social space assay to explore social behavior defects in the drosophila model of autism spectrum disorder

果蝇 自闭症谱系障碍的遗传和行为特征模型

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.

作者聚焦：探索果蝇的自闭症谱系障碍症状 — 遗传模型和行为测试

自闭症谱系障碍果 蝇 模型的行为评估范式

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.

探索自闭症谱系障碍果 蝇 模型中的社交行为缺陷的社交空间测定

首先，将雄性果蝇与冰上的混合果蝇种群分开，并将它们放入收集瓶中。要准备竞技场，请将两个三角形亚克力垫片平放在矩形玻璃板的顶部，使亚克力垫片的直角与玻璃板的角对齐。然后将两个矩形亚克力垫片平放在玻璃板上，与两个三角形垫片的底座对齐。确认四个亚克力垫片围绕着矩形玻璃板上未被垫片覆盖的三角形竞技场。将标尺的贴纸放在其中一个三角形垫片上，确保从顶部可以看到它。现在，将第二个矩形玻璃板放在亚克力垫片的顶部，使其与底部的玻璃板对齐。使用四个小活页夹固定窗格和垫片。对于测定，取下右下角的粘合剂夹，并将矩形垫片稍微向外移动以形成间隙。要将苍蝇从收集瓶转移到社交空间领域，请轻轻地将它们吸入创造的空间。立即滑动矩形垫片并将活页夹固定回原位。将腔室直立，在软垫上轻轻敲打 3 次，让所有果蝇都安顿在底部。将腔室夹在直立位置并启动计时器。20 分钟后拍摄一张清晰的竞技场照片。使用 ImageJ 进行分析以列出 fly-inter 最近邻距离。突变苍蝇的最近邻距离明显高于对照苍蝇，表明它们更倾向于与其他苍蝇保持更大的距离。

Research

JoVE Journal

Neuroscience

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

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 ...

科研

神经科学

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

果 蝇 的攻击性测定分析自闭症谱系障碍 （ASD） 相关的沟通和社会行为缺陷

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

研究果蝇自闭症谱系障碍的求爱和梳理行为测定

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

嗅觉习惯化分析使用 果蝇 模型获得对自闭症谱系障碍的遗传和行为见解

探索自闭症谱系障碍果 蝇 模型中的社交行为缺陷的社交空间测定

探索自闭症谱系障碍果蝇模型中的社交行为缺陷的社交空间测定