olfactory habituation assay to gain genetic and behavioral insights into autism spectrum disorder using drosophila models

Drosophile Modèles de traits génétiques et comportementaux des troubles du spectre autistique

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.

Pleins feux sur l’auteur : Explorer les symptômes du trouble du spectre autistique chez les mouches des fruits - Modèles génétiques et tests comportementaux

Paradigmes pour l’évaluation comportementale dans le modèle de trouble du spectre autistique de la drosophile

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.

Test d’habituation olfactive pour obtenir des informations génétiques et comportementales sur les troubles du spectre autistique à l’aide de modèles de drosophile

Pour commencer, procurez-vous les mouches préparées pour le test d’habituation. Placez un millilitre de l’odorisant préféré dilué avec du liquide de paraffine légère dans un tube microfuge de 1,5 millilitre. Agitez le tube pendant 10 minutes et couvrez-le d’une pellicule plastique uniformément perforée.À l’aide d’un fil, suspendez les tubes odorisants dans des flacons séparés contenant des mouches. Couvrez bien la bouteille de coton et enveloppez-la de papier kraft pour éviter une fusion de l’odeur et de la vapeur. Étiquetez les bouteilles de contrôle et les bouteilles contenant des odeurs comme étant respectivement naïves et exposées aux odeurs.Conservez ces flacons d’induction pendant trois jours dans un incubateur. Pour le test du labyrinthe en Y, fixez le labyrinthe en Y à l’adaptateur, fixez le fond du labyrinthe en Y au flacon d’entrée, puis fixez-le aux chambres d’escalade aux deux bras du labyrinthe en Y. À l’aide de tubes en silicone inodores, connectez l’extrémité effilée de chaque chambre d’escalade avec le flacon de réactif contenant l’odorisant.Avec la pompe à vide, conduisez l’odorisant des bouteilles de gaz vers les deux bras du Y et laissez-le saturer pendant 15 minutes. Introduisez doucement les mouches étoilées de chaque fiole dans la fiole d’entrée de la configuration du labyrinthe en Y. Après une brève acclimatation, connectez le flacon d’entrée à l’adaptateur Y-maze pour commencer le test.Laissez les mouches grimper dans les bras du labyrinthe en Y et rester piégées dans les deux chambres de collecte pendant plus d’une minute. Une fois terminé, remettez les mouches au fond de la fiole d’entrée et changez la position des bras du labyrinthe en Y pour éviter les biais latéraux. Notez le nombre de mouches grimpant sur chacun des deux bras du labyrinthe en Y pendant la durée de l’opération.Les mouches renversées ne se sont pas habituées après une exposition de trois jours à un odorant par rapport aux mouches de type sauvage.

Research

JoVE Journal

Neuroscience

Olfactory Habituation Assay to Gain Genetic and Behavioral Insights into Autism Spectrum Disorder Using Drosophila Models

Drosophila Models for Genetic and Behavioral Traits of Autism Spectrum Disorder

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

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

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

Recherche

Neurosciences

Aggression Assay in Drosophila to Analyze Autism Spectrum Disorder &#40;ASD&#41;&#45;Related Communication and Social Behavior Deficit

Test d’agressivité chez la drosophile pour analyser le déficit de communication et de comportement social lié aux troubles du spectre autistique (TSA)

Social Space Assay to Explore Social Behavior Defects in the Drosophila Model of Autism Spectrum Disorder

Un test d’espace social pour explorer les défauts du comportement social dans le modèle de drosophile du trouble du spectre autistique

Courtship and Grooming Behavior Assay to Study Autism Spectrum Disorder in Drosophila

Test de comportement de parade nuptiale et de toilettage pour étudier le trouble du spectre autistique chez la drosophile