We thank Daniel Veit for technical assistance and Pedro Gouveia at Electricidade Em P&#242; (electricidadeempo.net) for customizing the tracking software for our demands. We&#160;also thank Tom Retzke for support during the filming process. This study was supported by the Max Planck Society.

The construction and technical details of Flywalk have been described elsewhere26 (in case of any problems of establishing this set up, further information can be obtained from MK). Here we focus on detailed instructions on the handling of the paradigm that will help to obtain reliable results.
1. Fly Handling
<ol>
	<li>Rear flies in low to medium density cultures on food medium27 under a 12 hr:12 hr light:dark regime at 23-25 &#176;C and 70% relative humidity. To this end, allow 20-30 newly emerged adult flies to reproduce in a big food vial for 1 week, then discard adult flies and wait for offspring to emerge.</li>
	<li>Collect 30-40 newly emerged (age &#60;24 hr) adult flies and age them on in new a vial containing food medium27 for 3-5 days.</li>
	<li>Twenty-four hr before the start of the behavioral experiment: Transfer all 30-40 previously collected 3-5 d old (see 2.2) flies to a new vial containing a moist rubber foam plug or a moist tissue paper using an aspirator. 
	NOTE: Do not anaesthetize flies using CO2.</li>
</ol>
2. Preparation of the Flywalk Setup&#160;
<ol>
	<li>Use 250 ml bottles as humidifiers. Fill humidifiers with 100 ml of distilled water.</li>
	<li>Prepare odor vials.
	<ol>
		<li>Prepare 500 &#181;l 10-3 dilutions of the pure odors ethyl acetate, ethyl butyrate, isopentyl acetate and 2,3-butanedione in the solvent mineral oil.</li>
		<li>Attach two ball check valves per odor vial. Note that check valves allow for uni-directional airflow only. Therefore, connect check valves in such a way that air can enter the vial on one side and leave it on the other side.</li>
		<li>Remove the lid of a 200 &#181;l PCR reaction tube. Pipette 100 &#181;l of every odor dilution into a separate reaction tube and place the tubes in separate odor vials. Also prepare one odor vial containing only the solvent mineral oil.</li>
		<li>Tightly seal the odor vials by closing them using stainless steel plugs and rubber gaskets.</li>
		<li>Connect the 5 odor vials (4 containing odors and 1 containing mineral oil) to the odor delivery system. Make sure to connect them in the right flow direction. A wrong connection will not only compromise the planned experiment, but it may also contaminate the delivery system.&#160;</li>
	</ol>
	</li>
	<li>Check for leaks by sealing the outlet of the mixing chamber of the stimulus device. Make sure, that all airflows before the stimulus device now progressively drop to zero. If not, check for leaks which can now be identified by the hissing sound of air leaving the system.</li>
	<li>Carefully transfer 15 individual flies to 15 individual glass tubes using an aspirator and close glass tubes on both sides using the corresponding adapters.&#160; 
	Note: Because the system must be hermetically sealed for successful experiments, ensure that adapters fit glass tubes tightly and note that glass tubes may break during this step. Take care to avoid injuries by wearing protective gloves and goggles.</li>
	<li>Connect the glass tubes to the Flywalk setup and, from here on, wait for at least 15 min before starting the experiment to allow flies to habituate to the new environment.</li>
	<li>After attaching glass tubes: check the readout of the downstream digital flow meters on computer 1 if the 16 airflows after the glass tubes add up to the airflow entering the system. Also check on computer 1, if humidity is between 60% and 80%.</li>
	<li>Design stimulus protocol controlling the sequence and timing of odor stimuli presented to the flies. To obtain e.g. the described data, present 4 odors and the control (mineral oil) singly and all possible ternary and the quarternary mixtures of the odors simultaneously for 40 times each. Set pulse duration to 500 msec at an interstimulus interval of 90 sec and randomize stimulus sequence.</li>
	<li>Switch on the light source (LED-cluster; &#955; = 630). Make sure to provide enough light for efficient tracking without increasing the temperature inside the glass tubes.</li>
	<li>Set up a region of interest of the tracking system by dragging a frame across the area to be monitored in such a way that all 15 glass tubes are included and approximately 5 mm of the edges of the tubes are excluded.</li>
	<li>Set up 14 parallel separation lines between individual tubes in the tracking system by changing their Y-positions in the corresponding script to keep individual flies identifiable throughout the experiment. Make sure to position them in such a way that there is always one glass tube between two such separator lines, because only one fly will be tracked between any set of two lines.</li>
	<li>Make sure to set camera parameters in such a way that flies are reliably tracked throughout the glass tubes. If flies are lost at the edges of the region of interest, increase brightness or gain of the tracking software. Avoid mechanical vibrations of the tracking system. Track using commercial software according to manufacturer&#39;s protocol.</li>
	<li>Start experiment by starting the stimulus protocol. Record flies&#180; XY-coordinates at 20 fps (frames per sec) and log in combination with the odor valve status in text files.</li>
</ol>
3. Data Analysis
NOTE: The following steps in the data analysis are automatized using custom-written routines programmed in R. Because these steps are crucial to obtain meaningful results the analysis will nevertheless be presented in a step-by-step manner. The raw data for the analysis are .csv-files containing synchronized information on odor valve status, pulse number in the experiment and 15 fly x-positions in cm on a common time axis for one odor stimulation cycle. Custom code for data analysis can be provided upon request.
<ol>
	<li>Open .csv-file, find time-point of valve opening signified by a change in the column representing the valve status.</li>
	<li>Calculate the linear function of odor position of the form 
	f(t) = s*t + i 
	where t is time in the stimulation cycle, s is the wind speed (here 20 cm/sec) and the intercept i can be calculated using the time-point the odor enters the tubes at position 0 (valve opening plus delay).</li>
	<li>Find the time point at which odor and fly x-position intersect for every fly and set this time-point to 0. Note: This way fly positions are aligned to each individual&#180;s encounter with the odor.</li>
	<li>Exclude flies sitting at the very edges of the region of interest.</li>
	<li>Calculate speed from X-positions by dividing displacement along the x-axis by the time interval (100 msec) and repeat procedure for every stimulation cycle.</li>
	<li>To obtain speed time-courses as shown in Figure 2E calculate mean speed time-course for every fly and odor and from those the mean time-course for a given odor.</li>
	<li>To obtain net displacement as shown in Figure 3C calculate the net displacement within 4 sec after the odor pulse for every tracking event and afterwards the mean net displacement per fly and odor.</li>
</ol>
4. Cleaning Procedure
<ol>
	<li>Clean Glass Tubes
	<ol>
		<li>Remove flies and adapters from glass tubes and soak glass tubes in detergent.</li>
		<li>Rinse glass tubes under running distilled water and dry them using pressurized air.</li>
		<li>Heat glass tubes at 200 &#176;C for 8 hr.</li>
	</ol>
	</li>
	<li>Clean Odor Delivery System
	<ol>
		<li>Remove all odor vials and tubing from the central mixing chamber.</li>
		<li>Remove tubing adapters from the mixing chamber.</li>
		<li>Clean mixing chamber by rinsing it with laboratory cleaning solution and solvents (e.g. ethanol, acetone). Perform these steps under the laboratory hood.</li>
		<li>Dry mixing chamber using pressurized air and heat it at 200 &#176;C for 8 hr.</li>
	</ol>
	</li>
	<li>Clean Odor Vials and Check Valves
	<ol>
		<li>Remove steel plug (discard rubber gasket) and check valves from odor vials and soak all components in laboratory cleaning solution.</li>
		<li>Sonicate components in an ultrasound bath and rinse them with distilled water.</li>
		<li>Clean all components except check valves with ethanol and acetone. Perform these steps under the laboratory hood.</li>
		<li>Dry components using pressurized air and heat them at 200 &#176;C for 8 hr.</li>
		<li>Clean check valves on the inside by flushing them with ethanol and acetone using a syringe (consider flow direction). Perform these steps under the laboratory hood wearing laboratory goggles. Because acetone attacks rubber parts, immediately dry check valves by flushing them with pressurized air.</li>
		<li>Remove residual odors by pulsing air through check valves for several days. Use an incubator at 60 &#176;C and a 1 sec air on/1 sec air off regime for this cleaning step.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Although the Flywalk system appears rather sophisticated at first glance, once set up and running it is easy to use and produces very robust results. To stress the consistency of the results produced with the bioassay it may be said that the representative results shown here were obtained almost 2 years after some of the results shown in a previous study29 with a modified setup using a new tracking software and light source. Nevertheless, the attractant responses are - despite slightly higher response amplitud...

The overarching goal of any neuroethological research is to establish a causal link between the activity states of single neurons or neuronal circuits and the behavior of an organism. To achieve this goal neuronal activity and behavior should be monitored under identical stimulus conditions and these stimulus conditions should ideally be similar to those the nervous system under scrutiny evolved to make sense of. Particularly when it comes to behavioral bioassays, these requirements have historically proven quite demanding in Drosophila melanogaster olfactory neuroethology.
Once released from the source, odor plumes rapidly break up into thin filaments with turbulent diffusion caused by air movement being the main determinant of odor distribution1. As a result, an insect navigating towards an odor source experiences intermittent stimulation with odor packages interspersed with variable intervals of clean air. Both walking and flying insects - including Drosophila- have been demonstrated to exploit this intermittent stimulation regime for navigation by surging upwind upon plume encounter and predominantly moving cross-wind in the absence of odors2-5. Whereas stimulation procedures in physiological experiments largely mimic those an insect may experience in its natural environment by either providing single puffs of odors interspersed with extended periods of clean air or dynamic stimulation sequences6-11, many behavioral bioassays used in Drosophila neuroethology such as trap assay, open-field arenas or T-maze rely on odor-gradients12-15. However, because odor gradients by definition are variable in concentration depending on the distance from the odor source, a particular behavior cannot be attributed to a precise odor concentration using these paradigms. In addition, the slope of an odor gradient critically depends on the physicochemical properties of the odorant. A gradient of a highly volatile compound will be shallower than that created by a less volatile compound and therefore also harder to track for an organism relying on measuring concentration differences in space as the only means of navigation16-20, which may lead to a misinterpretation of olfactory preferences particularly in choice assays. This effect is also highly detrimental when investigating behavior towards odor mixtures because it leads to different blend component ratios at every point in space and therefore again precludes a clear correlation between physiology and behavior.
While vinegar flies tend to aggregate on fermenting fruit, they are solitary in their navigation towards food sources and oviposition sites. Nevertheless, rather than testing individual animals many behavioral paradigms used in Drosophila neuroethology examine the odor-guided behavior of cohorts of flies and attraction is scored as the fraction of flies choosing the odor over a control stimulus. These cohort experiments have contributed greatly to the understanding of fly neuroethology and many of the observations made by using them could be confirmed in single-fly experiments. However, it has been observed that flies can influence each other&#180;s decision21 and in extreme cases the evaluation of an odor can switch from indifference to avoidance depending on population density22. Additionally, results from these kinds of experiments often provide only the endpoint of a sequence of behavioral decisions rather than observing what the fly is doing while it is doing it, which would be desirable when attempting to correlate behavior with neuronal activity. These rather low-resolution cohort experiments are contrasted by high-resolution single-fly methods such as tethered flight arenas and treadmills which allow for a direct observation of behavioral responses at the time the stimulus is presented20,23,24. Nevertheless, cohort experiments are still popular, because they are very efficient and provide robust results even at comparably low sample sizes because inter-individual and inter-trial variability are partially averaged out due to the observation of populations over extended periods of time. While tethered flight and treadmill probably provide the gold standard concerning stimulus presentation and temporal resolution, the arenas used are designed for single animals and it is therefore time-consuming to obtain sample sizes necessary for a statistical analysis. Several other approaches have recently been developed that allow an efficient acquisition of high-resolution behavioral data in combination with a well-defined stimulus regime. These include unsupervised 3D-tracking of multiple vinegar flies in a windtunnel in combination with an accurate 3D-model of the odor plume5, tracking of multiple individual flies in choice chambers supplied with airstreams from both sides25 and the Flywalk paradigm26.
In Flywalk, 15 individual flies are situated in small glass tubes and continuously monitored by an overhead camera under red-light conditions. Odors are added to a continuous airstream of 20 cm/sec and travel through the glass tubes at a constant speed. The airstream is humidified by passing it through 250 ml bottles containing distilled water (humidifiers) before entering the odor delivery system. The flies&#180; positions are recorded within a square region of interest (ROI) encompassing most of the length of the odor tubes (but excluding the outer edges of the tubes (approximately 5 mm at each side) where the flies cannot move further up- or downwind) around the time of odor presentation (Figure 1A, B). Fly identities are kept constant by the tracking system throughout the experiment on the basis of their Y-positions (i.e. their glass tube limits). Odor stimulation is achieved using a multi-component stimulus device which allows the presentation of up to 8 single odors and all possible mixtures thereof26,29 (Figure 1B). The course of an experiment is controlled by a computer regulating the odor delivery system and collecting temperature and humidity information (computer 1, Figure 1C). This computer also controls a datalogger (start/stop recording) on a second computer which continuously tracks fly positions at 20 frames per sec (computer 2). Fly positions, odor valve status (i.e. time-point of valve opening), odor ID, temperature and humidity around odor stimulation cycles are logged on computer 2. This way information on odor and fly positions are synchronized and exported as .csv-files which can be further processed and analyzed using custom-written analysis routines. Because the whole system is computer-controlled, no human intervention is necessary during an experimental session.

<table><tbody><tr><td>Flywalk setup</td><td>Custom</td><td></td><td>details available upon request</td></tr><tr><td>stimulus device</td><td>Custom</td><td></td><td>details available upon request</td></tr><tr><td>LED cluster</td><td>Custom</td><td></td><td>details available upon request</td></tr><tr><td>HD Pro Webcam C920</td><td>Logitech, Lausanne, Switzerland</td><td></td><td></td></tr><tr><td>2 Computers</td><td></td><td></td><td></td></tr><tr><td>Flywalk Reloaded v1.0 software</td><td>Electricidade Em P&amp;oacute; (electricidadeempo.net)</td><td></td><td></td></tr><tr><td>Labview 11.0 software</td><td>National Instruments, Austin, TX</td><td></td><td></td></tr><tr><td>Standard fly food</td><td>Custom</td><td></td><td></td></tr><tr><td>Standard fly vials</td><td>Greiner bio-one GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Standard fly vials</td><td>Greiner bio-one GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>aspirator</td><td>Custom</td><td></td><td></td></tr><tr><td>mineral oil</td><td>Sigma-Aldrich (www.sigmaaldrich.com)</td><td></td><td></td></tr><tr><td>odors</td><td>Sigma-Aldrich (www.sigmaaldrich.com)</td><td></td><td></td></tr><tr><td>200 &amp;micro;l PCR reaction tubes</td><td>Biozym Scientific GmbH, Oldendorf, Germany</td><td></td><td></td></tr></tbody></table>

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.

Because flies are allowed to distribute freely within their glass tubes between odor pulses and the odor pulse travels through the glass tubes at a constant speed flies encounter the odor at different times depending on their x-position at the time of stimulation. As a result, the onsets of the upwind trajectories evoked by a 500 msec pulse of an attractive 10-3 dilution of ethyl acetate are delayed by about 1 sec for flies at the downwind end of their glass tubes compared to those of flies sitting closer to t...

Watch this Scientific Journal Video about High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm at JoVE.com

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.

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 ...

high-resolution-quantification-odor-guided-behavior-drosophila

Research

JoVE Journal

Neuroscience

9.3K Views.  Max Planck Institute for Chemical Ecology.  De automatische tracking systeem Flywalk wordt gebruikt voor hoge-resolutie kwantificering van geur-begeleide gedrag in Drosophila melanogaster. 

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

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 ...

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.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://www.jove.com/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

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

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