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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Autism Spectrum Disorder (ASD) is associated with impaired social and communicative behavior and the emergence of repetitive behavior. For studying the interrelation between ASD genes and behavioral deficits in the Drosophila model, five behavioral paradigms are described in this paper for assaying social spacing, aggression, courtship, grooming, and habituation behavior.

Abstract

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in social interaction and ability for communication, enhanced restricted or repetitive behaviors, and also, in some cases, learning disability and motor deficit. Drosophila has served as an unparalleled model organism for modeling a great number of human diseases. As many genes have been implicated in ASD, fruit flies have emerged as a powerful and efficient way to test the genes putatively involved with the disorder. As hundreds of genes, with varied functional roles, are implicated in ASD, a single genetic fly model of ASD is unfeasible; instead, individual genetic mutants, gene knockdowns, or overexpression-based studies of the fly homologs of ASD-associated genes are the common means for gaining insight regarding molecular pathways underlying these gene products. A host of behavioral techniques are available in Drosophila which provide easy readout of deficits in specific behavioral components. Social space assay and aggression and courtship assays in flies have been shown to be useful in assessing defects in social interaction or communication. Grooming behavior in flies is an excellent readout of repetitive behavior. Habituation assay is used in flies to estimate the ability for habituation learning, which is found to be affected in some ASD patients. A combination of these behavioral paradigms can be utilized to make a thorough assessment of the human ASD-like disease state in flies. Using Fmr1 mutant flies, recapitulating Fragile-X syndrome in humans, and POGZ-homolog row knockdown in fly neurons, we have shown quantifiable deficits in social spacing, aggression, courtship behavior, grooming behavior, and habituation. These behavioral paradigms are demonstrated here in their simplest and straightforward forms with an assumption that it would facilitate their widespread use for research on ASD and other neurodevelopmental disorders in fly models.

Introduction

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurological disorders. It includes a range of complex neuro-developmental disorders characterized by multi-contextual and persistent deficits in social communication and social interaction and the presence of restricted, repetitive behavioral and activity patterns and interests1. According to World Health Organization (WHO), 1 in 100 children is diagnosed with ASD worldwide with a male-to-female ratio of 4.22. The disease becomes evident in the second or third year of life. ASD children show a lack of interest in social-emotional reciprocity, non-verbal communication, and relationship skills. They exhibit repetitive behaviors like stereotyped motor movement, inflexible and ritualized routine following, and intense focus on restricted interests. ASD children show a high degree of response towards touch, smell, sound, and taste whereas pain and temperature response is comparatively low1. The penetrance of this disorder is also different among different patients suffering from ASD and hence, the variability increases.

Current clinical diagnosis of ASD is based on behavioral assessment of the individuals as there is no confirmatory biomarker-based or common genetic test that covers all forms of ASD3. Deciphering the genetic and neurophysiological bases would be helpful in targeting treatment strategies. In the last decade, a large body of research has resulted in the identification of hundreds of genes that are either deleted or mutated or whose expression levels are altered in ASD patients. Ongoing research emphasizes the validation of the contribution of these candidate genes using model organisms like the mouse or fruit-fly, in which, these genes are knocked out or knocked down followed by tests for ASD-like behavioral deficits and elucidation of underlying genetic and molecular pathways causing the anomalies. A mouse model recapitulating Copy Number Variations (CNVs) in the human chromosomal loci 16p11.2 shows some of the ASD behavioral defects4,5,6. Prenatal exposure to a teratogenic drug valproic acid (VPA) is another mouse model depicting traits resembling human ASD7,8. In addition, there exists a range of mouse models that exhibit genetic syndrome-associated autism, for example, single-gene syndromic models caused by mutations in Fmr1, Pten, Mecp2, Cacna1c, and single-gene non-syndromic models caused by mutations in genes like Cntnap2, Shank, Neurexin, or Neuroligin genes5.

Fruit-fly (Drosophila melanogaster) is another prominent model organism for studying the cellular, molecular, and genetic bases of a plethora of human disorders9, including ASD. Drosophila and humans share highly conserved biological processes at the molecular, cellular, and synaptic levels. Fruit-flies have been used successfully in ASD studies10,11,12 to characterize genes linked to ASDs and decipher their exact role in synaptogenesis, synaptic function and plasticity, neural circuit assembly, and maturation; fly homologs of ASD-associated genes were found to have roles in the regulation of social and/or repetitive behavior11,13,14,15,16,17,18,19,20,21. The fruit-fly has also worked as a model for the screening of ASD genes and their variants15,22,23. The biggest challenge in ASD research in flies is that, unlike other disease models, there is no single ASD fly model. To understand the impact of mutations or knocking down of a specific ASD gene, a researcher needs to validate whether the behavioral phenotypes sufficiently mimic the symptoms of ASD patients and then, proceed towards understanding the molecular or physiological underpinnings of the phenotypes.

Hence, the detection of ASD-like phenotypes is vital to ASD research in the fly model. A handful of behavioral techniques have emerged over the years that enable us to detect abnormalities like deficits in social behavior/interaction, communication, repetitive behaviors, and responsiveness to stimuli. In addition, several modifications and upgrades of these behavioral techniques have been made in different labs to suit specific requirements such as upscaling, automation of assays, readouts, quantification, and comparison methods. In this video article, the most basic versions of five behavioral paradigms are demonstrated, which, in combination, can be used to detect ASD-like behavioral outcomes in the easiest way.

Aggression is an evolutionarily conserved innate behavior affecting survival and reproduction24. Aggressive behavior towards conspecifics is influenced by 'motivation for socialization'25,26 as well as 'communication'27, both being compromised in ASD-affected individuals. Aggressive behavior is well described in Drosophila and its quantifiability through the robust aggression assay28,29,30 and a well-understood genetic and neurobiological basis31 makes it a suitable behavioral paradigm32 for assessing the ASD phenotype in a fly model. Aggression is affected by social isolation away from a social environment, which leads to enhanced aggression; the same has been observed when male flies are housed in isolation for a few days33,34. Another behavioral assay that quantifies sociability in flies is the Social Space Assay35, which measures distances between nearest neighbors and interfly distances in a small group of flies, making it perfectly suited for testing the roles of ASD gene orthologs in fly12,21,36,37 as well as environmentally induced ASD fly models38,39.

The Drosophila courtship assay is another behavioral paradigm frequently used to assay for alteration in social and communication skills upon circuit or genetic manipulation, including Autism related genes18,19,21,40. Repetitive patterns of behavior are prevalent in ASD patients, which is recapitulated in flies by grooming behavior-a series of distinct, stereotyped actions performed for cleaning and other purpose. It has been successfully used to assay for the impact of ASD gene mutations in flies21,41 as well as exposure to chemicals38,39. Multiple advancements and automation in the assay have been described before16,41,42,43; here, we are demonstrating the most basic assay pattern, which is easy to adopt and quantify.

ASD is known to impact the ability for habituation, learning, and memory in some patients44,45,46,47,48,49,50, ASD model organisms51,52 and also causes deficits in different olfactory behaviors50. Drosophila light-off jump habituation has been used previously to screen for ASD genes23. Habituation can be assayed by a simple method of olfactory habituation assay53,54,55. We describe the method to induce olfactory habituation and assay the outcome using a classic Y-maze-based binary odor-choice assay56 that can be used to detect defects in habituation in ASD gene mutant or gene knockdown condition. To assess whether the impact of a mutation (or gene knock-down) or a pharmacological treatment on the behavior of a fly amounts to an ASD-like phenotype, one can use a combination of these 5 assays described here.

Protocol

See the Table of Materials for details related to all materials and reagents used in this protocol.

1. Aggression assay

  1. Preparing the aggression assay arena
    1. Take a standard 24-well plate (Figure 1A) and use each well of the plate as a single 'arena' (Figure 1B) for fly aggression. Fill half of each well with regular fly food and allow it to dry overnight.
      NOTE: Optionally, a focal point for aggression may be included in the arena, such as a dot of yeast paste or a decapitated female to ensure male aggression.
    2. Take any transparent plastic/acrylic sheet, enough to cover the surface of about three to four wells. Perforate the sheet to make a small aperture of ~2.5 mm diameter for inserting individual flies into the wells through it.
  2. Preparing the fly aspirator
    1. Take a 30-50 cm long rubber/silicone tube (Figure 1C1).
    2. Take a 200 µL (or 1,000 µL) pipette tip and chop off ~1 cm from the narrow tip. Insert the cut end at one end of the rubber tube; this tip will be the 'mouth end', used for mouth-based aspiration.
    3. Take another pipette tip and cut the narrow end of this tip to make an opening sufficient for a fly to enter through it. Insert the base of this pipette tip into the tail end of the tube; this will be the 'fly end' of the aspirator, from where the fly will get aspirated (Figure 1C2).
    4. Insert a piece of mesh cloth or a thin layer of cotton on the 'fly end' of the tube-at the junction between the tube and tip. Ensure that a fly aspirated into the pipette tip remains trapped in the tip, in front of the mesh.
    5. Seal the junctions between the pipe and the pipette tips using parafilm.
  3. Preparing single-housing tubes and group-housing vials
    1. Preparation of single-housing tubes
      1. Add nearly 500 µL of freshly prepared fly food to a 2 mL microfuge tube (Figure 1D). Use a mini centrifuge to pull down the food to the bottom part of the tube.
      2. Set the food to solidify at room temperature by keeping the lid open overnight. Cover the tubes with a fine piece of cloth to prevent stray flies from entering the tubes.
      3. Perforate the lids of the microfuge tubes with needles for air circulation, and close the lids after the food is solidified.
    2. Preparation of group-housing vials
      1. Take regular fly vials and fill a bare minimum of the vials with freshly prepared food (Figure 1D).
  4. Preparing the flies before the experiment
    1. Maintain fly lines of the desired genotypes in regular glass/plastic bottles containing standard fly food in an incubator at 25 °C on a 12 h light/dark cycle.
    2. Collect newly eclosed (0 to 24 h) flies of the desired genotype and sex-separate them under carbon dioxide anesthesia using a stereomicroscope.
    3. 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 regular 'group-housing vials' creating a social condition. Store all tubes and vials at 25 °C for 5 days.
      NOTE: Maintain a temperature of 24-25 °C and ~50% humidity in the behavior room. The fly strains used were: w1118 and FMR trans-heterozygote mutant flies (Fmr1Δ50M/Fmr1Δ113M)57. Fmr1 flies, which were kept in isolation for 5 days, show significantly decreased aggression bouts towards another male (Figure 1E) .
  5. Performing the aggression behavior experiment
    1. Perform the aggression experiment during the ZT0-ZT3 time window as the flies show peak activity during this time of the day.
      NOTE: ZT=Zeitgeber time in a standard 12 h light/dark cycle; ZT0= lights on, that is, the start of light phase, ZT3= 3 h after the start of the light phase, and so on. ZT12= lights off.
    2. Transfer two male flies from either the single- or group-housing chambers by the mouth-aspiration method to the aggression arena through the hole in the lid; move the hole away immediately to ensure that the flies cannot escape.
    3. Allow the flies to acclimate within the arena for 1-2 min. Start a timer for 20 min. Video record the flies for the entire 20 min by placing a camera or a mobile phone exactly vertically above the arena using a stand. Ensure that the types of aggressive bouts are visible in the video.
      NOTE: Ensure clear illumination of the arena and avoid any glare/reflection from the lid directly falling onto the lens of the camera.
    4. Repeat the experiment for 15-20 pairs of flies for each genotype and every type of housing condition.
      ​NOTE: Unconscious bias can be negated by double blinding of the control and test flies as well as video files belonging to multiple genotypes.
  6. Analyzing the aggression assay data
    1. Transfer the video files to a computer with a sufficiently large screen so that the aggressive bouts are visible.
    2. Play the video and count the total number of aggressive bouts in a time span of 20 min58,59.
    3. Compile and organize data from each genotype and each housing pattern in a spreadsheet and perform data analysis using any statistical software. Perform a two-tailed t-test and plot the data as box and whiskers.

2. Social space assay

NOTE: The assay protocol, arena, and analysis described here have been described previously60,61.

  1. Preparing the social space assay (SSA) arena
    1. Place both the triangular acrylic spacers (height = 8.9 cm, base = 6.7 cm, and thickness = 0.3 cm) flat on top of a rectangular glass pane (13.5 cm x 10.4 cm, thickness 0.3 cm) such that the right angles of the acrylic spacers are aligned with the corners of the glass pane (Figure 2A).
    2. Place two rectangular acrylic spacers (6.7 cm x 1.5 cm, thickness = 0.3 cm) flat on the glass pane, aligned with the bases of the two triangular spacers. After this arrangement, confirm that the four acrylic spacers surround a triangular arena (~2.16 sq. cm61) on the rectangular glass pane that is not covered by spacers (Figure 2A,B). Place a sticker of a ruler (Figure 2C) on one of the triangular spacers and ensure that it is visible from the top.
    3. Now place a second rectangular glass pane (13.5 cm x 10.4 cm, thickness 0.3 cm) on top of the acrylic spacers such that it aligns with the glass pane at the bottom; acrylic spacers would end up sandwiched between the glass space, leaving a triangular space in the middle between two glass panes. Use four small binder clips to hold the panes and spacers.
  2. Preparation of the flies for SSA
    1. Collect newly eclosed (0 to 24 h) flies of the desired genotype and sex-separate them under cold anesthesia using a stereomicroscope.
      NOTE: Chill the cold anesthesia apparatus (a Petri dish may be used) in a -20 °C incubator. Place the flies into empty vials, submerge these vials into ice (in an ice bucket) till the flies become immobile, place them on the cold Petri dish, and start sorting.
    2. Store the male and female flies separately for 24 h in food vials in a 25 ˚C incubator with a 12 h light/dark cycle.
      NOTE: For the experiments demonstrated here, the fly strains used were w1118 and FMR trans-heterozygote mutant flies (Fmr1Δ50M/Fmr1Δ113M).
  3. Performing the SSA experiment
    1. Maintain the same experimental conditions as the aggression assay (temperature: 24-25˚C, humidity ~ 50%) and perform the experiment during the ZT0-ZT3 time window.
    2. Remove the bottom right binder clip and slightly shift that rectangular spacer outwards so that a gap (~0.5 cm) is created between the two rectangular spacers.
    3. Transfer flies (collected on the previous day) from the food vial into an empty vial. Transfer them into the social space arena (the triangular area) by gentle aspiration through the space created between the rectangular spacers. Immediately close the space by sliding the rectangular spacer and binder clip back in their positions and ensure that no space is left for the flies to escape.
    4. By holding the chamber in the upright position, gently pound the chamber 3x onto a soft pad to ensure that all the flies are at the base of the chamber at the onset of the experiment.
    5. Clamp the SSA chamber in the upright position and start the timer. Take a clear photo of the arena after 20 min when the flies settle at their positions in the arena and show minimum movement. Avoid glare and irregular illumination.
    6. Repeat the experiment 3x for the same population of flies by pounding the flies and repeating the steps from steps 2.3.5 to 2.3.7 (internal replicates). Repeat 3x with a different population of flies of the same genotype/condition (independent repeats).
      ​NOTE: Freeze the assay chamber to discard the flies from the chamber. Wipe the glass and plastic surfaces with ethanol to remove all odors after one round of experiment is done with one group of flies.
  4. Analyzing the social space assay data
    1. Analyze the images in ImageJ62 software and list the inter-fly nearest-neighbor distances as previously described61.
    2. Perform statistical analysis and plot graphs using statistical software.
      NOTE: The fly strains used for the SSA assay described here were w1118 and Fmr1 trans-heterozygote mutant flies (Fmr1Δ50M/Fmr1Δ113M)57. Fmr1 flies show significantly increased distance from the nearest neighbors (Figure 2D).

3. Courtship assay

  1. The courtship chamber
    1. Assemble all four pieces of the courtship chamber (Figure 3A) together in the order shown in Figure 3B. Each perforation of the central disk, sandwiched between the lid and the base pieces, will work as the 'courtship chamber' (Figure 3C).
  2. Obtaining premated females
    1. Start and maintain multiple culture bottles of CS (Canton S, wild type) flies with ~60-100 male and female flies in each food bottle.
    2. When adults emerge, discard all adult flies and transfer the flies emerging from these bottles every 2-3 h to new food vials supplemented with a tiny amount of yeast paste.
      ​NOTE: Avoid overcrowding of the vials. Be careful not to transfer old flies, larvae, or pupae to the mating vial.
    3. Incubate these "mating vials" for 4 days to ensure that all females have mated.
  3. Preparation of single-housing tubes for males
    1. Add nearly 500 µL of fly food to each 2 mL microfuge tube. Use a minicentrifuge machine to pull down the food to the bottom part of the tube.
    2. Allow overnight solidification of the food, cut the lid of the microfuge tube, cover it with parafilm, and perforate the parafilm with a needle for air circulation.
  4. Collection and isolation of virgin males
    1. Set up crosses with 10-15 males and 20-30 virgin females of the desired genotypes in separate food vials.
    2. Collect virgin males of the desired genotypes from the progeny and keep them individually in 'single-housing tubes' using the aspirator. Keep collecting newly eclosed males every 5-6 h (up to 40-50 males for each genotype) and isolate them in individual single-housing tubes.
    3. Re-seal the lid of the tube with the parafilm.
  5. Performing the courtship assay experiment
    1. After 5 days of isolation of the test males, perform the courtship assay during the ZT0-ZT3 time window.
    2. Set up the video recording devices well in advance focusing on the assay workspace.
    3. With the help of an aspirator, collect one premated female (6-10 days old) from the mating vials and insert into a courtship chamber.
    4. Using the aspirator, gently transfer a male (control/test) fly from the single-housing tube to the courtship chamber containing the single premated female through the transfer hole. Rotate the lid quickly to close the chamber.
    5. Video record the behavior of the flies for 15 min.
  6. Video data analysis and statistics
    1. Transfer the video files to a computer; note the duration of time spent by the male in courtship during 15 min by manually going through the video.
    2. Calculate the courtship index (CI) for each male fly, which is the fraction (or percentage) of time spent by the male in courting the female during 15 min.
    3. Count the total number of copulation attempts in 15 min.
    4. Note the duration of time lag shown by the male before its first attempt to court as the courtship latency (CL).
      NOTE: It is recommended to analyze 40-60 males per condition/genotype to achieve statistical power and determine the consistency of the CI results.

4. Grooming behavior assay

  1. Grooming behavior assay arena
    1. Use a small circular chamber with a volume of ~0.4 cm3 as an arena for recording the grooming behavior.
      NOTE: The same courtship arena described in section 3 may be used for grooming behavior assay.
    2. As grooming behavior involves recording of movement of finer organs like legs, use high-resolution or high-contrast video recording. To follow this protocol, use a diffused glass-covered LED panel as a uniformly illuminated surface of dimensions 20 cm x 20 cm. Place the courtship chamber on top of the panel to ensure light passes from the bottom through the chamber.
  2. Preparing the flies before the experiment
    1. Maintain experimental genotype flies in food bottles at 25 °C with a 12 h light/dark cycle.
    2. Collect newly eclosed (0 to 24 h) flies of the desired genotype and sex-separate them under cold anesthesia using a stereomicroscope as described in the social space assay.
    3. Isolate the male flies in single-housing tubes (as used in the aggression assay) for 24 h.
      NOTE: For the experiment demonstrated here, the fly strains used were: W1118 and FMR trans-heterozygote mutant flies (Fmr1Δ50M/Fmr1Δ113M).
  3. Performing the grooming behavior assay
    1. Maintain a temperature of 24-25 °C and humidity ~ 50%; perform the experiment during the ZT0-ZT3 time-window16.
    2. Transfer a single-housed male fly from a single-housing tube into the grooming arena by using an aspirator. Immediately slide away the hole in the lid to ensure that the flies cannot escape.
    3. Place the grooming arena on a diffused LED panel and allow the fly to acclimate within the arena for 1 min. Video record the fly for 10 min by placing the camera exactly vertically above the arena mounted on a stand. Ensure that the types of grooming bouts are visible and quantifiable in the video.
      NOTE: The grooming behavior includes rubbing of the head, eyes, antennae, proboscis thorax, abdomen, genitalia, and wings with legs (first pair: T1, second pair: T2, or third pair of legs: T3). Grooming behavioral parameters that have been taken into account in this study are as described in Andrew et al. 41 and demonstrated in Figure 4.
    4. Repeat the experiment for each genotype.
    5. Analyzing the grooming behavioral data
      1. Analyze the videos and calculatethe following four parameters: grooming Index (percentage of time spent in grooming); grooming latency (time until first grooming bout); grooming-bout number; and mean grooming-bout duration (total bout duration/bout number).
      2. Mark a single grooming bout as finished when a fly either stops showing any of the parameters and remains motionless for 2 s or stops the bout and walks at least 4 steps.

5. Assay for olfactory habituation

NOTE: As shown in Figure 5, the final assembly needs to be done on the day of Y-maze assay54,56.

  1. Preparing flies for habituation
    1. Raise flies of the desired genotypes for all the experiments in an incubator with an ambient temperature of 25 oC and 70% humidity under standard 12 h light: 12h h dark cycle (LD).
    2. Collect 0-12 h old, newly eclosed flies and transfer ~30-40 flies in a fresh medium bottle with a tightened cotton stopper.
    3. Assign codes to each bottle to keep the experimenter blind about the genotypes under experimentation.
  2. Induction of olfactory habituation
    1. Place 1 mL of the preferred odorant diluted with paraffin liquid (light) in a 1.5 mL microfuge tube. For the control, just use 1 mL of paraffin liquid light in a 1.5 mL microfuge tube. Vortex the contents in the tube for 10 min to ensure uniform mixing and then cover it with evenly perforated plastic wrap.
      NOTE: In the video, 20% ethyl butyrate will be used.
    2. Use wire to suspend the tubes containing the diluted odorant or only the diluent liquid in separate media bottles containing flies.
    3. Cover the bottles well with cotton and then wrap with kraft paper to prevent diffusion of the odorant vapor. Label the control and odor-containing bottles as naïve and odor-exposed (habituated), respectively. Maintain these induction bottles for 3 days in an incubator with the above-mentioned conditions.
  3. Preparation of flies and the Y-maze apparatus
    1. Transfer the flies from the induction bottles to vials containing only water-soaked filter paper.
    2. Starve them for approximately 16-18 h at room temperature prior to the experiment to increase motivation.
    3. Ensure that the components of the Y-maze apparatus are clean and odor-free; assemble the four parts in a vertical fashion. Attach the climbing chambers to the Y-maze, which is then connected to the top of the adaptor. Firmly attach the bottom of the Y-maze to the entry vial that serves as the central stem at the bottom as shown in Figure 5.
    4. Connect the tapering end of each climbing chamber with the reagent bottles containing odorant (10-3 dilution with distilled water) using odor-free silicone tubes.
    5. Pump the odorant at an equal flow rate (~120 mL/min) from the gas bottles to the two arms of the Y with the help of a vacuum pump and allow it to saturate for 15 min for consistency.
  4. Performing the classical Y-maze assay
    1. Gently introduce the starved flies of each vial into the entry vial of the Y-maze setup.
    2. Allow them to acclimate for a brief period; connect the entry vial with the Y-maze adapter to begin the test; and let the flies climb the Y-maze arms and get trapped in the two collection chambers. Set the time duration of each test to 1 min.
    3. 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. Take four readings for the same set of flies.
    4. Record the number of flies climbing each of the two arms of Y-maze within the time duration.
    5. Repeat the assay for at least 8 batches to obtain a representative dataset.
  5. Analysis and interpretation of collected data
    1. Quantify the results by calculating the Performance Index (PI), which can be represented as the difference between the number of flies choosing the air arm (A) and the number of flies choosing the odor arm (O) as a fraction of the total number of participant flies.
    2. Use statistical tests (unpaired Student's t-test) to check if the PI of naïve versus odor-exposed flies are significant.

Results

Aggression assay
As a fly ASD model, Fmr1 mutant flies have been used63,64. w1118 males were used as control and Fmr1 trans-heterozygote Fmr1Δ113M/Fmr1Δ50M57 male flies as experimental flies; adult males were housed in isolation tubes for 5 days. Homotypic males (same genotype, same housing conditions) were introduced in the aggression arena and their behavior ...

Discussion

Drosophila is used as a fine model organism for research in human neurological disorders due to a high degree of conservation of gene sequences between fly and human disease genes9. Numerous robust behavioral paradigms make it an attractive model for studying phenotypes manifested in mutants recapitulating human diseases. As hundreds of genes are implicated in autism spectrum disorder (ASD), no common ASD model exists in any model organism. Hence, for each mutant, researchers must first e...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

We are immensely thankful to Mani Ramaswami (NCBS, Bangalore) and Baskar Bakthavachalu (IIT Mandi) for the habituation and odor choice assay setup, Pavan Agrawal (MAHE) for his valuable suggestions on the aggression assay, Amitava Majumdar (NCCS, Pune) for sharing his courtship assay chamber prototype and Fmr1 mutant fly lines, and Gaurav Das (NCCS, Pune) for sharing the MB247-GAL4 line. We thank Bloomington Drosophila Stock Center (BDSC, Indiana, USA), National Institute of Genetics (NIG, Kyoto, Japan), Banaras Hindu University (BHU, Varanasi, India), and National Center for Biological Science (NCBS, Bangalore, India) for Drosophila lines. Work in the laboratory was supported by grants from SERB-DST (ECR/2017/002963) to AD, DBT Ramalingaswami fellowship awarded to AD (BT/RLF/Re-entry/11/2016), and institutional support from IIT Kharagpur, India. SD and SM receive Ph.D. fellowships from CSIR-Senior Research Fellowship; PM receives a Ph.D. fellowship from MHRD, India.

Materials

NameCompanyCatalog NumberComments
Aggression arena:
Standard 24-well plate made of transparent polystyrene12 cm x 8 cm x 2 cm. Diameter of a single well= 18 cm. Sigma-aldrich #Z707791; depth = 1 cm
Transparent plastic/acrylic sheetAlternative: a perforated lid of a cell culture plate
Social Space Assay:
Binder clips19 mm
Glass sheets and acrylic sheets of customized sizesThickness = 5 mm
Courtship assay:
Nut and bolt with threading
Perspex sheets of customized shapesi) Lid: A custom-made round transparent Perspex disk (2-3 mm thickness, 70 mm diameter) with one loading hole at the peripheral region and another screw hole at the center (diameter ~ 3 mm for each); ii) A second transparent thicker Perspex disk (3-4 mm thickness, 70 mm diameter), with 6-8 perforations of diameter 15 mm, equidistant from the center; iii) Base: Same as lid except without the loading hole
Grooming assay:
Diffused glass-covered LED panel10–15-Watt ceiling mountable LED panel
Habituation and Y-maze assay
Climbing chambersx2, Borosilicate glass
Adapter for connecting Y-maze with entry vialPerspex, custom made, measurements in Figure 5A
Clear reagent bottlesBorosil #1500017
Gas washing stopperBorosil #1761021
Glass vialOD= 25 mm x Height= 85 mm; Borosilicate Glass
Odorant (Ethyl Butyrate)Merck #E15701
Paraffin wax (liquid) lightSRL #18211
Roller clampsPolymed #14098
Silicone tubesOD = 0.6 cm, ID = 0.3 cm; roller clamps for flow control
Vacuum pumpHana #HN-648 (Any aquarium pump with flow direction reversed manually)
Y-mazeBorosilicate glass
Y-shaped glass tube (borosilicate glass)Custom made, measurements in Figure 5A
Common items:
Any software for video playback (eg.- VLC media player)https://www.videolan.org/vlc/
Computer for video data analysis
Fly bottlesOD= 60 mm x Height= 140 mm; glass/polypropylene
Fly vialsOD= 25 mm x Height= 85 mm; Borosilicate Glass
Graph-pad Prism softwarehttps://www.graphpad.com/scientific-software/prism/www.graphpad.com/scientific-software/prism/
ImageJ softwarehttps://imagej.net/downloads
Timer
Video camera with video recording set upCamcorder or a mobile phone camera will work
For Fly Aspirator:
CottonAbsorbent, autoclaved
ParafilmSigma-aldrich #P7793
Pipette tips200 µL or 1000 µL, choose depeding on outer diameter of the silicone tube
Silicone/rubber tubelength= 30-50 cm. The tube should be odorless
Composition of Fly food:
Ingredients (amount for 1 L of food)
Agar (8 g)SRL # 19661 (CAS : 9002-18-0)
Cornflour (80 g)Organic, locally procured
D-Glucose (20 g)SRL # 51758 (CAS: 50-99-7)
Propionic acid (4 g)SRL # 43883 (CAS: 79-09-4)
Sucrose (40 g)SRL # 90701 (CAS: 57-50-1)
Tego (Methyl para hydroxy benzoate) (1.25 g)SRL # 60905 (CAS: 5026-62-0)
Yeast Powder (10 g)HIMEDIA # RM027
Fly lines used in the experiments in this study:
Wild type (Canton S or CS)BDSC # 64349
w1118BDSC # 3605
w[1118]; Fmr1[Δ50M]/TM6B, Tb[+]BDSC # 6930
w[*]; Fmr1[Δ113M]/TM6B, Tb[1]BDSC # 67403
MB247-GAL4 (Gaurav Das, NCCS Pune, India)BDSC # 50742
LN1-GAL4NP1227, NP consortium, Japan
row-shRNABDSC # 25971

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Autism Spectrum DisorderDrosophilaBehavioral AssessmentFly ModelsCommunication DeficitsSocial BehaviorRepetitive BehaviorBehavioral ParadigmsAggression AssaySocial Space AssayGroomingHabituation AssayNeurodevelopmental DisordersGene KnockdownsMolecular Pathways

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