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.

Drosophila Models for Genetic and Behavioral Traits of Autism Spectrum Disorder

The authors have no conflicts of interest to disclose.

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><tbody><tr><td>&lt;strong&gt;Aggression arena:&lt;/strong&gt;</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>&lt;strong&gt;Social Space Assay:&lt;/strong&gt;</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>&lt;strong&gt;Courtship assay:&lt;/strong&gt;</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>&lt;strong&gt;Grooming assay:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diffused glass-covered LED panel</td><td></td><td></td><td>10&amp;ndash;15-Watt ceiling mountable LED panel</td></tr><tr><td>&lt;strong&gt;Habituation and Y-maze assay&lt;/strong&gt;</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 &lt;strong&gt;Figure 5A&lt;/strong&gt;</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 &lt;strong&gt;Figure 5A&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Common items:&lt;/strong&gt;</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>&lt;strong&gt;For Fly Aspirator:&lt;/strong&gt;</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 &amp;micro;L or 1000 &amp;micro;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>&lt;strong&gt;Composition of Fly food:&lt;/strong&gt;</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>&lt;strong&gt;Fly lines used in the experiments in this study:&lt;/strong&gt;</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>&lt;em&gt;w&lt;sup&gt;1118&lt;/sup&gt;&lt;/em&gt;</td><td></td><td></td><td>BDSC # 3605</td></tr><tr><td>&lt;em&gt;w[1118]; Fmr1[&amp;Delta;50M]/TM6B, Tb[+]&lt;/em&gt;</td><td></td><td></td><td>BDSC # 6930</td></tr><tr><td>&lt;em&gt;w[*]; Fmr1[&amp;Delta;113M]/TM6B, Tb[1]&lt;/em&gt;</td><td></td><td></td><td>BDSC # 67403</td></tr><tr><td>&lt;em&gt;MB247-GAL4 &lt;/em&gt;(Gaurav Das, NCCS Pune, India)</td><td></td><td></td><td>BDSC # 50742</td></tr><tr><td>&lt;em&gt;LN1-GAL4&lt;/em&gt;</td><td></td><td></td><td>NP1227, NP consortium, Japan</td></tr><tr><td>&lt;em&gt;row-shRNA&lt;/em&gt;</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.

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 ...

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

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.

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 ...

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

Research

JoVE Journal

Neuroscience

1.5K Views.  Indian Institute of Technology Kharagpur. 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. 

Video: Author Spotlight: Exploring Autism Spectrum Disorder Symptoms in Fruit Flies — Genetic Models and Behavioral Tests

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Behavioral Assessment of Manual Dexterity in Non-Human Primates

The corticospinal (CS) tract is the anatomical support of the exquisite motor ability to skillfully manipulate small objects, a prerogative mainly of primates1. In case of lesion affecting the CS projection system at its origin (lesion of motor cortical areas) or along its trajectory (cervical cord lesion), there is a dramatic loss of manual dexterity (hand paralysis), as seen in some tetraplegic or hemiplegic patients. Although there is some spontaneous functional recovery after such lesion, it remains very limited in the adult. Various therapeutic strategies are presently proposed (e.g. cell therapy, neutralization of inhibitory axonal growth molecules, application of growth factors, etc), which are mostly developed in rodents. However, before clinical application, it is often recommended to test the feasibility, efficacy, and security of the treatment in non-human primates. This is especially true when the goal is to restore manual dexterity after a lesion of the central nervous system, as the organization of the motor system of rodents is different from that of primates1,2. Macaque monkeys are illustrated here as a suitable behavioral model to quantify manual dexterity in primates, to reflect the deficits resulting from lesion of the motor cortex or cervical cord for instance, measure the extent of spontaneous functional recovery and, when a treatment is applied, evaluate how much it can enhance the functional recovery.
The behavioral assessment of manual dexterity is based on four distinct, complementary, reach and grasp manual tasks (use of precision grip to grasp pellets), requiring an initial training of adult macaque monkeys. The preparation of the animals is demonstrated, as well as the positioning with respect to the behavioral set-up. The performance of a typical monkey is illustrated for each task. The collection and analysis of relevant parameters reflecting precise hand manipulation, as well as the control of force, are explained and demonstrated with representative results. These data are placed then in a broader context, showing how the behavioral data can be exploited to investigate the impact of a spinal cord lesion or of a lesion of the motor cortex and to what extent a treatment may enhance the spontaneous functional recovery, by comparing different groups of monkeys (treated versus sham treated for instance). Advantages and limitations of the behavioral tests are discussed. The present behavioral approach is in line with previous reports emphasizing the pertinence of the non-human primate model in the context of nervous system diseases2,3.

As manual dexterity is a prerogative mainly of primates, behavioral tasks have been developed in macaque monkeys. Four reach and grasp prehension tasks, measuring hand manipulation ability and force, allow to establish functional recovery after a lesion of the central nervous system and to test the effect of a treatment.

The corticospinal (CS) tract is the anatomical support of the exquisite motor ability to skillfully manipulate small objects, a prerogative mainly of ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Behavior

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Summary

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Strategies for Assessing Autistic-Like Behaviors in Mice

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Conditions Affecting Social Space in Drosophila melanogaster

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster