Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes

Our research evaluates grooming behaviors in Drosophila melanogaster after RNAi-mediated Atg8a knockdown to model GABARAP deficits observed in Tourette syndrome patients. We aim to understand aAtg8a's role in behavioral development, providing insights into the regulatory functions of its human counterpart in neuro behavioral processes. Current experimental challenges with the manual annotation method include the complex analytical process, such as annotating and reviewing footage, and the subjectivity involved in manual annotation.

Automated methods also face challenges, including the requirement for substantial computer science expertise to implement and execute effectively. We have found that the gene Atg8a plays a role in the development of behavior in fruit flies beyond what has been previously established. More specifically, we found that it regulates both grooming and learning, suggesting that Atg8a orthologs may play similar roles in other organisms.

This technique allows for the direct quantification of grooming frequency and distribution in Drosophila. It is especially useful in labs unable to utilize automated methods and allows for more detailed analysis than indirect, dye-based methods. Overall, our research advances the use of Drosophila as a model organism for studying neurodevelopmental disorders that exhibit obsessive-compulsive behavior such as Tourette syndrome.

It also adds to the increasing evidence that autophagy-related proteins play a role in behavioral development. To begin, place four three-well dishes side by side on a white surface and cover each dish with a glass slide. Position a camera above the dishes.

Ensure there is no glare on the slides that could limit the visibility of flies in the dish and adjust the plate position or dim the lights as needed to eliminate glare. Now mark the position of the dishes and the camera on the bench where the recording is taking place. Place four-to nine-day-old flies on an ice block covered with parafilm and a paper towel to lightly anesthetize them.

Once anesthetized, move the flies to observation chambers as quickly as possible using a paint brush. Place one fly in each well of the three-well dish, ensuring minimal disturbance to avoid provoking stress-induced behavior changes. After acclimatizing the flies 30 minutes post anesthesia, record the flies using a camera, then open the terminal and type cd PiSpy.

Type python3 PiSpy. py, and enter the desired recording length and frame rate. Select the desired resolution and click on Quick Capture.

Now use the Preview Camera function in Pi Spy to ensure the flies are in focus and sufficiently visible. Record the flies for 10 minutes at a recommended frame rate of 24 frames per second and save the file. To analyze the video, import the file into the ELAN 6.8 software for detailed annotation and in-depth analysis of grooming behaviors.

Select Tier, Add New Tier, type location in the dish in the tier name, and add to assign each row to annotate the footage of individuals manually. To annotate behaviors, double click the tier to be annotated, right click, and drag the cursor along the selected tier for a period during which a single grooming bout is exhibited. Left click to select new annotation and double click the newly highlighted time period to type the abbreviation for the behavior observed at that time point.

Once the video is completely annotated, obtain a breakdown of each behavior by clicking on View, followed by Annotation Statistics. Identify anterior grooming as grooming performed at the anterior region of the fly body. Identify posterior grooming as grooming performed at the anterior-posterior end of the fly body.

Grooming bouts significantly increased in Atg8a RNAi mutants compared to control groups. Time spent grooming was also significantly elevated in Atg8a RNAi mutants compared to controls. No significant differences were observed in the distribution of anterior versus posterior grooming behaviors.