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Spontaneous Alternation in SimBA

The SimBA spontaneous alternation interface allows users to compute detailed alternations data from pose-estimation data and user-defined ROIs.

Before analyzing Spontaneous Alternation in SimBA

i) To analyze spontaneous alternation data in SimBA, we need to work with a project which contains a single animal. Also, the tracking data first has to be processed the up-to and including the Outlier correction step described in Part 2 - Step 4 - Correcting outliers. Thus, before proceeding to calculate ROI based measures, you should have one CSV file for each of the videos in your project located within the project_folder\csv\outlier_corrected_movement_location sub-directory of your SimBA project.

ii) Use the SimBA ROI tools to define four different ROIs in each video: One ROI which encompasses the center of the maze, and three or more arms that defines the arms of the maze. E.g., in this example image below, I have drawn 4 ROI rectangles that encompasses the three arms and the center of the maze:

Analyzing Spontaneous Alternation in SimBA

Onces the ROIs have been defined, click on the SPONTANEOUS ALTERNATION button in the ROI tab, and you should see the following pop-up:

  1. In the ARM DEFINITIONS frame, use the MAZE ARM 1-3: dropdown menus select the names of your ROIs that represents your three maze arms.

  2. In the ARM DEFINITIONS frame, use the MAZE CENTER dropdown menu to select the name of your ROI that represents the center of your maze.

  3. The ANIMAL SETTINGS frame contains settings that helps us control how an animal and entries/exits are defined:

  • POSE ESTIMATION THRESHOLD: Each body-part tracked through pose-estimation data has a detetion probability/confidence score associated with it in each frame. Use this dropdown menu to filter out body-parts detected below a certain confidence. Setting this value to 0 means that all body-parts in all frames will be used to detect the animal.

  • ANIMAL AREA (%): When detecting the location of the animal within the maze, SimBA will draw a "convex hull" or polygon around the animal that encapsulates all of the tracked body-parts, similar to how propriatory tools like AnyMaze compute spontaneous alternations.

    The ANIMAL AREA (%) represents the percent of this area that have to enter, or exit, the maze arms or the maze center for it to count as an entry or an exit. Smaller ANIMAL AREA (%) values are likely to results in more arm entries and exits being counted. The below image may not be the most suited as it doesnt show a 3-arm maze, but it can help to visualize how ANIMAL AREA (%) represents a threshold value:

  • ANIMAL BUFFER (MM): As mentioned above, SimBA will draw a "convex hull" or polygon around the animal that encapsulates all of the tracked body-parts. Sometimes, you may want to extend the size if this polygon, and give it a little wriggle room, especially if you don't pose-estimate the outer boundaries of your animals. For example, in this image below, the original detected animal polygon is shown at the top, and the buffered polygon with N % is shown at the bottom:

  • SAVE DETAILED DATA: Beyond the standard metrics (alternation rate, alternation count, same-arm return errors, alternate arm return errors), we may want data for specific arm combinations and exact frame count for when alternations or errors happened towards those arms and combinations. Setting this dropdown to TRUE will create one CSV file for each video inside the logs folder of your SimBA project within a subdirectory named something like detailed_spontaneous_alternation_data_20240327141628.

  • VERBOSE: This dropdown is helful for general troubleshooting. Setting thir dropdown to TRUE will print more information within each step of the processing pipeline.

  1. Once the above has been selected, click the RUN ANALYSIS button. You can follow the progress in the SimBA main window and the operating system terminal window. Every video represented in the project_folder/csv/outlier_corrected_movement_location will be analysed. Once complete, a CSV file will be stored in the logs folder of your SimBA project named something like spontaneous_alternation_20240327145612.csv. This file will have one row per video and columns representing alternation rate, alternation count, same-arm return error count, alternate arm return errors count. For an example file of expected output file with one analysed video, click HERE.

If you have selected SAVE DETAILED DATA, you will also get two further CSV files for each analysed video, documenting the frame counts when different errors and alternation sequences occured. For an example file of expected output of the detailed data, click HERE. You will also get another CSV file for each analyzed video, documenting the sequence of arm entries, together with the frame and time the animal entered the specific arm. For an example of the expected file, click HERE.

  1. We may want to visualize the spontanous alternation for some videos to make sure the alternation counts and errors as counted by SimBa matches up with manual scoring. To do this, go to the RUN VISUALIZATION sub-menu and select the video you want to visualize spontaneous alternation for. Next, click CREATE VIDEO. A video will be stored in the /project_folder/frames/output/spontanous_alternation directory of your SimBA project. Overlay on the video, is the convex hull polygon of the animal according to your settings, the ARM and CENTER ROIs, the current 3-arm sequence, and current alternation and error counts. Click play on the video below for an example.
F1.HAB.mp4