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 border="0" cellpadding="0" cellspacing="0" width="866">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flywalk setup</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td style="width:207px;">details available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stimulus device</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td>details available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED cluster</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td>details available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HD Pro Webcam C920</td>
			<td style="width:325px;">Logitech, Lausanne, Switzerland</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 Computers</td>
			<td style="width:325px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:216px;">Flywalk Reloaded v1.0 software</td>
			<td style="width:325px;">Electricidade Em P&#243; (electricidadeempo.net)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Labview 11.0 software</td>
			<td style="width:325px;">National Instruments, Austin, TX</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard fly food</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:216px;">Standard fly vials</td>
			<td style="width:325px;">Greiner bio-one GmbH, Frickenhausen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard fly vials</td>
			<td style="width:325px;">Greiner bio-one GmbH, Frickenhausen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">aspirator</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mineral oil</td>
			<td style="width:325px;">Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">odors</td>
			<td style="width:325px;">Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:216px;">200 &#181;l PCR reaction tubes</td>
			<td style="width:325px;">Biozym Scientific GmbH, Oldendorf, Germany</td>
			<td style="width:119px;"></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

9.2K Görüntüleme.  Max Planck Institute for Chemical Ecology.  Otomatik izleme sistemi Flywalk Drosophila melanogaster koku güdümlü davranış yüksek çözünürlüklü ölçümü için kullanılır. 

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

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 ...

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, ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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 ...

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 ...

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 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.

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.

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

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Drosophila Grooming Behavior

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

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

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

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.