The overall goal of this procedure is to utilize a modified juvenile play test in order to view inch worming behavior, a novel motor stereotyping. This is accomplished by first socially isolating each mouse 12 hours before the testing commences with independent food and water. The second step is to set up the video camera at a 45 degree angle to the testing enclosure.
Next, the isolated mice are placed in pairs within the enclosure where they're video recorded for an 11 minute duration in the dark. The final step is to analyze inch worming behavior from the video to measure duration, frequency, and latency to inch worm. Ultimately, this method allows for the analysis of motor neuropathies and can be used as an indicator for the testing of novel therapeutic interventions.
This method can help answer key questions in the autism field, such as the incidents and regulation of motor stereotypies. Visual demonstration of this procedure is critical to learning the task because the behavioral phenotype is difficult to identify and the inch worming behavior occurs in a rapid and repetitive fashion. Prepare the BTBR and black six mice for the procedure by isolating them in separate cages for 12 hours overnight.
Prior to testing the BTBR mice used in this study display all the main aspects of autism spectrum disorders found in mice black six mice are used as controls the day of the experiment. Place the isolated mice in the behavioral analysis room for 30 minutes prior to testing so that the animals can habituate to the novel environment. Then place a 30 by 30 centimeter plexiglass box with a removable lid diagonally onto the table.
In the testing room, backlight the box using an infrared light to minimize glare. Next position, a video camera that possesses a high zoom function with infrared capabilities at a 45 degree angle so that it can record the entire floor of the enclosure and the mouse. Focus the camera on the enclosure so that the mice are able to be visualized throughout the entire box.
Once the box and camera are set up, cover the entire floor of the box with sawdust bedding so that it is approximately one inch deep. Place two BTBR or black six mice within the testing enclosure at the same time, and turn the lights off in the testing room. Then start the video camera to record and exit the room to prevent, experiment or influence on the behavior of the mice.
Record the activity for 11 minutes. Once the recording is complete, reenter the testing room and return the mice to their original home cages. Dispose of the sawdust bedding.
Wash the entire testing box with 70%ethanol, and then place new sawdust bedding in the bottom of the testing Enclosure approximately one inch deep. Repeat the entire procedure for the rest of the interest strain pairs and return mice to their original home cages. When finished, to verify the reliability of the inch worming data, repeat the procedure on subsequent days with different BTBR and black six interest strain pair combinations.
This will ensure that the observed behavior is not influenced by a specific partner in the enclosure. Manually watch and score each video twice, once for each mouse to ascertain the total duration frequency and latency to inch worm. In the videos.
Analyze each video for 10 minutes, starting at the three second time point until 10 minutes. Three seconds. Define inch worming as the synchronous, inward and outward movement of the animals four and hind feet with a minimum of one co-occurring inward and then subsequent outward movement that effectively displaces the bedding Score the inch warming duration as the total time each mouse spends engaged in the inch warming behavior.
To score this aspect of inch worming, use a timer that can be started and stop. Start timing when the mouse begins to inchworm and stop the timer when the mouse stops. If the mouse begins to inch worm again, restart the timer and run it until the mouse stops again.
This will generate an inch warming duration score that is between zero and 10 minutes. Define inch warming frequency as the number of times the mouse engages in the inch warming behavior. In the 10 minute observation period, a single count is awarded each time the mouse starts and stops.
Inch worming. Define latency to inch worm as the time that passes prior to the first onset of inch worming. Start the timer when the video starts and stop the timer when the mouse inchworms for the first time.
If the mouse does not display the inch worming behavior, the latency to inchworm will be 10 minutes. Next, calculate the total frequency and duration of inch worming for each mouse in the video. The average of the two mice is used as the total frequency and duration for that video.
Then determine the time required for the first mouse in the pair two inch worm for the latency two inch worm For that specific pair of mice, remove mice that fail to inchworm for the duration of the testing period from the sample population to ensure that the results are pertinent to a population of mice that display the inch worming behavior. Calculate the average duration, frequency and latency to dig for each experimental group in the analysis and then use Innova or T tests to compare data between strains in the experimental groups. Following the second day of testing, use a repeated measures Innova to ensure that there were no subsequent trial effects or pairing effects in the data shown.
Here are the average frequency, duration, and latency to inchworm displayed by pairs of BTBR and black six mice. The BTBR strain displayed a significantly higher frequency and duration of inch worming as well as a significantly shorter latency than the black six control strain. When these tests were repeated with alternate pairings, no significant differences were found, ensuring that the effects were not dependent on either specific mouse pairings or the repetition of the tests While attempting this procedure, it is important to remember to only score in swarming as the synchronous inward and outward movement of the animal's for and hind feet Following mastery of this procedure.
Other methods like drug administration can be added to the paradigm to look at additional questions such as therapeutic targets for stereotypic behaviors.
