The authors want to thank Dr. Tadmiri Venkatesh for his encouragement to prepare this manuscript, Dova Brenman and Selwyn Chui for their constructive comments, Elyssa Burg and Wayne Rasband for designing the macro in ImageJ for all interfly distances and for nearest neighbor distances. The authors also want to thank the reviewers for their constructive comments.
A.A.A, M.C.C. and A.F.S were responsible for research design; S.N.J., A.A.A, M.N., Z.R., and A.J.M. performed the experiments. A.A.A, M.N., A.J.M. and M.C.C and A.F.S. analyzed the data; A.R.M. and A.F.S. wrote the manuscript.
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S.; by a Mathematics, Biology, Chemistry, and Geology majors scholarship for teachers-in-training and by a Louis Stokes Alliance for Minority Participation scholarship to A.A.A.

1. Equipment and Reagents Created In-house (See List of Materials for Others)
<ol>
	<li>Prepare a Drosophila cold anesthesia apparatus as previously described 8.</li>
	<li>Prepare a fly aspirator as previously described 13.</li>
	<li>Prepare social space chambers and holders.
	<ol>
		<li>Order or make glass panes and acrylic spacers to create the social space chambers. Each social space chamber consists of two square glass panes (17.6 cm by 17.6 cm, with a thickness of 0.3 cm), two right triangle acrylic spacers (with a height of 16.5 cm, a base of 8.9 cm, with a thickness of 0.3 cm), and two rectangular spaces (9 cm by 1.5 cm with a thickness of 0.3 cm) (Figure 1A).</li>
		<li>Assemble the glass and acrylic social space chamber such that it is identical to the chamber in Figure 1B. To do so, start by placing one triangular spacer flat on top of one square pane of glass such that the right angle of the triangular spacer is aligned with one of the corners of the square pane. Next, place the second triangular spacer flat on top of the square pane, mirroring the first triangular spacer. 
		NOTE: Make sure to wear gloves so as not to contaminate the pieces with oils and scents from your hands.</li>
		<li>Place two small rectangular spacers flat on top of the square pane, along the side of the pane that is not covered. The acrylic spacers on top of the square plane should now form a triangular arena.</li>
		<li>Place a second square pane of glass on top of the acrylic spacers such that it is aligned above the original pane of glass below.</li>
		<li>Use four binder clips to secure the panes and spacers together to form the social space chamber. Place one clip near the corner, over the long side of each of the triangular spacers, securing them inside the glass panes. On these same sides, place a clip in the adjacent corner, over each of the rectangular spacers, securing them inside the glass panes.</li>
		<li>Assemble a support stand and a flask clamp such that the clamp will maintain the social space chamber in an upright position, as the social space chamber rests on its edge on top of the work surface.</li>
		<li>For each repeat in the experiment, assemble a social space chamber and support stand.</li>
	</ol>
	</li>
	<li>Prepare a climbing assay response apparatus to check the climbing ability of the flies tested.
	<ol>
		<li>Use a countercurrent apparatus, as previously described 14,15.</li>
	</ol>
	</li>
	<li>Ensure homogeneous lighting conditions by performing the experiment on a work surface covered with a white bench cover and in front of a white background.</li>
</ol>
2. Preparing the Flies before the Experiment
<ol>
	<li>Maintain flies in bottles containing standard Drosophila fly food. Keep them in an incubation chamber at 25 &#176;C on a 12 hr light/dark cycle.</li>
	<li>One to two days prior to the experiment, collect and sex flies under cold anesthesia, as previously described 8.
	<ol>
		<li>Chill the cold anesthesia apparatus at -4 &#176;C. Place flies into 50 ml plastic tubes. Fully submerge the plastic tubes containing flies into ice in an insulated ice bucket, wait at least 5 min for flies to become immobile.</li>
		<li>Place the flies on the polyethylene sheet of the cold anesthesia apparatus. At RT, use a stereomicroscope to separate males from females. Maintain flies, in groups, in vials containing standard Drosophila fly food, not exceeding 40 flies per vial.</li>
	</ol>
	</li>
	<li>Two hours before the beginning of the experiment, ensure that the temperature of the room where the experiment will be performed is between 24&#8211;25 &#176;C and the humidity is approximately 50%.</li>
	<li>Transfer the flies into new vials containing food, and place them for 2 hr on the work surface where the experiment will be performed. 
	NOTE: For these experiments, the fly strains used were Drosophila melanogaster strains: Canton-Special or Canton-S (CS), w1118Cs10 (or W - w1118 outcrossed 10 times to Canton-S), Oregon and Samarkand flies were all from our laboratory stocks 16; Elwood flies were collected in Fall 2011 in the Elwood neighborhood of Huntington, on Long Island, New York, USA 8. Apart when specified otherwise, the results presented were obtained with Canton-S flies (in shades of red).</li>
</ol>
3. Performing the Experiment
<ol>
	<li>Perform the experiment between 12 p.m. and 3 p.m. (Zeitgeber time &#8212; the time in hours after the onset of light &#8212; of ZT 4 to 7).</li>
	<li>Prepare the social space chamber for the transfer of flies.
	<ol>
		<li>Place a social space chamber flat on a work surface, with the side containing rectangular spacers closest to your body.</li>
		<li>Remove one clip nearest your body and slide one rectangular spacer outwards, creating a gap of approximately 1 cm between the rectangular spacers.</li>
		<li>Using tape and a marker, label the social space chamber in an upper corner with sex, strain, and repeat number. Take care to ensure the tape does not cover part of the inner triangular arena.</li>
	</ol>
	</li>
	<li>Transfer the flies into the social space chamber.
	<ol>
		<li>Transfer flies from their vial containing food into a new, empty vial. Aspirate flies from the empty vial and transfer them into the social space chamber.
		<ol>
			<li>Inhale to draw the flies into the tip of the aspirator. Place the tip into the 1 cm gap between the rectangular spacers of the social space chamber and exhale at a consistent rate to force the flies into the inner triangular arena.</li>
			<li>Immediately slide the rectangular spacer back into place, closing the base of the inner triangular arena, and place the binder clip back on.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>On a pounding pad, which is located on different bench than where the experimental work takes place, hold the social space chamber upright such that the rectangular spacers are on the bottom side. Pound three times to ensure all flies have fallen to the bottom of the arena. 
	NOTE: Due to the acrylic spacer sticking out of the smallest chamber size (Figure 1D), bang elbows on work surface while maintaining a secure hold on apparatus to ensure the flies have fallen to the bottom.</li>
	<li>Start a timer.</li>
	<li>Place the social space chamber on the work surface and use the support stand and flask clamp to keep it upright. Place a ruler or a sticker of known length flat against the social space chamber, but not covering any part of the inner triangular arena.
	<ol>
		<li>For horizontally positioned experiments, do not place the social space chamber in the support stand; instead lay the social space chamber flat on the work surface.</li>
	</ol>
	</li>
	<li>When the flies have settled, usually after around 30 min, take a picture of the social space chamber. Ensure that the frame contains the entire inner triangular arena, the ruler, and the label.</li>
	<li>Repeat the experiment for each genotype and condition (ideally 3 internal replicates and 3 independent repeats).</li>
</ol>
4. Analyzing the Social Space Data
<ol>
	<li>Import the following Macros in ImageJ 17
	<ol>
		<li>ImageJ is available here: http://rsbweb.nih.gov/ij/.</li>
		<li>You can create a &#39;Measure Distances&#39; command in the Plugins menu by saving the macros below in a file named &#39;Measure_Distances.txt&#39; in the ImageJ/plugins folder, in the macro subfolder, and using the Help&#62;Update Menus command.&#39;</li>
		<li>Copy in a .txt file the macros that are provided in supplementary data and were originally published in 3.</li>
	</ol>
	</li>
	<li>Upload the pictures to a computer, and open one picture using ImageJ.
	<ol>
		<li>Create a scale by drawing a line from the 0 cm to 1 cm marks on the ruler in the picture, and under the Analyze tab, choose the Set Scale feature to set a scale of 1 cm for that distance (same approach for the sticker of known length). Choose the Global option before applying the scale to the picture.</li>
		<li>Crop the picture using the Crop feature under the Image tab such that it includes all of the flies, while removing as much of the rest of the picture as possible.</li>
		<li>Make the picture black and white by choosing 8-bit from the Type option under the Image tab.</li>
		<li>Remove all background noise from the picture by going to the Threshold feature under the Adjust option of the Image tab. Drag the sliders to enhance or remove the contrast so that the body of each fly can be clearly seen without any other markings in the image. If there are markings that are not flies, capture them with the rectangular tool and delete them.</li>
		<li>Set the measurements by using the Set Measurements tool under the Analyze tab. Select Area, Center of Mass, Centroid, and Display Label. Next, choose the Analyze Particles feature under the Analyze tab to create a numbered list of all the black spots representing flies.
		<ol>
			<li>When using Analyze particles; set size 0.01-0.1 (do NOT choose pixel unit); circularity 0.00-1.00; show outlines; display results, and add to manager.</li>
			<li>Ensure that each black spot (each particle) is an accurate representation of the flies that are in the original picture, by comparing the numbered list to the original picture. 
			NOTE: If some flies that are very close together being counted as one particle, manually draw a white line to separate the two flies on the binary image.</li>
		</ol>
		</li>
		<li>While the list is selected, use the Nearest Neighbor &#8212; List Distances macro under the second Macros option in the Plugins tab to create a new list containing the distances, in cm, from each fly to their nearest neighbor.</li>
	</ol>
	</li>
	<li>Copy the nearest neighbor distances from ImageJ and paste into a column of a spreadsheet program.
	<ol>
		<li>Compile all of the data from each repeat onto the same spreadsheet and organize by genotype and condition.</li>
	</ol>
	</li>
	<li>Perform data analysis using statistical software. In this experiment, Graph Pad Prism (version 6 for MacOSX, GraphPad Software, San Diego California USA, www.graphpad.com) was used to conduct one-way ANOVA, and Kruskal-Wallis Tests, Tukey&#8217;s and Dunnet&#8217;s Post-Hoc Tests.</li>
	<li>The distribution of the distances follows a non parametric distribution 1, and data are represented as box and Tukey&#8217;s whiskers (to eliminate outliers).</li>
</ol>
5. Climbing Assay (Control Behavior)
<ol>
	<li>Use a climbing assay as a control to test flies climbing ability 14,15,18.
	<ol>
		<li>Use the countercurrent apparatus such that three trials can be run in parallel.</li>
	</ol>
	</li>
	<li>Transfer 50 to 100 na&#239;ve flies (flies that have not been tested before) in three different test vials, and snap them into the 1st, 3rd and last slots at the bottom of countercurrent apparatus, with fresh empty vials in the opposite locations.</li>
	<li>Place fresh, empty vials in the empty slots at the bottom of the apparatus.</li>
	<li>Tap down the apparatus three times, such that all the flies are at the bottom of each tube, to initiate the experiment.</li>
	<li>Start the timer for 15 sec (time sufficient for ~100% of 3&#8211;7 days old Canton-S to reach the top vial &#8212; see Figure&#160;3F).</li>
	<li>Slide the upper portion of the counter-current apparatus, to displace the top vials by one slot.</li>
	<li>Collect flies by tapping the apparatus down, to bring all flies in the bottom vials.
	<ol>
		<li>1st and 2nd tubes (or 3rd and 4th, 5th and 6th) correspond respectively to the flies that did not reach to top and the flies that reached the top vial.</li>
	</ol>
	</li>
	<li>Calculate the Performance Index (PI).
	<ol>
		<li>Count the number of flies in each vial.</li>
		<li>The PI is the percentage of flies able to climb on the upper tube.</li>
		<li>The means of the PI follow a normal distribution1, and data are represented as column graphs of mean plus or minus standard error to the mean (SEM). 
		NOTE: Alternatively, the PI can be calculated as the percentage of flies able to reach the upper part of the lower vial (for older flies, 5 sec is the time sufficient to quantify a difference in climbing ability between infected and non infected 12 days old Canton-S quantified from a still picture: see results). Others have used different measures of performance in startle-induced negative geotaxis, that are also appropriate to quantify climbing ability 19-22.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

In this protocol, a detailed procedure for the quantification of social space was described. Some crucial steps to ensure the experiment is successful are: 1) always use gloves when cleaning and setting up the apparatus, to keep your own oils and scents off the inner chamber of the apparatus, 2) ensure flies are collected at least one day before the experiment to reduce any potential effects of cold anesthesia, 3) 2 hr before the experiment provide new vials containing fresh food to ensure the flies are not starved, and ...

Social interactions are crucial to the proper development and health of individuals within a group as a whole, and can be observed across numerous species, from humans (Homo sapiens) to simpler organisms such as fruit flies (Drosophila melanogaster) 5,6. An individual fruit fly or human share common means to process sensory information during these interactions, whether it be: auditory, visual, olfactory, tactile, or gustation. We and others hypothesize that there is a potentially shared neurocircuitry underlying behavioral responses to social interactions and that the neuronal cells and genes involved might be evolutionary conserved 7. Once the initial interaction has occurred, social space between the interacting individuals will either increase (social avoidance 8) or decrease (group formation/aggregation 5). More complicated interactions, like aggression or courtship, can then take place.
Neither sophisticated tools and methods, nor large investments in time and training are required to quantify this simple form of social behavior, making it a powerful analytical tool. Here, we explain a straightforward protocol that quantifies inter-fly distance, or social space, to assess social interaction in stable groups of Drosophila melanogaster, as used in the following studies 1-4,9. Social space refers to a measure of the distance between a fly and its closest neighbor 10. Social space is consistent for a given population of D. melanogaster when experimental conditions are preserved (averaging approximately within 1-2 body lengths), and varies with respect to the social experience of the flies, increasing if the individual has been kept in isolation 1. Proper vision is necessary to maintain normal social distance, but not classical odorants or cVA perception 1. Measure of social space can thus be used as a diagnostic tool to analyze social interactions and quantify social behavior in D. melanogaster 1. We describe here in details how to perform this quantification, and to what extent common experimental variables affect this behavior.
We show that the orientation of the chamber in which the assay is performed, as well as the number of flies &#8212; to an extent &#8212; do affects social space. It was previously shown that chamber geometry affects spontaneous exploratory movement of flies 11,12, and this phenomena may ultimately impact where they decide to settle. However, as long as the fly density (fly / cm2) and chamber orientation is kept the same, the social space of the flies also remains constant. The robustness of this assay is illustrated by the fact that independent laboratories using different chamber sizes, shape, and orientation can replicate the result displayed by mutants of the white gene (affecting eye pigmentation), which is an increase social space (vertical triangle or horizontal circle in 1, horizontal square with airflow in 3).
Our results also indicate that maintaining the time at which the social space experiment is performed is crucial to the consistency of the results, as we show that males, but not females, are further apart in the evenings. However, the differences seen between daytime and evening hours are not due to activity differences of the flies, and we discuss arguments indicating that activity levels are not correlated with social space.
Finally, there are genetic underpinnings to the determination of social space, as indicated by the white mutant already described 1,3, and the differences between various inbred and wild-caught strains of flies that we present here.
Therefore, this assay makes an excellent tool for studying the effects of genetic as well as environmental factors.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope&#160;</td>
			<td>Nikon&#160;&#160;</td>
			<td>SMZ-645</td>
			<td>Any other standard scope for fly handling would work</td>
		</tr>
		<tr>
			<td>Small paint brushes&#160;</td>
			<td></td>
			<td></td>
			<td>for pushing flies</td>
		</tr>
		<tr>
			<td>Jazz-mix</td>
			<td>Fisher</td>
			<td>33545</td>
			<td>any other standard drosophila food would work</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Alarm Timer/Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sharpie pens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium size binder clips</td>
			<td>Staples</td>
			<td></td>
			<td>to hold the chambers together: 1-1/4&#34; (32mm) medium clips with 5/8&#34; capacity</td>
		</tr>
		<tr>
			<td>small SupportStands</td>
			<td>Carolina&#160;</td>
			<td>707161</td>
			<td rowspan="2">to hold the chambers in a vertical orientation</td>
		</tr>
		<tr>
			<td>Buret clamps</td>
			<td>Carolina</td>
			<td>707362</td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Nikon</td>
			<td>&#160;Coolpix S8000&#160;</td>
			<td>to take the still pictures</td>
		</tr>
		<tr>
			<td>small ruler</td>
			<td></td>
			<td></td>
			<td>to be able to scale the picture</td>
		</tr>
		<tr>
			<td>trifold board and white bench cover</td>
			<td></td>
			<td></td>
			<td>to provide a white background, and a homogeneous light.</td>
		</tr>
		<tr>
			<td>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 6</td>
			<td>GraphPad Software Inc.</td>
			<td>Prism 6 for Mac OS X</td>
			<td>Any statistical analysis software with t-test, one-way and two ANOVA would work</td>
		</tr>
	</tbody>
</table>

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.

The social space chamber can be used as a tool to quantify the social behavior of Drosophila melanogaster. Acrylic spacers and glass panes are clipped together to form an inner triangular arena that provides a two-dimensional area in which flies can form stable groups without the presence of many potentially confounding cues. When flies are transferred to the vertical arena, they are startled by being tapped down, and they respond by an escape behavior: negative geotaxis. They climb to the vertex of the upright ...

Watch this Scientific Journal Video about Conditions Affecting Social Space in Drosophila melanogaster at JoVE.com

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.

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 ...

conditions-affecting-social-space-in-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

12.1K Views.  University of Western Ontario. 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.

Video: Conditions Affecting Social Space in Drosophila melanogaster

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 ...

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

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

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

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

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 ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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

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

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.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

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

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

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

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