The overall goal of this behavioral assay is to identify the presence of higher order repetitive behaviors in mice. This assay can help validate potential mouse models and autistic like behavior, such as the inbred C 58 J mouse strain. The main advantages of this technique is that it does not require any mouse training and it is not particularly anxiety provoking.
Begin by filling the testing arena. A clear plastic rectangular cage with a half inch of corn cob bedding. Select four novel objects that are different in shape and color.
For example, a pink toy brick, a red monkey, a white tile with blue writing, and a standard white die. Ensure that these objects are approximately the same size and constructed with high density plastic to facilitate cleaning and resist chewing. In the rectangular arena, place these objects three centimeters from the corners in a random and counter-balanced order throughout testing.
Record the placement of each object as a different number from one to four. Finally, position a camera directly above the testing arenas so that the entire arena is recorded during the acclimation and rest periods. Begin testing at the beginning of the light cycle in a room illuminated with fluorescent lighting at approximately 100 lux.
Ensure that lighting is uniform across the testing arena to standardize appearance while video recording. Then, place a note card of known dimensions in the bottom of the testing arena for later use as a calibration standard. And push the record button on the video camera.
Next, transfer a test mouse into the empty testing arena for 10 minutes to serve as an acclimation period and continue recording. After the acclimation period, leave the mouse in the arena and quickly add the four novel objects to the arena. Record mouse behavior for an additional 10 minutes during this testing period.
Finally, after the testing period has completed, return the test mouse to its home cage. Clean and dry the novel objects as well as the testing arena with unscented dish soap and water. Begin by setting up the project coding scheme in the behavioral logging software.
Within the project setup box, set data acquisition to offline, offline observation. Within the behavior coding box, program scrabble, digging, rearing, grooming, and sniff object one, two, three, and four as state events. Program jumping as a point event.
To program the secondary keyboard, open the keyboard software, click on the secondary keyboard button displayed on the screen, type in the appropriate keystroke combination, and click okay. Once the secondary keyboard has been programmed, close the software and let the program run in the background of the computer. After the project has been set up in the software, prepare to score the number and duration of each of the following repetitive behaviors.
Rears, digs, self-grooms, and jumps. To score a video, click file, open project, observe, observation, and new. Choose a name for the new file and then select the video media file to score.
Begin scoring the repetitive behaviors by clicking on the begin observation button. Use two keystrokes for all state events. One to signify the initiation and the other for the end of the behavior.
And use only one keystroke for point events. Next, use the software to score the number of times and length of duration the mouse sniffed each object, which is defined as any time a mouse moves its nose within 0.5 centimeters of an object. Each time a mouse sniffs an object, record the corresponding position number of the object and manually record the string of position numbers until the end of the 10 minute testing period.
Count a position twice if a mouse sniffs an object, looks away, then sniffs the object again. After the 10 minute testing video has been scored for sniffs, visualize the data by clicking on analyze, behavior analysis, and new. Then, once the data appear on the screen, export into a separate spreadsheet.
Next, record the total distance the mouse traveled within the arena during testing. To do this, use the note card to calibrate each video by setting a calibration line along each end of the note card and inputting the appropriate length within the software's calibration screen. After the lines are drawn, input the known length and width of the note card that correspond to each line.
Next, within arena settings, select the entire arena. Within trail control settings, select a 10 minute duration. And in detection settings, choose a dark object on a light background.
Once these settings have been programmed, score the videos by clicking acquisition and open acquisition. In the acquisition control box, click new trial. And then start trials.
After 10 minutes have elapsed, stop the program and observe the data. Finally, click analyze and calculate statistics. After the data appear on the screen, export into a separate spreadsheet.
The percentage of the number of times the three most common three pattern sequences were repeated were observed across three different inbred mouse strains. The C 58 females displayed a strong adherence to their preferred patterns in the round arena, but the not the rectangular arena. While male mice did not differ from each other in either arena.
Further more, male and female C 58 mice in the round arena showed a stronger preference for their most preferred object than the other two strains. While no differences in object preference were shown in the rectangular arena. Once mastered, this technique can be completed in under one hour, with 15 minutes needed for data acquisition and 20 minutes needed for behavioral scoring.
While attempting this procedure, it is important to remember that mice are very sensitive to changes in their surroundings. Please ensure that the testing environment remains consistent throughout the experiment. Following this procedure, other methods like the Barnes Maze or Team Maze can be used to further validate the presence of higher order repetitive behaviors.
After watching this video, you should have a good understanding of how to use novel object exploration to identify the presence of higher order repetitive behaviors in mice.
