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

초파리 자폐 스펙트럼 장애의 유전 및 행동 특성에 대한 모델

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.

저자 스포트라이트: 초파리의 자폐 스펙트럼 장애 증상 탐구 - 유전자 모델 및 행동 검사

자폐 스펙트럼 장애의 초파리 모델에서 행동 평가를 위한 패러다임

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.

자폐 스펙트럼 장애의 초파리 모델에서 사회적 행동 결함을 탐색하기 위한 사회적 공간 분석

시작하려면 얼음 위의 파리 혼합 개체군에서 수컷 파리를 분리하여 수집 바이알에 넣습니다. 경기장을 준비하려면 아크릴 스페이서의 직각이 유리판의 모서리와 정렬되도록 직사각형 유리판 위에 두 개의 삼각형 아크릴 스페이서를 평평하게 놓습니다. 그런 다음 두 개의 삼각형 스페이서의 바닥과 정렬된 유리판에 두 개의 직사각형 아크릴 스페이서를 평평하게 놓습니다.4개의 아크릴 스페이서가 스페이서로 덮이지 않은 직사각형 유리판의 삼각형 경기장을 둘러싸고 있는지 확인합니다. 삼각형 스페이서 중 하나에 눈금자의 스티커를 놓고 위에서 볼 수 있도록 합니다. 이제 아크릴 스페이서 위에 두 번째 직사각형 유리창을 놓고 하단의 유리창과 맞춥니다.4개의 작은 바인더 클립을 사용하여 창과 스페이서를 고정합니다. 분석을 위해 오른쪽 하단 바인더 클립을 제거하고 직사각형 스페이서를 바깥쪽으로 약간 이동하여 틈을 만듭니다. 수집 바이알에서 파리를 사회적 공간 영역으로 옮기려면 생성된 공간을 통해 파리를 부드럽게 흡인합니다.즉시 직사각형 스페이서를 밀어 바인더 클립을 제자리에 다시 고정합니다. 챔버를 똑바로 잡고 부드러운 패드를 세 번 부드럽게 두드려 모든 파리가 바닥에 정착하도록 합니다. Clamp 챔버를 수직 위치에 놓고 타이머를 시작합니다.20분 후 경기장의 선명한 사진을 찍습니다. ImageJ로 분석하여 파리 간 최근접 이웃 거리를 나열합니다. 돌연변이 파리는 대조군 파리보다 훨씬 더 가까운 이웃 거리를 보여주며, 이는 다른 파리와 더 큰 거리를 선호하는 것을 나타냅니다.

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 ...

연구

신경과학

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

자폐 스펙트럼 장애(ASD) 관련 의사소통 및 사회적 행동 결핍을 분석하기 위한 초파리 의 공격성 분석

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

초파리의 자폐 스펙트럼 장애를 연구하기 위한 구애 및 그루밍 행동 분석

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

초파리 모델을 사용하여 자폐 스펙트럼 장애에 대한 유전 및 행동 통찰력을 얻기 위한 후각 습관화 분석