Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer independent manner. Our system allows a detailed investigation of social behaviors and their their dynamics in small rodent models and supports accurate comparisons between strains, conditions and treatments. This system may be used for any type of social discrimination tests, such as discriminating between males and females.
Moreover, there are no limits to the sizes of the subjects. The analysis requires some experience for selecting the optimal parameters for tracking. For new users I recommend monitoring the tracking on the computer screen for the initial evaluation.
At least one hour before the analysis place the home cages of all of the mice in the experimental room for acclimation. At the end of the acclimation period insert two empty chambers into the arena at two opposite corners of the arena. Then, locate the arena within the acoustic chamber and place the subject mice in the middle of the arena for 15 minutes of habituation.
During this period, place the two social stimuli mice in different chambers located outside of the arena for habituation. And a 5x5x5 centimeter plastic toy with a distinct shape and color in another chamber. To perform the SP test, start recording with a video camera.
Immediately remove the two empty chambers and place the social and object stimuli chambers in the corners of the arena that had been empty during the habituation. Allow the subjects to interact with the stimuli for five minutes before stopping the recording, and removing the stimuli containing chambers from the arena. Then, leave the subject in the empty arena for 15 minutes.
To perform a social novelty preference test start the video recording, and insert the chamber containing the same social stimulus animals as used for the SP test and the chamber containing the novel social stimulus mouse into the two corners of the arena that were not used for the SP test. Allow the subjects to interact with the stimuli for five minutes before stopping the recording and removing the subjects and chambers from the arena. Then, clean the arena and chambers with running water and 10%ethanol.
To use the track rodent graphic user interface to analyze the mouse behavior, open MATLAB and choose the Track Rodent Directory, right click to select Add to Path, Selected Folders and Subfolders to add all of the subfolders to the working path. Enter TrackRodent into the command window and press Enter. Select Load Session File to upload single or multiple video files.
A movie inspector that allows a frame by frame inspection of the video clip will immediately be opened for the first file in the list. To inspect additional video files click Inspect Movie to open the video inspector and select a specific video file of interest at any time. Select the species tested if necessary and click Exclude Area to exclude all of the areas that my interrupt the tracking in accordance with the colors of the subject and arena.
To define each chamber as a stimulus area for automatic detection of it's investigation by the subject, check the polygon or elliptical box and click Stimulus X to allow the stimulus areas to be marked. Then, select the desired algorithm from the list and enter the starting and ending frame numbers for the analysis in the corresponding edit boxes. To evaluate the automatic detection of the subject borders for a given threshold enter a value into the relevant threshold field and press Enter.
For starting a behavioral analysis of all of the selected files, move to the first file and click Start. Finding the right threshold is critical and requires experience. It is recommended that new users use the non-fast algorithms and monitor the analysis online to ensure that they work properly.
At the end of the analysis a results file will be saved for each movie in the same directory of the movie files. To inspect the results of each movie file click Load Results File and select the MAT files generated by the behavioral analysis. Move between the toggle buttons to examine the mouse location trace, stimuli exploration along session and total stimuli exploration time.
For population analysis in the TrackRodent folder in MATLAB enter TrackRodentPopulationSummary as one word in the command window. Click Enter and choose Results Files to upload multiple TrackRodent results files. Fill in the numbers of the last frame for analysis, test name, stimulus one name, and stimulus two name and select all of the appropriate boxes for the desired analyses from the optional list.
Check the Export box to extract the results to a spreadsheet in a single file. Then, click Start and wait until the program reports in the status line the analysis parameters and the data have been saved. In this experiment, the mean total investigation time of the population of subjects toward each of the two stimuli depicts a clear preference of the subjects toward the social stimulus.
The social preference was maintained by the subject throughout the session although the preference seemed slightly stronger early in the session. The investigation time for each stimulus can be categorized according to the bout duration into short, medium and long bouts in addition to the total investigation time. As observed, there was no difference between the stimuli in the short bouts while the medium and long bouts showed clear social preferences, suggesting that in C57 Black 6J mice short bouts reflect curiosity, while long bouts reflect an interaction between the subject and the stimuli.
In addition, heat maps of the investigation bouts toward the social stimulus and object during the course of the test can be generated for the whole population of subjects with the colors coding the bout duration. Importantly, the rate of transitions made by subjects from one stimulus to another was highest during the first two minutes of the test before gradually declining. The same tendency was apparent in the distribution of mean transition rate over time.
This method can be used with any mouse connected to electrophysiological recording or optogenetic stimulating systems by a wire. For this, we use a special algorithm. Our system is currently in use by several laboratories worldwide for various paradigms requiring small rodent tracking.
It may be used for assessing behavioral dynamics in any discrimination test.
