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.

See the Table of Materials for details related to all materials and reagents used in this protocol.
1. Aggression assay
<ol>
	<li>Preparing the aggression assay arena
	<ol>
		<li>Take a standard 24-well plate (Figure 1A) and use each well of the plate as a single &#39;arena&#39; (Figure 1B) for fly aggression. Fill half of each well with regular fly food and allow it to dry overnight. 
		NOTE: ...

&lt;i&gt;Drosophila&lt;/i&gt; Models for Genetic and Behavioral Traits of Autism Spectrum Disorder

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...

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 &#39;motivation for socialization&#39;25,26 as well as &#39;communication&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aggression arena:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard 24-well plate made of transparent polystyrene</td>
			<td></td>
			<td></td>
			<td>12 cm x 8 cm x 2 cm. Diameter of a single well= 18 cm. Sigma-aldrich #Z707791; depth = 1 cm</td>
		</tr>
		<tr>
			<td>Transparent plastic/acrylic sheet</td>
			<td></td>
			<td></td>
			<td>Alternative: a perforated lid of a cell culture plate</td>
		</tr>
		<tr>
			<td>Social Space Assay:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binder clips</td>
			<td></td>
			<td></td>
			<td>19 mm</td>
		</tr>
		<tr>
			<td>Glass sheets and acrylic sheets of customized sizes</td>
			<td></td>
			<td></td>
			<td>Thickness = 5 mm</td>
		</tr>
		<tr>
			<td>Courtship assay:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nut and bolt with threading</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perspex sheets of customized shapes</td>
			<td></td>
			<td></td>
			<td>i) 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</td>
		</tr>
		<tr>
			<td>Grooming assay:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diffused glass-covered LED panel</td>
			<td></td>
			<td></td>
			<td>10&#8211;15-Watt ceiling mountable LED panel</td>
		</tr>
		<tr>
			<td>Habituation and Y-maze assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Climbing chambers</td>
			<td></td>
			<td></td>
			<td>x2, Borosilicate glass</td>
		</tr>
		<tr>
			<td>Adapter for connecting Y-maze with entry vial</td>
			<td></td>
			<td></td>
			<td>Perspex, custom made, measurements in Figure 5A</td>
		</tr>
		<tr>
			<td>Clear reagent bottles</td>
			<td></td>
			<td></td>
			<td>Borosil #1500017</td>
		</tr>
		<tr>
			<td>Gas washing stopper</td>
			<td></td>
			<td></td>
			<td>Borosil #1761021</td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td></td>
			<td></td>
			<td>OD= 25 mm x Height= 85 mm; Borosilicate Glass</td>
		</tr>
		<tr>
			<td>Odorant (Ethyl Butyrate)</td>
			<td></td>
			<td></td>
			<td>Merck #E15701</td>
		</tr>
		<tr>
			<td>Paraffin wax (liquid) light</td>
			<td></td>
			<td></td>
			<td>SRL #18211</td>
		</tr>
		<tr>
			<td>Roller clamps</td>
			<td></td>
			<td></td>
			<td>Polymed #14098</td>
		</tr>
		<tr>
			<td>Silicone tubes</td>
			<td></td>
			<td></td>
			<td>OD = 0.6 cm, ID = 0.3 cm; roller clamps for flow control</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td></td>
			<td></td>
			<td>Hana #HN-648 (Any aquarium pump with flow direction reversed manually)</td>
		</tr>
		<tr>
			<td>Y-maze</td>
			<td></td>
			<td></td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>Y-shaped glass tube (borosilicate glass)</td>
			<td></td>
			<td></td>
			<td>Custom made, measurements in Figure 5A</td>
		</tr>
		<tr>
			<td>Common items:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Any software for video playback (eg.- VLC media player)</td>
			<td></td>
			<td></td>
			<td>https://www.videolan.org/vlc/</td>
		</tr>
		<tr>
			<td>Computer for video data analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly bottles</td>
			<td></td>
			<td></td>
			<td>OD= 60 mm x Height= 140 mm; glass/polypropylene</td>
		</tr>
		<tr>
			<td>Fly vials</td>
			<td></td>
			<td></td>
			<td>OD= 25 mm x Height= 85 mm; Borosilicate Glass</td>
		</tr>
		<tr>
			<td>Graph-pad Prism software</td>
			<td></td>
			<td></td>
			<td>https://www.graphpad.com/scientific-software/prism/www.graphpad.com/scientific-software/prism/</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td></td>
			<td></td>
			<td>https://imagej.net/downloads</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera with video recording set up</td>
			<td></td>
			<td></td>
			<td>Camcorder or a mobile phone camera will work</td>
		</tr>
		<tr>
			<td>For Fly Aspirator:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td>Absorbent, autoclaved</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td>Sigma-aldrich #P7793</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td></td>
			<td></td>
			<td>200 &#181;L or 1000 &#181;L, choose depeding on outer diameter of the silicone tube</td>
		</tr>
		<tr>
			<td>Silicone/rubber tube</td>
			<td></td>
			<td></td>
			<td>length= 30-50 cm. The tube should be odorless</td>
		</tr>
		<tr>
			<td>Composition of Fly food:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ingredients (amount for 1 L of food)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar (8 g)</td>
			<td></td>
			<td></td>
			<td>SRL # 19661 (CAS : 9002-18-0)</td>
		</tr>
		<tr>
			<td>Cornflour (80 g)</td>
			<td></td>
			<td></td>
			<td>Organic, locally procured</td>
		</tr>
		<tr>
			<td>D-Glucose (20 g)</td>
			<td></td>
			<td></td>
			<td>SRL # 51758 (CAS: 50-99-7)</td>
		</tr>
		<tr>
			<td>Propionic acid (4 g)</td>
			<td></td>
			<td></td>
			<td>SRL # 43883 (CAS: 79-09-4)</td>
		</tr>
		<tr>
			<td>Sucrose (40 g)</td>
			<td></td>
			<td></td>
			<td>SRL # 90701 (CAS: 57-50-1)</td>
		</tr>
		<tr>
			<td>Tego (Methyl para hydroxy benzoate) (1.25 g)</td>
			<td></td>
			<td></td>
			<td>SRL # 60905 (CAS: 5026-62-0)</td>
		</tr>
		<tr>
			<td>Yeast Powder (10 g)</td>
			<td></td>
			<td></td>
			<td>HIMEDIA # RM027</td>
		</tr>
		<tr>
			<td>Fly lines used in the experiments in this study:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wild type (Canton S or CS)</td>
			<td></td>
			<td></td>
			<td>BDSC # 64349</td>
		</tr>
		<tr>
			<td>w1118</td>
			<td></td>
			<td></td>
			<td>BDSC # 3605</td>
		</tr>
		<tr>
			<td>w[1118]; Fmr1[&#916;50M]/TM6B, Tb[+]</td>
			<td></td>
			<td></td>
			<td>BDSC # 6930</td>
		</tr>
		<tr>
			<td>w[*]; Fmr1[&#916;113M]/TM6B, Tb[1]</td>
			<td></td>
			<td></td>
			<td>BDSC # 67403</td>
		</tr>
		<tr>
			<td>MB247-GAL4 (Gaurav Das, NCCS Pune, India)</td>
			<td></td>
			<td></td>
			<td>BDSC # 50742</td>
		</tr>
		<tr>
			<td>LN1-GAL4</td>
			<td></td>
			<td></td>
			<td>NP1227, NP consortium, Japan</td>
		</tr>
		<tr>
			<td>row-shRNA</td>
			<td></td>
			<td></td>
			<td>BDSC # 25971</td>
		</tr>
	</tbody>
</table>

paradigms for behavioral assessment in drosophila model of autism spectrum disorder

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.

Author Spotlight: Exploring Autism Spectrum Disorder Symptoms in Fruit Flies &amp;#8212; Genetic Models and Behavioral Tests

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, coat shape, 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 described 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.

Aggression assay 
As a fly ASD model, Fmr1 mutant flies have been used63,64. w1118 males were used as control and Fmr1 trans-heterozygote Fmr1&#916;113M/Fmr1&#916;50M57 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 ...

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.

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in ...

paradigms-for-behavioral-assessment-drosophila-model-autism-spectrum