial location and use a sequential hole to hole search in a ring shaped fashion until the hole is found.
In the Damsel in Distress model, older mice cross more squares than younger mice. In the presence of a trapped female however, male mice of both ages spend less time exploring the arena. Juvenile males spend more time digging during their time in the arena in the presence of a trapped female mouse than when alone suggesting anxiety or pro-social behavior toward the trapped female.
By contrast, male mice touch the tube containing the trapped female and initiate trapped female contact significantly more frequently as adults than as juveniles. While attempting this procedure, it's important to remember to keep the conditions constant between and within the trials to ensure that the environmental variables do not confound the results of the study. Following the initial use of these behavioral assays, the same animals can be retested at various time points during development to answer questions about long term memory and possible changes in social behaviors.
