Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The overall goal of this procedure is to measure locomotion and behavioral activity of mice in an open field environment. The first step is to acclimate the mice to the open field chamber for four consecutive days. Following acclimation behavioral activity is recorded using specialized software.

Each day, the data are exported and reviewed for quality control purposes. After testing, the data are analyzed for multiple behavioral parameters. Ultimately, open field activity is used to show differences between control and test animals in a non-invasive manner.

The advantage of this non-invasive method is that it provides an overall assessment of general quality of life in a longitudinal manner. This mimics the six minute walk test used as a clinical outcome measure and provides insight into potential off target drug effects For the best results. Make sure that all animals are properly acclimated and that the testing is performed at the same time each day, and a quiet temperature and humidity controlled room with even illumination.

To begin, bring the mice into the testing room and allow them to acclimate for approximately 10 minutes. Leave the room during this time. Next, return to the room and turn on the activity chambers.

Even though data are not being collected at this time, this will mimic the testing environment. The activity chamber used in this protocol contains a center divider that divides the chamber into quadrants. Make sure that the divider is properly aligned inside the chamber.

Next, gently remove each mouse from their home cage and immediately place them into an empty quadrant. Once all animals are loaded, place the lid on top and leave the room. Allow animals to acclimate to the test chamber for 60 minutes following the acclimation period.

Return to the room and remove the lid. Gently return each mouse to their home cage. Clean each chamber thoroughly with disinfectant and paper towels between each session.

Repeat these steps for four consecutive days one week prior to initial data collection. Take care to randomly assign the animals to a new box each session to avoid habituation and track the box assignments throughout the duration of the study. To measure open field activity, bring mice into the testing room to acclimate for 10 to 30 minutes.

Leave the room during this time. After this time, return to the room and turn on the activity chambers. Next, open the accompanying computer software that will record the animal's movement.

Set the primary data collection parameters in the computer software to collect six, 10 minute blocks of data and then enter the appropriate date, file name and mouse ID numbers. Once all parameters are set, run a pre beam check to verify that all infrared beams are functioning properly. If the chamber does not pass the pre beam check, it is most likely due to poor alignment of the center divider.

If this happens, realign the divider until the sensors are unblocked and the system is ready. When all test chambers are ready, gently remove the mouse from its home cage and place it into the test chamber. Two animals can be placed in the test chamber at the same time during data collection.

For example, one animal can be placed in the front left quadrant and one in the back right quadrant. Place the lid on top of the chambers once all animals are loaded, and then start the program to begin data collection. The program will start recording activity levels according to the data collection parameters set earlier.

Leave the testing room for the duration of the testing period. Return to the room when data collection is complete. Save the data and then return each animal to their respective home cage.

Clean all units with a disinfectant and paper towels before testing another group of animals. After testing, export the data to a spreadsheet. The computer software calculates multiple parameters over the data collection period.

For each mouse, review all data for quality control purposes. Repeat these steps for four consecutive days. Take care to randomly assign the animals to a new box each session and track the box assignments throughout the duration of the study.

Lastly, calculate the mean horizontal activity. Vertical activity, total distance traveled, total movement time and total rest time from the four days of data collection. These results show typical open field activity levels of male and female ddy two J laminate alpha two deficient congenital muscular dystrophy, mice and control mice over time, horizontal activity was higher in control mice as was vertical activity and total distance traveled.

DY two J mice also showed a complete loss of vertical activity by 19 weeks of age, which reflects their hind limb paralysis and in turn, inability to rear at six weeks of age during the peak necrotic phase. In the animal model of Duchenne muscular dystrophy, MDX mice show lower levels of horizontal activity, vertical activity, and total distance traveled. Differences in open field activity were also demonstrated between mice of different strains.

BL 10 mice had higher horizontal activity, vertical activity, and total distance traveled compared to L six mice. Because behavioral activity is prone to large variations, open field activity may not be a suitable primary endpoint measure. However, it can be a very valuable secondary outcome measure in preclinical studies when conducted with additional functional histological and molecular assays.