Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Autism spectrum disorder affects children, leading to impaired communication, social behavior, and enhanced repetitive behavior. Drosophila is an increasingly popular model for autism research to decipher underlying molecular pathways. We describe a set of standard techniques for behavioral characterization of fly autism models.

For decoding the molecular underpinnings of autism spectrum disorder in flies, its behavioral manifestations must be precisely established. Quantifying human behavioral traits of autism in Drosophila melanogaster is quite a challenging task. There is no single behavioral paradigm in flies that addresses all the behavioral anomalies of this disorder.

We have described a set of five well-established behavioral paradigms in Drosophila, namely aggression assay, social space assay, courtship, grooming, and habituation assay. All of these can be used in ASD fly models to characterize and quantify the level of impairments in communicative behavior, repetitive behavior, and social behavior. We describe these five techniques in their simplest form, so that every lab with minimal fly research facility can adopt them with ease and perform experiments on fly model of autism spectrum disorder and contribute to the advancement of our understanding of this disease.

To begin, take a standard 24-well plate and fill half of each well with regular fly food. Let the food dry overnight. Make a small 2.5 millimeter hole on a transparent plastic or acrylic sheet, and place it over a well plate.

Using a stereo microscope, separate newly enclosed flies of the desired genotype based on their sex. Insert half the male flies individually into single housing tubes. Keep the other half of the male flies in a group of 10 with the female flies in a regular group housing vial, creating a social condition.

Store all vials at 25 degrees Celsius for five days. Mouth aspirate two male flies from either the single or group housing chambers to the aggression arena plate through the hole in the lid. Move the hole away immediately to ensure that the flies cannot escape.

After one to two minutes of acclimatization, start a timer for 20 minutes. Make sure to video record the flies. Play the video on a computer, and count the total number of aggressive bouts for each fly.

The number of aggressive bouts in 20 minutes was significantly reduced in the case of mutant males than in the control males. To begin, separate the male flies from a mixed population of flies on ice and put them in a collection vial. To prepare the arena, place two triangular acrylic spacers flat on top of a rectangular glass pane, such that the right angles of the acrylic spacers are aligned with the corners of the glass pane.

Then place two rectangular acrylic spacers flat on the glass pane aligned with the bases of the two triangular spacers. Confirm that the four acrylic spacers surround a triangular arena on the rectangular glass pane that is not covered by spacers. Position a sticker of a ruler on one of the triangular spacers, ensuring that it is visible from the top.

Now, place a second rectangular glass pane on top of the acrylic spacers to align it with the glass pane at the bottom. Using four small binder clips, secure the panes and spacers. For the assay, remove the bottom right binder clip, and slightly shift the rectangular spacer outwards to create a gap.

To transfer flies from the collection vial into the social space arena, gently aspirate them through the created space. Immediately slide the rectangular spacer and secure the binder clip back in its position. Holding the chamber upright, gently pound it three times onto a soft pad to let all the flies settle at the base.

Clamp the chamber in the upright position and start the timer. Take a clear photo of the arena after 20 minutes. Analyze with ImageJ to list the inter-fly nearest neighbor distances.

The mutant flies show significantly higher nearest neighbor distances than the control counterparts, indicating a preference for greater distancing from other flies. To begin, assemble all four pieces of the courtship chamber together. Using the aspirator, gently transfer a five-day isolated test male fly from the single housing tube to the courtship chamber containing the single pre-mated female.

Rotate the lid quickly to close the chamber. Record the behavior of the flies for 15 minutes to calculate the courtship index and total number of copulation attempts. After setting up the assay arena, transfer a single housed male fly into the arena with an aspirator.

Immediately slide away the hole in the lid to prevent the fly from escaping. Place the grooming arena on a diffused LED panel, and let the fly acclimatize for one minute. Video record the fly for 10 minutes.

Analyze the videos and calculate the grooming index, grooming latency, grooming bout number, and mean grooming bout duration. Mutant flies showed a significantly lower courtship index as well as a reduced number of attempts to copulate. Latency to first grooming was significantly decreased in the mutant flies, whereas the mean grooming bout duration and grooming index were significantly increased.

To begin, obtain the flies prepared for habituation assay. Place one milliliter of the preferred odorant diluted with paraffin liquid light in a 1.5 milliliter microfuge tube. Vortex the tube for 10 minutes, and cover it with evenly perforated plastic wrap.

Using a wire, suspend the odorant tubes in separate media bottles containing flies. Cover the bottle well with cotton and wrap it with craft paper to prevent diffusion of the odorant vapor. Label the control and odor-containing bottles as naive and odor-exposed respectively.

Maintain these induction bottles for three days in an incubator. For the Y-maze assay, attach the Y-maze to the adapter, fix the bottom of the Y-maze to the entry vial, and then attach the climbing chambers to the two arms of the Y-maze. Using odor-free silicone tubes, connect the tapering end of each climbing chamber with the reagent bottle containing the odorant.

With a vacuum pump, drive the odorant from the gas bottles to the two arms of the Y and let it saturate for 15 minutes. Gently introduce the starved flies of each vial into the entry vial of the Y-maze setup. After brief acclimatization, connect the entry vial with the Y-maze adapter to begin the test.

Let the flies climb the Y-maze arms, and get trapped in the two collection chambers for over one minute. Upon completion, tap back the flies to the bottom of the entry vial and switch the position of the arms of the Y-maze to avoid side bias. Record the number of flies climbing each of the two arms of Y-maze within the time duration.

The row knockdown flies did not get habituated after a three-day odorant exposure as compared to the wild type flies.