Name: quantification of drosophila melanogaster grooming behavior for evaluation of excessive grooming phenotypes
Uploaded: 2025-03-21T13:10:00.000+00:00
Duration: 5 min 8 s
Description: Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit ...

Thanks to John Young for feedback on experimental design, Eric Luth for manuscript revision, and Madeleine Hatfield for figure design assistance. This work was funded by Simmons University and the Department of Biology.

NOTE: An overview of the protocol is presented in Figure 1.
1. Preparation for filming
<ol>
	<li>Place four three-well dishes side by side on a white surface, covering each with a glass slide.</li>
	<li>Position a camera (here, a Raspberry Pi system, but any camera capable of high-definition video is sufficient) above the dishes.
	<ol>
		<li>Check to ensure that all dishes are in frame and focus.</li>
		<li>Check to ensure that there is no glare on the slides that will limit the visibility of flies in the dish. Eliminate the glare by adjusting the plate position or dimming the lights in the recording space.</li>
		<li>Use high resolution (minimum 1920 x 1080 pixels) to accurately visualize fly limbs.</li>
	</ol>
	</li>
	<li>Mark the position of the dishes and camera on the bench where the recording is taking place. 
	NOTE: This will limit time spent on step 2 in future use of the apparatus. Performing the assays at the same time and in the same space will limit variability in behavior based on factors beyond intended manipulations.</li>
	<li>Lightly anesthetize the 4-9 day-old flies with cold on an ice block covered with parafilm and a paper towel (CO2 anesthetization will have a significant impact on behavior).
	<ol>
		<li>Tap the flies onto the block, and once anesthetized, move the flies to observation chambers as quickly as possible using a paintbrush. 
		NOTE: This transfer should happen as quickly as possible, as excessive exposure to anesthetization of any kind can negatively impact flies.</li>
	</ol>
	</li>
	<li>Place a single fly in each well.</li>
	<li>Allow files to acclimate to their new environment for 30 min post-anesthetization. Do not touch or disturb the wells containing flies after this point, as added stressors will impact behavior.</li>
</ol>
2. Recording the flies
<ol>
	<li>After the 30-min acclimation, record the flies using a camera.</li>
	<li>Use a Raspberry Pi camera run through PiSpy11 for behavioral data collection. 
	NOTE: The software is free and available on GitHub (https://github.com/gpask/PiSpy),&#160;and a Pi system can easily be set up for under $300. Other cameras, if available already, are also sufficient for recording footage</li>
	<li>Start a recording with Pi with the following command flow.
	<ol>
		<li>Open the terminal and type cd PiSpy. Then, type python3 PiSpy.py, enter the desired recording length, and enter the desired frame rate. Select the desired resolution and click Quick Capture.</li>
		<li>Use the Preview Camera function to ensure the flies are in focus and sufficiently visible.</li>
	</ol>
	</li>
	<li>Record the flies for 10 min at a recommended frame rate of 24 fps and save the file (PiSpy automatically saves to the videos folder).</li>
</ol>
3. Video analysis (Figure 2)
<ol>
	<li>To analyze the video, import the file into the Elan 6.8 software12. 
	NOTE: This software, while originally developed for psycholinguistic tallows for detailed annotation of videos and in-depth analysis of the grooming behaviors</li>
	<li>Assign each row as illustrated below to annotate the footage of individuals manually.
	<ol>
		<li>Do this with the following command flow: Tier &#62; Add New Tier &#62; Type Location in the dish on Tier Name &#62; Add.</li>
	</ol>
	</li>
	<li>Depending on the needs of a study, note and quantify various specific behaviors (anterior and posterior grooming, walking, sleeping, and standing).
	<ol>
		<li>Make annotations following the steps below.
		<ol>
			<li>Double-click the tier to be annotated. Right-click and drag the cursor along the selected tier for a period in which a single grooming bout is exhibited. Left-click, select New Annotation and double-click the newly highlighted time period. Type the abbreviation for the behavior exhibited at that time point.</li>
		</ol>
		</li>
		<li>Observe the videos slowly, scrub through them with a mouse, and annotate. Zoom into a single fly well during annotation to prevent false identification of behaviors. These could include tapping on the edge of the glass by the fly or shifting the legs, which might initially look like grooming.</li>
	</ol>
	</li>
	<li>Once the video is completely annotated, obtain a breakdown of each behavior. Ensure that the same abbreviation or annotation is used throughout the videos to use this feature. 
	NOTE: Elan 6.8 will provide the number of times a behavior occurs, the total duration of behaviors, and the mean time spent on each behavior.
	<ol>
		<li>Define grooming bouts as 2 s of uninterrupted grooming. Identify each behavior observed as follows. Define the parameters for anatomical regions prior to the conduction of the assay.
		<ol>
			<li>Anterior grooming: Identify grooming performed at the anterior region of the fly body as anterior grooming. 
			NOTE: This behavior will almost always involve a rubbing motion on an anterior part, like the head or proboscis, with the front two legs of the fly.</li>
			<li>Posterior grooming: Identify grooming performed at the anterior-posterior end of the fly body as posterior grooming. 
			NOTE: Additionally, wings were included in the posterior region for simplicity. This behavior will almost always involve a rubbing motion with the central and most posterior set of legs on the fly. A good indicator that posterior grooming is occurring will be that, at first glance, the fly may look as if it is missing its most posterior legs, as depicted in Figure 2D.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Overall, this easily executable and cost-effective assay allows for robust characterization of Drosophila melanogaster grooming behavior. This technique gives insight into the frequency, time spent engaging in, and anatomical distribution of a large number of previously identified grooming behaviors. A good indicator that grooming will occur or is underway is changes in leg placement, specifically raising any of the 6 legs. Taking note specifically of behaviors after leg movement can assist in identifying grooming behavior. This assay allows for behavioral annotation ranging from general total grooming to stereotyped grooming behaviors specific to an anatomical region, such as those targeting the wings, head, or legs of the fly. Any of these can be included or ignored to fit the needs of a study. In the representative data provided here, we use obsessive grooming as a representative measure of obsessive/compulsive behaviors associated with neurodevelopmental conditions like Tourette Syndrome. Other data may also be collected, such as information on walking patterns, time spent sleeping, and frequency of falls within the dish.
Aside from the availability of a camera or the desired camera that would be purchased, the assay is cost-effective and utilizes tools often found within lab spaces. There can be additional adjustments made, especially in constructing observation chambers to fit study needs or available supplies.
It is vital to avoid using standard carbon dioxide anesthetic in conducting experiments, as this can significantly impact the behavior of the flies16. Instead, we suggest the use of cold anesthetic with a 30-min recovery time in which the flies are left undisturbed in the observational chambers. Ensure that all chambers are effectively covered by a slide to ensure that no flies escape. Additionally, all data should be collected on flies at the same stage of adult life (aim for 4-9 days) and should be recorded at the same time within the 12/12-h light/dark cycle, as fly activity varies throughout the day. Neutral lighting and the elimination of direct overhead lighting are also ideal for video collection, as they prevent glare on the slides that will limit the view of flies in some observation chambers. Conducting assays in the same space for each trial/replicate can additionally prevent variation based on environmental factors.
Given the higher degree of subjectivity that can be introduced to this kind of behavioral observation, making annotating research blind to the genotype/treatment of the flies is ideal in executing the assay. It is also important to note that manual annotation of videos can be time-consuming, especially if many individuals or different variables are being assessed. Allow for the thorough analysis of 12 biological replicates to take roughly 12-14 h. This may be changed based on how the footage is annotated specifically. This study utilizes Elan 6.8, given that it is free to use and allows for the sub-categorization of behaviors; however, other software may be used if they are capable of annotation.
With the rise in automated processes to evaluate Drosophila grooming, manual methods of grooming like the one presented remain important to utilize. Current methods of automation present various advantages, including decreased time spent on analysis and a lack of annotator bias. Despite this, there are limitations to this kind of method that can be mediated by the use of manual methods of grooming observation.
Primarily, establishing automated processes can prove difficult in some labs, especially those at smaller undergraduate institutions with limited budget or computational expertise. In labs without a member or collaborator with the computer science skill to set up or train automated software, these methods can be inaccessible. Available methods are often complex, both in the hardware and software required, making them difficult and expensive to adapt to the specific needs of the study17.
Additionally, having a method of manual annotation is vital for times when it is needed, and it can be used in conjunction with automated methods. Drosophila&#160;mutants may display atypical behaviors that could easily be mistaken for grooming if non-grooming motor behavior abnormalities are present. This may lead to misidentification of behaviors by either a manual annotator or a computer. While studies evaluating automated annotation of Drosophila flies found that it has high accuracy rates for identification of grooming behavior18, manual methods are still necessary for verification of automated annotation accuracy to limit error.
While automated methods of grooming annotation represent a great step forward in behavioral neuroscience, beginner researchers would need to dedicate significant time and effort to learning software methods. This method presents a more straightforward method for labs lacking the expertise and time needed to initiate the use of automated annotation.
During observation, it is key to keep in mind that abnormal grooming phenotypes may be due to factors beyond a researcher&#39;s intended manipulation. Injury during fly transfer may occur, or greater motor deficits may be present in selected mutants. These individuals may display abnormal grooming phenotypes and should be watched closely to prevent false positive data. In cases of injury, the fly should be excluded from the data set.
Despite this assay&#39;s strengths, certain limitations make this assay non-ideal for certain projects. The procedure requires significant time and attention from investigators, which can significantly expand the time needed in order to get results. For this reason, automated methods may be better suited for larger-scale experiments or in labs with the computational skills and resources to execute automated observation. This would also prevent bias introduced by subjective viewing by human researchers. However, this can also be limited by blinding researchers to the samples present in a video. The assay can easily be modified to fit a study&#39;s specific needs and requires little supplies or reagents. With the ability to produce strong and reproducible results without requiring significant resources, this method provides a straightforward way in which excessive grooming may be studied in the fruit fly.

Drosophila melanogaster is a well-established model organism in behavioral and neurobiological studies, providing insight into mechanisms driving analogous human behaviors. Self-grooming in this organism is a highly regulated and well-defined behavior, following stereotypical patterns that are easily distinguished from one another1. The separate grooming behaviors exhibited by the fly can generally be classified by anatomical region2, most easily being defined as posterior or anterior. Drosophila grooming will initially focus on the anterior region and subsequently transition to the posterior end3. Under typical conditions, flies exhibit grooming behaviors to maintain cleanliness (e.g., by removing dust) and occur in response to exposure to potentially harmful external stimuli like pathogenic microbes4.
Abnormalities in grooming behavior, specifically spontaneous obsessive grooming, have been used in various model systems as an indicator of obsessive and/or compulsive behavior. Translational findings observing obsessive grooming behaviors in organisms such as rodents, birds, and canines have given insight into conditions eliciting similar compulsive behavior in humans5. These include conditions such as trichotillomania, obsessive compulsive disorder, and Tourette Syndrome6. Excessive grooming behavior has also been used as a benchmark in evaluating behavioral phenotypes in models of similar neurodevelopmental conditions in Drosophila melanogaster. Obsessive grooming behaviors have been observed in fly models of Fragile-X Syndrome (FSX) and associated autism spectrum disorder (ASD). Excess spontaneous grooming occurs under mutations of dfmr1, the ortholog to the ASD and FSX associated gene FMR17. There is additionally a notable change in grooming distribution between posterior and anterior ends in these mutants8. These changes are interpreted as reflective of obsessive and compulsive body-focused behaviors displayed by some patients with these conditions. In using the grooming assay described here, we observed grooming behaviors in flies after an RNAi-mediated knockdown of the Drosophila gene Atg8a produced by commercially available GAL4 drivers and UAS-RNAi lines9.
This method involves the manual annotation of footage taken of flies for specific grooming behaviors. Previous studies aiming to evaluate grooming behavior, such as those using indirect methods like dyes, while effective at quantifying the efficacy of grooming, do not allow for measurement of grooming duration or frequency10. This assay, however, allows for quantification of Drosophila grooming frequency and duration, both generally and by anatomical region. The method detailed here presents some advantages over current automated methods, as it is easily modified and can be conducted by individuals lacking a computational background. With the equipment required easily available in most laboratories we present a cost-efficient manner to evaluate for the presence of an excessive self-grooming phenotype (see Table of Materials). This makes the method readily accessible to primarily undergraduate institutions and easily adaptable to training environments or teaching laboratories.

<table><tbody><tr><td>16 GB Micro SD card</td><td>Adafruit</td><td>1294</td><td>For use in introducing the OS</td></tr><tr><td>Display</td><td>Adafruit</td><td>3578</td><td>Any display able to be connected by HDMI or flex cable is sufficient</td></tr><tr><td>Drosophila Atg8a-RNAi UAS line</td><td>Bloomington Stock Center</td><td>34340</td><td>Line used in representative data</td></tr><tr><td>Drosophila Ok6 Driver</td><td>N/A</td><td>N/A</td><td>Line used in representative data</td></tr><tr><td>Glass slides</td><td>Fisher Scientific</td><td>12-550-A3</td><td>Keeps flies in containment</td></tr><tr><td>Heatsinks</td><td>Adafruit</td><td></td><td>Prevents overheating of the computer</td></tr><tr><td>Lens</td><td>Adafruit</td><td>4563</td><td>Used with HD camera only</td></tr><tr><td>PiCamera</td><td>Adafruit</td><td>4561</td><td>The HD version was used here but a standard camera can be used under budget constraints</td></tr><tr><td>Raspberry pi 4</td><td>Adafruit</td><td>4292</td><td>Computer PiSpy is run on</td></tr><tr><td>Ribbion Cable</td><td>Adafruit</td><td>1648</td><td>For use in connecting camera and display</td></tr><tr><td>SB components Case</td><td>Adafruit</td><td>4301</td><td>Protects the computer</td></tr><tr><td>Spot Plate</td><td>Fisher Scientific</td><td>S99406</td><td>Pyrex versions were already avaliable for our use but cell culture dishes or plastic spot plates are sufficient as well</td></tr><tr><td>Tripod</td><td>Best Buy</td><td>6355959</td><td>For suspension and positioning of camera, any apparatus capable of this is sufficient</td></tr><tr><td>USB C Power Supply</td><td>Adafruit</td><td>1995</td><td>Supplies power to the computer</td></tr></tbody></table>

quantification of drosophila melanogaster grooming behavior for evaluation of excessive grooming phenotypes

Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit similar deviations in human behavior; for example, excessive grooming acts as a proxy for obsessive and compulsive behaviors present in conditions like Tourette Syndrome or obsessive-compulsive disorder. The grooming assay presented allows for the evaluation of abnormal self-grooming phenotypes in Drosophila melanogaster. Flies are recorded for a period of 10 min, and these recordings are observed and annotated blind for previously defined grooming behaviors. Quantitative measures of both grooming bout frequency and the time spent engaging in self-grooming can be obtained by manually annotating the footage. The assay is relatively inexpensive, requires few materials not already available in laboratory environments, and is easily adaptable to fit the specific needs of any given study aiming to observe grooming. Additionally, the low level of skill needed to perform the assay, as compared to computer science-heavy automated methods, makes the protocol well-suited for small labs and students. We discuss in detail the steps required to perform this assay and its present limitations.

This assay produces quantitative data measuring the time and frequency of grooming behaviors from annotated footage. A representative image of the setup and how behaviors are defined is outlined in Figure 2. Given the subjectivity introduced by video analysis, all annotations of videos should be blinded to the researcher performing the analysis.
This method was used to evaluate the role the Drosophila gene Atg8a has in the development of grooming behavior. Atg8a is orthologous to the human gene GABARAP, which has displayed decreased expression in some Tourette Syndrome (TS) patients9. TS is a condition characterized by involuntary movements and vocalizations known as tics that are commonly comorbid with other conditions that elicit obsessive/compulsive behavior13. We aimed to understand how this decrease in expression could impact the development of this neurodevelopmental condition by utilizing the common behavioral marker for TS and other neurodevelopmental models, obsessive grooming. By comparing the frequency and time spent grooming between RNAi mediated knockdown mutants of Atg8a produced through the Gal4-UAS system14, and their respective UAS lines and Gal4 drivers acting as controls. Overall, we found that there was a significant general increase in grooming behavior for Atg8a knockdown mutants15.
In exploring the distribution of grooming behaviors, we aimed to evaluate how obsessive grooming in the model described here compares to previously published neurodevelopmental condition Drosophila models, such as those evaluating the Autism Spectrum Disorder gene dfmr1. Studies found that there was not only an increase in grooming frequency7 but also that preferential grooming at the posterior end was observed under mutation of the dfmr18. We found, however, that Atg8a mutants, while displaying an overall increase in grooming, do not exhibit significant changes in the distribution of these behaviors from their control group counterparts. This finding allows us to differentiate our TS-like model from the ASD-like model by behavioral phenotype.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67708/67708fig01.jpg" /> 
Figure 1: Illustrated workflow of obsessive grooming assay <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67708/67708fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67708/67708fig02.jpg" /> 
Figure 2: Representative images and results of the study. (A) Loaded wells with sample flies as they will appear in the Elan software. (B) Illustrated representation of anatomical distribution classifications made for representative data. (C) Each row in an annotated .mov file is representative of a single fly in a well. Highlighted is the fly, abbreviated FL1, which indicates that the fly is the first in the farthest left column. (D) Drosophila exhibiting posterior grooming behavior abbreviated as PG in Elan 6.8. (E) Drosophila exhibiting anterior grooming behavior abbreviated as ANT in Elan 6.8.&#160;(F) Atg8a knockdown mutant flies had an overall increase in the number of grooming bouts initiated compared to the Ok6-Gal4 driver line (p = 0.0068) and the UAS Atg8a RNAi line (p = 0.0011). There was, however, no significant difference in grooming distribution either posteriorly or anteriorly in mutant flies as compared to these controls. Statistical analysis involved a Brown-Forsythe and Welch two-way ANOVA test meeting a significance level of at least P &#60; 0.001, followed by a Dunnet&#39;s multiple comparisons test. All analyses were performed in Prism 10.&#160;(G) Overall, there was a significant increase in the time the Atg8a knockdown mutant flies spent engaging in grooming behaviors as compared to the Ok6-Gal4 driver line (p = 0.0005) and the UAS Atg8a RNAi line (p = 0.0004). There was, however, no significant difference in grooming distribution either posteriorly or anteriorly in mutant flies as compared to these controls. Statistical analysis involved a Brown-Forsythe and Welch two-way ANOVA test meeting a significance level of at least P &#60; 0.001, followed by a Dunnet&#39;s multiple comparisons test. All analyses were performed in Prism 10. Panels F and G were adapted with permission from Hatfield and Johnson15. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67708/67708fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The method presented here involves the manual annotation of footage acquired of Drosophila melanogaster for specific grooming behaviors. It allows for quantification of both the number of grooming bouts and overall time spent grooming to evaluate for atypical self-grooming phenotypes.

Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes

Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit ...

quantification-drosophila-melanogaster-grooming-behavior-for

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

338 Views.  Simmons University. The method presented here involves the manual annotation of footage acquired of Drosophila melanogaster for specific grooming behaviors. It allows for quantification of both the number of grooming bouts and overall time spent grooming to evaluate for atypical self-grooming phenotypes.

Video: Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes

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 ...

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits&#160;within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to ...

Behavior

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

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 ...

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 ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Drosophila Models for Genetic and Behavioral Traits of Autism Spectrum Disorder

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 &#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.

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

High-Resolution Imaging and Analysis of Drosophila Eye Defects

A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It is a simple structure of repeating unit eyes, termed ommatidia, that is used to characterize genetic interactions and gene functions. Mutations that affect eye architecture can be easily detected and analyzed; hence, this system is frequently used in under-resourced institutions. Further phenotypic analysis often includes a Scanning Electron Microscope (SEM) to generate high-magnification images suitable for quantitative analysis. However, SEMs are expensive and require costly reagents; sample preparation spans days; and, often, they need full-time staff for sample preparation and instrument maintenance. This limits their utility at under-resourced institutions or during budgetary austerity. In entomology, the use of high-resolution digital imaging technology is a common practice for the identification and characterization of species. This paper describes a method that combines strategies and allows for high-resolution digital imaging of adult Drosophila structures and quantitative analysis using the open software ImageJ. The workflow is a rapid and student-friendly alternative that remedies the limitations of underfunded and under-resourced research facilities with a cost-effective and rapid approach to quantitative phenotypic analysis.

Here, we present a rapid, low-cost, workflow for high-resolution imaging of adult Drosophila eyes to quantify patterning and growth defects. We describe our protocol for sample preparation by point-mounting, high-resolution image acquisition, and image analysis.

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It ...

Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes

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Methods to Assay Drosophila Behavior

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Strategies for Assessing Autistic-Like Behaviors in Mice

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

Quantification of Drosophila Grooming Behavior

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures