This study was supported in parts by NIH R15ES029673 (AKM).

1. Preparation of passive avoidance apparatus
<ol>
	<li>Drill a 4 mm hole perpendicular to the wall surface of the 14 mL polypropylene culture tube and 8 mm away from the tube bottom. 
	NOTE: Use an electric drill and 5/32 drill bit for best results.</li>
	<li>Using a steel utility knife, cut off the upper part of the 14 mL polypropylene culture tube to create a 45 mm long tube bottom fragment. The bottom fragment serves as the lower compartment.</li>
	<li>Cut off the tip of 1,000 &#181;L blue pipette tip using a single edge razor blade to make the opening wide enough for a passage of a single fly. Cut off the narrowing part of the blue tip to create a 12 mm fragment. Insert this fragment firmly into a 4 mm hole of the lower compartment. This is used as a loading dock for transferring the flies.</li>
	<li>Cut a 15 mm piece of transparent vinyl tubing 5/8&#34; ID (see Table of Materials) to create a coupling. Insert upper and lower compartment into the coupling from opposite ends to securely attach the lower compartment to the upper compartment.</li>
	<li>Using a 2-prong adjustable clamp, attach the assembly to a vertical stand. Orient the assembly vertically with the shock tube as an upper compartment.</li>
	<li>Connect the shock tube wires to an electrical stimulator (see Table of Materials) to deliver electric shocks. The duration of the training period is 1 min. 
	&#8203;NOTE: To facilitate observation, position a piece of white paper behind the shock tube to serve as a white background of the apparatus. Put a lamp with a 75 W equivalent soft light bulb above the shock compartment. Place an adjustable arm magnifier lamp in front of the setup. A representation of the passive avoidance apparatus is shown in Figure 1.</li>
</ol>
2. Preparation of the flies for the passive avoidance procedure
<ol>
	<li>Immobilize 3-4 day old D. melanogaster or D. simulans flies using ice-cold block and transfer them into individual vials with food 24 h before the experiment (1 fly per vial) following the procedure described previously19. 
	NOTE: The experiments described here compared 3-4 day-old male vs. female flies in D. melanogaster and D. simulans.</li>
	<li>Before the behavioral experiments, code all the vials. For this, assign each group a letter, for example, &#34;A&#34;, &#34;B&#34;, &#34;C&#34;, etc., and each fly a number. Reveal this code only after all data have been recorded and analyzed. Use at least 20 flies per genotype or treatment to counter individual variations. 
	&#8203;NOTE: Performing the experiments and analyses &#34;blind&#34; allows excluding a bias in assessing the performance of the fly and data analysis.</li>
</ol>
3. Performing the first trial
<ol>
	<li>Using a fly mouth aspirator (see Table of Materials) described previously20, gently transfer a fly from the individual vial into the lower compartment via the loading dock. Gently suck one fly into the mouth aspirator by sucking air. Deposit the fly by lightly blowing into the loading dock. 
	NOTE: Avoid stressing the animal during catching and loading.</li>
	<li>Immediately after the fly is loaded into the lower compartment, start a 1 min timer and stopwatch. 
	NOTE: The stopwatch is used to measure latencies and tally counter to count the number of shocks.</li>
	<li>Press the stopwatch to record the first latency when the fly enters the shock tube by placing all paws on the grid. Turn on the stimulator to deliver an electric shock to the fly. The stimulation parameters are 120 volts, 1000 ms duration, 1 pulse/s (PPS), train duration 2000 ms.</li>
	<li>Deliver additional shocks if fly re-enters shock tube. Record the number of received shocks during a 1 min trial with a tally counter or an Arduino-based counter (see Table of Materials). If using the Arduino-based counter, please follow the steps below. 
	NOTE: An optional Arduino-based device AKM-007 (see Table of Materials) can be used to measure time, latency, the number of shocks, and the frequency and duration of grooming bouts for each animal by pressing and releasing the corresponding buttons on the device. The buttons on the device are assigned to measure latency, administer and record the number of shocks, and measure the frequency and duration of grooming bouts.
	<ol>
		<li>Press the Start button at step 3.2., and press the Shock button at step 3.3.</li>
		<li>To record the duration of a grooming bout, press the Grooming button at the beginning of a grooming bout on the device and release this button at the end of the grooming bout. 
		NOTE: The grooming bouts were measured throughout 1 min trial. Extensive grooming could be indicative of animal stress21,22. The Arduino-based device saves all data as CSV file to a memory card.</li>
	</ol>
	</li>
	<li>At the end of a 1 min trial, gently transfer the fly back to an individual vial. Write down the latency, the number of received shocks, and any notable changes in the behavior.</li>
	<li>Clean the lower and shock compartment with 70% ethanol, wipe with a lint-free cleaning tissue (see Table of Materials), and dry with the hairdryer. Repeat the trial with the next fly.</li>
	<li>After the behavioral experiments, clean the lower compartment with water and odorless detergent. Wipe the lower compartment and the shock compartment with 70% ethanol, and air-dry overnight.</li>
</ol>
4. Performing the second trial
<ol>
	<li>Perform the second trial by repeating the procedure described above (step 3) 24 h later. Test the flies in the same sequence as in the previous day.</li>
</ol>
5. Analysis of the results
<ol>
	<li>Calculate the average latency, the average number of shocks, and duration of grooming bouts for trial 1 and trial 2 for each experimental group of animals. Perform student t-test for two-group comparison or ANOVA for multiple comparisons with post-hoc analyses using Tukey&#39;s test23.</li>
</ol>

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The authors declare no conflicts of interest.

Avoidance of threatening stimuli is a crucial characteristic of adaptive behavior in various species from C. elegance to human32. Avoidance learning procedures which typically entail the escaping of an aversive event, are commonly used behavioral tasks to investigate learning and memory processes in laboratory rodents13 since the 1970&#39;s32. In active avoidance procedures, an indifferent stimulus or conditioned signal (CS) is followed by an aversive event or unconditioned signal (US), which animals learn to avoid by performing a specific behavioral task. In passive avoidance procedures, an animal needs to avoid the aversive US by associating a previously punished behavior with a specific environmental context33. Longer retention test latencies indicating better memory suggest that the animal developed a detailed representation of the training experience13. Passive avoidance training consists of a single trial; however, the brain mechanisms underlying the acquisition of this task are complex as the animal learns to associate various pieces of information, including environmental, spatial-positional, and aversive stimuli13. Altering these stimuli allows studying of episodic and contextual types of memory13.
The paradigm described here adopted passive avoidance behavior in rodents to adult fly as a model system. While several procedures have been developed to study learning and memory in flies, including olfactory7,8, visual9, and courtship conditioning10, these assays are rather time-consuming and require a complex setup. The protocol described here is a simple behavioral assay to study associative aversive learning in flies, which consists of 1 min trials and can be easily set up with a few available supplies. The protocol potentially allows for additional manipulation of the environmental cues, with the addition of olfactory, visual, and other contextual stimuli, by changing colors or geometrical patterns for the lower compartment or adding olfactory cues into the cap of the upper chamber. In addition, this assay can be easily adapted to study short-term memory, middle-term memory, and anesthesia-resistant memory by manipulating intervals between trials and subjecting flies to anesthesia.
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly lower&#160;in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.
Interestingly, as shown in Figures 3E and Figure 4E, the latencies in the third trial were split into two extremes (less than 30 s and 60 s). These differences, however, were not observed in the 4th trial indicating that the majority of the flies eventually acquired the task (Figure 2E). While the exact reasons for the individual differences in developing passive avoidance tasks are unclear, natural populations may contain polymorphism and mutations influencing learning and memory3,5,34,35. A recent study showed that polymorphism in the foraging (for) locus encoding a cGMP-dependent protein kinase (PKG) might mediate alternative strategies in learning foraging behavior34. Flies with the &#34;rover&#34; allele and higher activity have better short-term memory, while flies with the &#34;sitter&#34; allele and sedentary behavior show better long-term memory34.
The assay revealed that the WD significantly impaired learning and memory in flies, which was evident by shorter latencies and receiving more shocks in the second trial. The higher number of shocks in the first trial after the WD could indicate deficits in short-term memory and a failure to make associations between specific environmental context and an electric shock. Interestingly, flight exercises partially mitigated the negative effect of the WD as the WDE flies had received significantly fewer shocks in the second trial. This suggests that flight exercise could reverse the impact of metabolic overload and improve cognitive performance. These data are supported by our previous observation on the beneficial effects of flight exercise on fly physiology, reproduction, and behavior31.
One of the critical steps in the described protocol is transferring the flies from the individual vial into the lower chamber. Stressed flies would either enter the upper compartment too quickly or won&#39;t enter at all. The flies that did not enter the upper compartment on the first trial could still enter the upper compartment after being transferred back to an individual vial and given a 5 min break. However, it is better to exclude these flies from the experiment. Fly age is another crucial factor as the negative geotaxis became significantly weaker in 30-day old flies (data not shown). Thus more flies would not enter the upper chamber on the first trial and would be excluded from the experiment.
The advantage of the described assay is that it is a simple behavioral task that produces reproducible data in both D. melanogaster and D. simulans male and female flies. The assay could help the study of basic mechanisms underlying learning and memory impairments resulting from various genetic, pharmacological, and dietary manipulations. Moreover, since the retention trials can be performed at different times after initial training, short-term, intermediate, and long-term memory could be potentially interrogated separately.
There are also potential limitations with this one-trial task, including inter-subject variability and age differences (discussed above). The learning and memory could also be influenced by various factors, including the behavioral state at the time of learning, experimental manipulations, and responses to stress36. Since the US is given in the compartment with a copper grid, the entry of animals is sometimes difficult to assess unless the photo beams and infrared cameras could be installed for video tracking in the future.
Taken together, the assay presented here is a simple, reliable, and reproducible procedure that allows for studying memory mechanisms. It could potentially open new avenues for detailed analysis of memory consolidation in flies, including gene-drug interactions, in both contextual and episodic-like aspects of memory.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/63163">Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning</a>. The Representative Results and Discussion&#160;sections were&#160;updated.
In the Representative Results, the legend for Figure 5 was updated from:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A)&#160;Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable increase in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P&#60;0.05. One-way ANOVA with Tukey&#39;s multiple comparisons test.
to:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A)&#160;Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable decrease in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P&#60;0.05. One-way ANOVA with Tukey&#39;s multiple comparisons test.
In the Discussion, the third paragraph&#160;was updated from:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly higher in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.
to:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly lower&#160;in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/63163">Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning</a>. The Representative Results and Discussion&#160;sections were&#160;updated.

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

Learning and memory is an evolutionarily ancient adaptation mechanism to the environment, conserved from Drosophila (D.) to human1. The fruit fly is a robust model organism to study fundamental principles of learning and memory as it offers a wide range of powerful genetic tools to dissect intrinsic molecular mechanisms2. The pioneering genetic screening studies, which identified rutabaga3, amnesiac4, and dunce5 genes critical for learning and memory2, took advantage of olfactory conditioning as the fruit flies rely on their keen sense of smell to find food, potential mates, and to avoid predators6.
Olfactory conditioning has become a popular paradigm to study the mechanism of learning and memory, thanks to the introduction of olfactory T-maze by Tully and Quinn7,8. Subsequently, other methods to measure various types of learning and memory have been proposed, including visual conditioning9, courtship conditioning10, aversive phototaxis suppression assay11, and wasp-exposure conditioning12. However, most of these assays have a complex setup that must be custom-built at a university workshop or purchased through a vendor. The paradigm described here is based on a simple behavioral assay to study aversive associative learning in flies that can be easily assembled with a few available supplies.
The described paradigm is equivalent to passive (or inhibitory) avoidance behavior in laboratory mice and rats in which animals learn to avoid a compartment where they have previously received electric foot shock13. In murids, the procedure is based on their innate avoidance of bright light and preference for darker areas14. On the first trial, the animal is placed into the bright compartment, from where the animal quickly exits, stepping into a dark compartment, where an electric foot shock is delivered. Usually, a single trial is sufficient to form a solid long-term memory, resulting in significantly increased latency 24 h later. The latency is then used as an index of the ability of the animal to remember the association between the aversive stimulus and the specific environment15.
This work describes an analogous procedure using D. as a model system which offers several advantages over rodent models including cost-effectiveness, larger sample size, the absence of regulatory oversight, and access to powerful genetic tools16,17. The procedure is based on negative geotaxis behavior, which manifests in flies&#39; urge to climb up when they are placed on a vertical surface18. The setup consists of two vertical chambers. On the first trial, a fruit fly is placed into a lower compartment. From there, it usually exits within 3-15 s, stepping into the upper compartment where it receives an electric shock. During a 1 min trial, some flies may occasionally re-enter the upper compartment, which results in an additional electric shock. During the testing phase, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first day indicating that flies formed aversive associative memory about the upper compartment. The latency, number of shocks, and the duration and frequency of grooming bouts are then used to analyze the animal behavior and the ability to form and remember the association between the aversive stimulus and the specific environment. The representative results reveal that exposure to the Western diet (WD) significantly impairs passive avoidance behavior in male flies, suggesting that the WD profoundly impacts the fly&#39;s behavior and cognition. Conversely, flight exercise alleviated the negative effect of the WD, improving passive avoidance behavior.

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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.

Watch this Scientific Journal Video about Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning at JoVE.com

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.

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 ...

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3.6K Views.  East Carolina University. 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.

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

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Neuroscience

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

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 ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

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 ...

Drosophila Courtship Conditioning: A Method to Test Learning and Memory In Flies

Source: Koemans, T. S., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55808/" target="_blank">Drosophila Courtship Conditioning As a Measure of Learning and Memory</a>. J. Vis. Exp. (2017).
This video describes a classical conditioning assay that tests learning and memory in Drosophila, called courtship conditioning. The assay is based on the reduction of male courtship after experiencing a rejection by a non-receptive premated female. The example protocol shows a setup for the procedure that can be used to assess short- and long-term memory.

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

Summary

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Chapters in this video

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila Adult Olfactory Shock Learning

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

Drosophila Courtship Conditioning As a Measure of Learning and Memory

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Drosophila Courtship Conditioning: A Method to Test Learning and Memory In Flies