This method can help improve consistency in small animal Behavioral Testing by creating objective Motor Function Metrics. The advantage of this technique is that unlike conventional methods, the results are quantified by a computer system which cuts down on human error, effort, and time. Demonstrating this procedure will be Marina Yu and Sydney Lindner, Undergraduate Researchers from the Capadona Lab.
Begin filming and place the animal in the center of the grid, facing away from the researcher to begin the Grid Test. Allow the animal to run freely for three minutes while being video recorded from above. Stop filming when the three-minute time period has ended and return the animal to its home cage.
Clean the grid with a Chlorine Dioxide-based Sterilant, taking care to fully dry the surfaces before testing. Next, for the Ladder Test, place the animal in a clean temporary cage. Set the Ladder Apparatus up to span two cages, where the Start Cage is a clean cage and the End Cage is the animal's living cage, an incentive to complete the run.
Place the camera on a tripod, centered on the length of the ladder. Extend to the tripod so the lens of the camera is at the height of the ladder rung. Position the camera so that the rungs are exactly aligned with the lens, as this is important for the Slip Detection Algorithm in the BVAS Code.
Start the video recording and allow the animal to begin the run by holding their front paws over the first rung of the ladder. Allow the animal to move onto the ladder unassisted. Allow the animal to move from the start line to the finish line at their own pace while filming.
Finally, end the video recording and remove the animal from the ladder once the animal has completed the run. Begin by naming each video with the animal's designation for all Grid Testing videos. Take the videos now named after each animal and place the files in their own storage folder.
Then, create a new empty spreadsheet file to store the data for each study and testing mode. Next, install the latest BVAS version by double-clicking on the Installer icon. After installation is complete, double-click on the BVAS Program icon to bring up the BVAS Main Menu, which allows the following options:Grid Analysis, Ladder Analysis, Ladder Review, or Quit.
Within the BVAS Program, begin by selecting Grid Analysis from the top menu screen. Then, use the Ellipses button next to the upper input box to browse the file system to find the file of videos to analyze. Select the Ellipses button next to the lower input box to browse the file system to select the spreadsheet file in which to store the data.
Click go to start the Analysis Process. Examine the images of each video on the Reviewer screen. Select the Edit button if there are no green lines or the green line does not appear to be in the correct square around the testing area.
To edit the corners, select the Edit Corners button. Follow the instructions to the right of the representative image to select the Testing Area and hit Enter to complete the selection process. Lastly, select Done to save the selection and return to the Reviewer screen.
Then select the center Done button to start the Complete Analysis process, once the Grid Area for each video is selected correctly. Allow the program to run and upon completion, a Success prompt will appear. Next, select a Ladder Analysis from the top menu to open the Ladder Analysis Menu.
Select the upper input Ellipses button to select the folder with Ladder Videos named after each animal. Then, select the lower Ellipses button to select a Directory to save a folder with data from the Ladder Videos to review later. Select Go to begin the Analysis Process, and repeat the same steps for the Open Field Test Analysis.
Allow the program to run and upon competition, a Success prompt will appear. Within the BVAS Program, select a Ladder Image Review from the top menu to pull up the Ladder Review file select screen. Select the top Ellipses button to select the folder of data files, and select the bottom Ellipses button to select the spreadsheet file created for the study.
Then, press Go to start the Manual Review Process. Next, view the automatically opened Ladder Data Reviewer menu. If a Slip is detected, use the next frame and last frame buttons to check any consecutively recorded frames, if available.
Use the Slip Toggle to denote what foot of the rat has slipped. Leave the toggle on No Slip if the detection is a false positive. Click Save and Continue to move on to the next detection.
In the case of failure, use the toggle to denote whether the failure is due to Reversal or Stagnation. Finally, click Save and Continue to move on to the next detection. On the last detection, click Save and Continue to complete the analysis and save the data to the spreadsheet file chosen earlier.
Dismiss the Completion notice. In this study, the results for the Open Field Grid Test showed that Expert Reviewers were more consistent than Novice Reviewers were, but when using the BVAS, there was zero variance for both Expert and Novice Users. Similarly, the results for the Ladder Test showed that Expert Reviewers were more consistent than Novice Reviewers were, but when using the BVAS, there was, again, zero variance for both Expert and Novice Users.
For the Open Field Grid Test, trends were seen over the course of the study of increased Maximum Velocity achieved, increased Total Distance Traveled, and increased number of Gridlines crossed. The percentage of Right Turns largely hovered between 40 and 50%Lastly, for the Ladder Test, there was a decreasing trend in the time to cross the ladder, followed by an increase in time, starting around Week four. The software automatically detects and tabulates the number of Paw Slips during a Ladder Crossing.
And in Video Review Process, the user can sort the detected Slips into Left versus Right Paw Slips as shown in the bar graph. Once mastered, this technique can be completed in 30 to 40 minutes per animal per testing session. While attempting this procedure, it's important to film in a well-lit environment on the correct plane and with a high-contrast between the animal and the background.
Following this procedure, additional Behavior Tasks can be performed in order to answer specific questions in Disease or Injury Models. While we use this method to provide insight into Motor Deficit caused by Device Implantation in the brain, this analysis technique could be used in many other Disease or Injury Models. After watching this video, you should have a good understanding of how to film and analyze Behavioral videos using the Capadona Lab's BVAS Software, which provides a repeatable Behavior Quantification Technique.
