Name: a simple way to measure alterations in reward-seeking behavior using drosophila melanogaster
Uploaded: 2016-12-15T13:00:00
Description: Watch this Scientific Journal Video about A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster at JoVE.com

We thank U. Heberlein and A. Devineni for long-lasting discussions and technical advice. We also thank the Shohat-Ophir lab members, A. Benzur, L. Kazaz, and O. Shalom, for the help with demonstrating the method. Special appreciation goes to Eliezer Costi for establishing the fly systems in the lab. This work was supported by the Israel Science Foundation (384/14) and the Marie Curie Career Integration Grants (CIG 631127).

Note: General overview of the experimental design: The experimental design includes an adapted protocol for courtship suppression7-9 in which male flies are exposed to rewarding and nonrewarding experiences in 3 consecutive training sessions over the course of 4 d. At the end of the experience phase, the flies are tested in a two-choice voluntary ethanol consumption assay for 3&#160;- 4 d. The protocol herein includes several preparatory steps, some of which can be done in advance to be used in more than one experiment, while othe...

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R., Azanchi, R., Maung, Z., Hirsh, J., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+alcohol+reward.">A Drosophila model for alcohol reward.</a> Nat Neurosci. 14, 612-619 (2011).</li><li>Xu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+propensity+for+consuming+ethanol+in+Drosophila+requires+rutabaga+adenylyl+cyclase+expression+within+mushroom+body+neurons.">The propensity for consuming ethanol in Drosophila requires rutabaga adenylyl cyclase expression within mushroom body neurons.</a> Genes Brain Behav. 11, 727-739 (2012).</li><li>Devineni, A. 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Here, we illustrate the details of an integrated approach to measure alterations in reward-seeking behavior, based on previous work described by Devineni et al.6 and Shohat-Ophir et al5. The first section of the protocol uses different types of sexual interactions as the experience input, and the second section uses a two-choice feeding assay to assess the effect of experience on the preference to consume ethanol.
As shown by Devineni et al.<sup...

Modification of behavior in response to experience allows animals to adjust their behavior to changes in their environment1. During this process, animals integrate their internal physiological state with the changing conditions of the external environment and subsequently choose one action over another to increase their chances of survival and reproduction. Reward systems evolved to motivate behaviors that are required for the survival of individuals and species by reinforcing behaviors that enhance immediate survival, such as eating or drinking, or those that ensure long-term survival, such as sexual behavior or caring for offspring2. Artificial compounds such as drugs of abuse also affect reward systems by co-opting neural pathways that mediate natural rewards2.
During the last two decades, the fruit fly Drosophila melanogaster has been established as a promising model for studying the molecular and neuronal mechanisms that are shaping the effects of ethanol on behavior3,4.
Previously, we have identified a subset of peptidergic neurons in flies (NPF/NPF receptor (R) neurons) that couple natural rewards, such as sexual experience, to the motivation of obtaining drug rewards5. NPF expression is sensitive to both sexual experiences and to drug rewards, such as ethanol intoxication. Changes in NPF expression levels are converted to alterations in ethanol self-administration5, where high NPF reduces and low NPF increases the preference to consume ethanol. Activating NPF neurons is rewarding for flies, as they display strong preference for an odor paired with the activation, which is also reflected by reduced ethanol consumption. More importantly, activation of NPF neurons interferes with the ability of flies to form a positive association between ethanol intoxication and an odor cue. The causal link between the NPF/R system, reward memory, and ethanol consumption suggests that one can use ethanol self-administration as a measure for changes in reward states5.
In this publication we demonstrate an integrated approach for tapping into the fly natural reward system and assaying changes in reward states. The approach consists of two separate parts, a training protocol for manipulating natural reward-related experiences, followed by a two-choice capillary feeder assay (CAFE) to assess ethanol self-administration as an estimate for changes in reward states. The CAFE assay is analogous to the two-bottle choice assays used in rodent studies for drug self-administration and has been shown to reflect certain properties of addiction-like behavior in flies6.

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a simple way to measure alterations in reward-seeking behavior using drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

FPreviously, Devineni et al. showed that when fruit flies are given the choice to consume ethanol-containing food, they display a strong preference for ethanol-containing food over nonethanol containing food6. Shown here are some representative results we obtained when assaying the innate ethanol preference of na&#239;ve male flies that did not undergo the training protocol.
Na&#239;ve Canton S male flies wer...

Watch this Scientific Journal Video about A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster at JoVE.com

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The overall goal of this procedure is to measure ethanol self-administration in fruit flies as a proxy for changes in rewards states. This method can help answer key questions in neuroscience, such as, how experience is encoded in the brain, and leads to modification of behavior. The main advantage of this technique is that it is relatively simple, and does not require a complicated setup.The protocol is composed of two discrete parts. The first is exposing the flies to rewarding and non-rewarding experiences, and the second is assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts of the protocol can be used independently.To induce the modulation of experience as an initial step for further downstream assays, or, as an independent, two-choice feeding assay. Demonstrating the procedure will be Shir Zer, a master's student, and Julia Ryvkin, a graduate student, both from my lab. To begin the construction of the capillary feeder, first take a standard fly vial with small holes along the side of the vial.Use a razor blade to cut the plug of the vial in half, crosswise. Mark four points onto the surface of the plug, creating a square shape to position the capillary holders. With an 18-gauge needle, make four holes through the plug at the position of the marks.And make the holes wider using a micropipette. Using a razor blade, cut four micropipettes so that they firmly hold five microliter glass capillaries. Insert these into the holes in the prepared plug.Mark the position on the plug of the two capillaries that will contain ethanol. Next, arrange glass vials in microcentrifuge racks. Melt fly food, and then pour two milliliters into each of the vials.Allow the food to harden, and then cover the vials with plastic wrap. Begin by taking male flies that have been housed separately in smaller vials in seclusion. Assign these to two groups randomly.Those who will undergo rewarding, versus non-rewarding mating experiences. Beginning with the non-rewarding group, add a previously mated female to each vial. Moving on to the reward group, add a virgin female to each vial.Watch the mating encounters to ensure that males interacting with virgin females mate successfully within the first hour. And that males interacting with previously mated females do not mate. It is essential to observe carefully, to make sure that the mated cohort do indeed undergo mating, and that the rejected cohort do not manage to mate.After one hour, remove from the experiment any males from the rejected cohort that managed to mate, and males from the mated cohort that did not end up mating by the end of the first training session. Conclude the conditioning session by aspirating out the previously mated females from the non-rewarding condition, keeping these individuals for later trials. Finally, remove and discard the females from the mated male group.Let the flies rest for one hour, and then repeat the hour long conditioning assay two further times with an hour rest in between. These training sessions should be repeated again each day for three more days. After the final conditioning session on day four, aspirate the single males and place them in groups of six to eight into the capillary feeder vials, keeping flies in the same reward group together.Use a soft plug to keep the males in place during transfer. Once all flies are inserted, replace the soft plug with the capillary feeder plug. Wet the plugs by gently adding five milliliters of water to the top of each plug, avoiding spillage into the vial.Prepare the ethanol and water food solutions, and then place five microliter drops of these onto a strip of laboratory film. Allow the solution to fill the capillary until it reaches the five microliter mark. And then tap them gently on the strip to position the solution exactly at the premarked line.Seal the ends of the capillaries by dipping them into small droplets of mineral oil. Finally, insert the two ethanol containing and non-ethanol containing capillaries into the adapters in the plug. The exposed sides should protrude one millimeter or less from the plug surface.Keeping the opening for the capillaries close to the wet plug reduces the evaporation and allows easy access to the food, as flies normally sit on the plugs and climb down on the exposed capillary to feed. Additionally, prepare one mock vial without flies, which will be used as a control to assess natural evaporation. Finally, record the relative position of the liquid in each of the capillaries in vials with respect to the black line in millimeters.Set the vials into the experimental incubator for 24 hours. Measure and record the levels of the liquid in each removed capillary with respect to the black line. Once the experiment is in progress, replace the capillary at a set time, each day.Re-dampen the plugs with two milliliters of water, and then set the vials back into the incubator. Wild type Canton S strain male fruit flies collected upon eclosion and aged until four days old, were assayed over the course of four days for their innate preference to consume alcohol. Analysis of volumes consumed from ethanol and non-ethanol containing solutions, show that the flies exhibited a significant preference for the 15%ethanol food source.The CAFE experiment was repeated with mated versus rejected males. Both groups had been previously housed singly, and exposed to females that either accepted or rejected mating. Upon introduction in groups to the CAFE vials, the rejected group consumed more from the ethanol capillaries.This suggested that perceived lack of reward led to an increase in reward-seeking behavior. Conversely, successful mating increases internal reward levels, which in turn lowers ethanol consumption. While attempting this procedure, it's important to maintain a well-humidified environment for the entire experiment, with extra emphasis on the consumption part, watering the plugs every day, and keeping the capillary ends in close proximity to the wet plugs.

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Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

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 ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Conditions Affecting Social Space in Drosophila melanogaster

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

Conditions Affecting Social Space in Drosophila melanogaster

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

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

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.