Name: Measuring and Altering Mating Drive in Male Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2017-02-15T14:00:00.000+00:00
Duration: 7 min 2 s
Description: Learn to measure and manipulate male Drosophila melanogaster mating drive using a high-throughput behavioral assay. This method reveals genetic and neural mechanisms underlying motivation in a reductionist system.

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5 minutes

The authors thank Mike Crickmore, Dragana Rogulja, and Michelle Frank for comments on the manuscript. Pavel Gorelik provided technical support for manufacturing the behavioral arenas. This work was conducted in Mike Crickmore's lab and is also supported by the Whitehall Foundation (Principal Investigator: Dragana Rogulja). S.X.Z. is a Stuart H.Q. and Victoria Quan Fellow at Harvard Medical School.

NOTE: This protocol describes the preparation (Sections 1 - 3), execution (Section 4), and analysis (Section 4) of 2-D satiety assays. Then, using dopaminergic stimulation as an example, Section 5 shows how to combine thermogenetic stimulation with 2-D satiety assays to induce hypersexuality. Section 6 describes 3 ways to verify the results of 2-D satiety assays. Finally, Section 7 shows how to measure the recovery of mating drive in male flies.
1. Fabricating 8- and 32-chamber Behavioral Arenas
NOTE: Each behavioral arena consists of several layers of laser-cut plastic sheets held together by hex screws and thumb nuts at each of the four corners (Figure 1A).
<ol>
	<li>Fabricate the layers of the arena. Fabricate each layer using a laser cutter to cut sheets of acrylic plastic to the appropriate shape (see Figure 1B, 1C for products). 
	NOTE: Many research institutions have laser cutters in their machine shops or maker spaces. If the institution has access to a laser cutter, continue with the following steps. If not, then follow the instructions in Step 1.2 instead.
	<ol>
		<li>For each layer, open the design pattern in the laser cutter&#39;s software. 
		NOTE: The designs can be found in appropriately named PDF files (e.g., 8-Chamber Arena - Layer 2 - Doors.pdf) in Supplemental Material 1. This file also contains annotations for the type of plastic to be used (e.g., 1/8 inch (i.e.&#160;3.175 mm) clear acrylic). The exact thickness of each layer is not critical. The main requirement is to allow 0.12 inches (3 mm) or more of free vertical space in the chambers.</li>
		<li>Adjust the laser settings (power, focus, etc.) for the type and thickness of plastic to be used. Place plastic in the laser cutter bed and run the laser cutter. 
		NOTE: These settings will vary widely based on the laser cutter model and the strength of its laser. Find appropriate settings based on past experience or by making test cuts on scrap plastic. The deep hexagonal engravings are included in the design to recess the hex screw heads. This feature allows for one-handed tightening of the thumb nuts and allows the arenas to rest flat on the bench (without protruding screw heads). Nevertheless, making deep engravings is often hard to achieve, and time-consuming, using a laser cutter. This step is optional and can be skipped without affecting the behavioral results.</li>
		<li>When cutting the door frame (Layer 2, Figure 1A), also use appropriate laser settings for engraving the chamber numbers included in the design (as in Figure 1B, 1C).</li>
	</ol>
	</li>
	<li>Place an order with an online laser cutting fabricator. Upload all the PDF design files for the 8- and/or 32-chamber arena and specify the material type (e.g., 1/8 inch (i.e.&#160;3.175 mm) clear acrylic) based on the annotations in the files. The results will appear as in Figures 1B (8-chamber arena) and&#160;1C (32-chamber arena).</li>
	<li>Assemble arenas using four hex screws and four thumb nuts to align and hold together the plastic layers (Figure 1A). The arenas will appear as in Figure 1D, 1E (8-chamber) and Figure 1F, 1G (32-chamber). 
	NOTE: The food wall (Layer 4 in Figure 1A) is used for 2-D satiety assays only and is omitted in 32-chamber arenas, which are used in courtship assays.</li>
</ol>
2. Food Preparation for 2-D Satiety Assay
NOTE: Because a 2-D satiety assay spans 4.5 h, fly food is used in the arena to provide nutrition and water. This protocol uses, but is not restricted to, the conventional cornmeal-agar fly food.
<ol>
	<li>Partially assemble the 2-D satiety arena (8-chamber) with Layers 3 - 6 (see Figure 1A for layer assignments) and tighten it with thumb nuts and hex screws.</li>
	<li>Place fresh fly food into a clean, microwave-safe bottle. Add just enough water to cover the bottom surface of the bottle (Figure 2A).</li>
	<li>Microwave food on high for 30 - 45 s. Check progress and stir every 15 s until melted (Figure 2B). CAUTION! Fly food easily boils over.</li>
	<li>Use a blunted 1,000-&#181;L pipette tip (Figure 2C) to slowly transfer ~1 mL melted food into each chamber (Figure 2D).</li>
	<li>Allow food to resolidify at 4 &#176;C for ~10 min (Figure 2E) before completely assembling the arena (Figure 2F).</li>
	<li>Use the completed arena for experiments (see Section 4) or store at 4 &#176;C.</li>
	<li>Store the leftover food at 4 &#176;C and reuse.</li>
</ol>
3. Preparing Virgin Females for Behavioral Assays
NOTE: 2-D satiety assays use large numbers of virgin female flies (~120 - 160 for a full behavioral arena) that are difficult to collect using standard methods. This section describes an alternative approach using the hs-hid transgene on the Y chromosome28, which has been used successfully in courtship assays25,29. The stock used to generate white-eyed virgin females has the genotype w1118(X)/hs-hid(Y);+/+;+/+ (Bloomington stock number 24638).
<ol>
	<li>Flip flies into a new bottle once 20 - 50% of the pupae have eclosed (see Figure 3A for example) and there are enough males (5 - 10) to propagate the stock.</li>
	<li>Heat shock the original bottle in a 37 &#176;C hot water bath for 1 h to sufficiently activate hs-hid to kill males (Figure 3B). After the heat shock, only females will eclose from these bottles and so will remain virgin until the assays. 
	NOTE: Make sure all sides of the bottle are evenly heated (see Figure 3A for water line). Extend this time for up to 2 h if 1 h is not sufficient to euthanize all males.</li>
	<li>Isolate collected females for at least 3 d before experiments to ensure that they are old enough to be sexually receptive25,29. 
	NOTE: Y-chromosome-less males occasionally arise from this stock. These males have white eyes and are unaffected by the heat shock because they do not contain the hs-hid transgene. They cannot produce sperm and therefore cannot decrease receptivity in females30,31.</li>
</ol>
4. Performing and Scoring 2-D Satiety Assays
NOTE: It is important to control the age of the male flies as mating drive changes with age. This protocol uses males that are 3 - 4 d old25.
<ol>
	<li>Set the incubator to the desired temperature (e.g., 23 &#176;C for standard, RT experiments) and humidity (&#62;30%). For incubators without humidity control, leaving a cup of water in the incubator is sufficient.</li>
	<li>Place a 2-D satiety assay arena in the incubator for ~30 min to equilibrate the temperature and to prevent condensation in the chambers during the course of the experiment. The rotating doors should be in the open position.</li>
	<li>Use a fly aspirator32 to aspirate 15 - 20 virgin females into each chamber of the arena (Figure 4). 
	NOTE: It is important that the females and males are not anesthetized on the same day as the assay. In our experience, doing so may impact female receptivity and male mating behavior.</li>
	<li>Aspirate 1 male into each chamber.</li>
	<li>Place the loaded arena in the incubator under a standard consumer camcorder (Figure 5) and film for 4.5 h.</li>
	<li>Score videos by noting when each male fly starts and ends each mating (i.e. copulation). This step may be time-consuming at first. With practice, one can score the videos at 5x speed or higher. 
	NOTE: Since flies mate for ~20 min at ~23 &#176;C (shorter duration at higher temperatures)29, one can skim through the first 15 min of each mating.</li>
	<li>After logging all mating times, sum the number of matings for each fly (see representative Results).</li>
	<li>Use the provided code to generate an ethogram. See Supplemental Material 2 for the code, instructions, and example data.</li>
	<li>Use CO2 or cold temperature to anesthetize the flies before removing them.</li>
</ol>
5. Using Thermogenetic Manipulation to Reverse Satiety in 2-D Satiety Assays
NOTE: This protocol tests whether thermogenetic stimulation of a defined neuronal population can overcome satiety. The experimental flies express the heat-sensitive cation channel TrpA1 in defined populations of neurons (UAS-TrpA1). The steps below apply to the stimulation of dopaminergic neurons (TH-Gal4), which have been shown to promote mating drive25. These steps can be used in conjunction with other neuronal drivers to discover other populations that promote mating drive.
<ol>
	<li>To thermogenetically induce mating behaviors in satiated flies, increase the temperature in the incubator (e.g., from 23 &#176;C to 28.5 &#176;C) after completing a full 4.5 h 2-D satiety experiment.</li>
	<li>After the incubator reaches the desired temperature, continue heat stimulation and score the matings (i.e. copulations) of the males for 1 h (see Representative Results). 
	NOTE: The temperature and length of stimulation may vary depending on the genotype so it is necessary to troubleshoot the optimal conditions that prevent overstimulation of neurons while still producing a robust phenotype.</li>
</ol>
6. Using Courtship and Locomotion to Verify Satiety
NOTE: This section describes 3 optional methods that can be used to verify the results of 2-D satiety assays. They are not required for every assay.
<ol>
	<li>Use courtship assays to verify satiety.
	<ol>
		<li>Aspirate one male and one virgin female into each of the 32 chambers in a courtship arena.</li>
		<li>Film the flies for ~20 min using a standard consumer camcorder.</li>
		<li>Score the courtship index (CI) to quantify courtship25; this is the fraction of time spent performing courtship behavior (singing, following, etc.) and mating (i.e. copulation) over a 5 min window. This window starts from the first instance of courtship behavior. If a male fly does not start courtship in the first 15 min, its CI is 0. Na&#239;ve WT flies should show a CI greater than 0.9, whereas a fully satiated fly should have a CI below 0.2. Other courtship measures may also be considered23,33.</li>
	</ol>
	</li>
	<li>Scoring courtship in 2-D satiety assays
	<ol>
		<li>Score the amount of time that a male fly spends courting, over a time period of a 2-D satiety assay (e.g., the first h). Courtship is defined as the male performing any step of the ritual (singing, following, etc.). Unlike in Section 6.1.3, this step does not include time the male spends mating.</li>
		<li>Divide the courtship time above by the total time the male was not mating (i.e.&#160;not copulating) in the same time period. For example, if a male fly courts for 15 min and mates for 20 min over the first h, the ratio is 15 min / (60 min - 20 min) = 0.375. The ratio is shown as &#34;Fraction of nonmating time in courtship&#34; in Representative Results.</li>
	</ol>
	</li>
	<li>Using locomotion assay to evaluate exhaustion
	<ol>
		<li>Partially assemble the 32-chamber arena with all parts but the contrast sheet.</li>
		<li>Aspirate one male fly (no female flies) into each of the 32 chambers in a courtship arena.</li>
		<li>Film the flies for ~10 min using a standard consumer camcorder.</li>
		<li>Track the flies with the MTrack2 plugin in ImageJ (http://valelab.ucsf.edu/~nstuurman/ijplugins/MTrack2.html).</li>
	</ol>
	</li>
</ol>
7. Recovering Mating Drive after 2-D Satiety Assay
<ol>
	<li>Perform a 2-D satiety assay as described in Section 4, but aspirate the male flies out at the end of the assay.</li>
	<li>Isolate the male flies at 23 &#176;C for the desired number of days.</li>
	<li>Perform a 2-D satiety assay with the recovered males and score their matings (i.e.&#160;copulations) for just 1 h(see Representative Results).</li>
</ol>

<ol><li>Sturtevant, A. H., Bridges, C. B., Morgan, T. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16576369">The spatial relations of genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 5 (5), 168-173 (1919).</li>
<li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCampos-Ortega%2C+JA+%22The+Embryonic+Development+of+Drosophila+melanogaster%22">The Embryonic Development of Drosophila melanogaster</a>. , Springer. Berlin Heidelberg: Berlin, Heidelberg. (1985).</li>
<li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systems+Approaches+to+Biological+Rhythms+in+Drosophila%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Systems Approaches to Biological Rhythms in Drosophila.</a> Methods in Enzymology. 393, 61-185 (2005).</li>
<li>Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rho+GTPases+in+neuronal+morphogenesis%5BTitle%5D)+AND+%22Nature+reviews.+Neuroscience%22%5BJournal%5D)">Rho GTPases in neuronal morphogenesis.</a> Nature reviews. Neuroscience. 1 (3), 173-180 (2000).</li>
<li>Schmucker, D., Clemens, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+Dscam+Is+an+Axon+Guidance+Receptor+Exhibiting+Extraordinary+Molecular+Diversity%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Drosophila Dscam Is an Axon Guidance Receptor Exhibiting Extraordinary Molecular Diversity.</a> Cell. 101 (6), 671-684 (2000).</li>
<li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HLH+proteins%2C+fly+neurogenesis%2C+and+vertebrate+myogenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">HLH proteins, fly neurogenesis, and vertebrate myogenesis.</a> Cell. 75 (5), 827-830 (1993).</li>
<li>Stockinger, P., Kvitsiani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+circuitry+that+governs+Drosophila+male+courtship+behavior%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Neural circuitry that governs Drosophila male courtship behavior.</a> Cell. 121 (5), 795-807 (2005).</li>
<li>Yu, J. Y., Kanai, M. I., Demir, E., Jefferis, G. S. X. E., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+Organization+of+the+Neural+Circuit+that+Drives+Drosophila+Courtship+Behavior%5BTitle%5D)+AND+%22Current+biology%22%5BJournal%5D)">Cellular Organization of the Neural Circuit that Drives Drosophila Courtship Behavior.</a> Current biology. 20 (18), 1602-1614 (2010).</li>
<li>Zhou, C., Pan, Y., Robinett, C. C., Meissner, G. W., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+Brain+Neurons+Expressing+doublesex+Regulate+Female+Receptivity+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Central Brain Neurons Expressing doublesex Regulate Female Receptivity in Drosophila.</a> Neuron. 83 (1), 149-163 (2014).</li>
<li>Rideout, E. J., Dornan, A. J., Neville, M. C., Eadie, S., Goodwin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+sexual+differentiation+and+behavior+by+the+doublesex+gene+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Nature+neuroscience%22%5BJournal%5D)">Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster.</a> Nature neuroscience. 13 (4), 458-466 (2010).</li>
<li>Manoli, D. S., Foss, M., Villella, A., Taylor, B. J., Hall, J. C., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Male-specific+fruitless+specifies+the+neural+substrates+of+Drosophila+courtship+behaviour%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour.</a> Nature. 436 (7049), 395-400 (2005).</li>
<li>Kimura, K. I., Ote, M., Tazawa, T., Yamamoto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fruitless+specifies+sexually+dimorphic+neural+circuitry+in+the+Drosophila+brain%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain.</a> Nature. 438 (7065), 229-233 (2005).</li>
<li>Cachero, S., Ostrovsky, A. D., Yu, J. Y., Dickson, B. J., Jefferis, G. S. X. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+dimorphism+in+the+fly+brain%5BTitle%5D)+AND+%22Current+biology%22%5BJournal%5D)">Sexual dimorphism in the fly brain.</a> Current biology. 20 (18), 1589-1601 (2010).</li>
<li>Clowney, E. J., Iguchi, S., Bussell, J. J., Scheer, E., Ruta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodal+Chemosensory+Circuits+Controlling+Male+Courtship+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Multimodal Chemosensory Circuits Controlling Male Courtship in Drosophila.</a> Neuron. 87 (5), 1036-1049 (2015).</li>
<li>Kallman, B. R., Kim, H., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excitation+and+inhibition+onto+central+courtship+neurons+biases+Drosophila+mate+choice%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Excitation and inhibition onto central courtship neurons biases Drosophila mate choice.</a> eLife. 4, e11188(2015).</li>
<li>von Philipsborn, A. C., Liu, T., Yu, J. Y., Masser, C., Bidaye, S. S., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+control+of+Drosophila+courtship+song%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neuronal control of Drosophila courtship song.</a> Neuron. 69 (3), 509-522 (2011).</li>
<li>Zhou, C., Franconville, R., Vaughan, A. G., Robinett, C. C., Jayaraman, V., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+neural+circuitry+mediating+courtship+song+perception+in+male+Drosophila%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Central neural circuitry mediating courtship song perception in male Drosophila.</a> eLife. 4, e08477(2015).</li>
<li>Kohatsu, S., Koganezawa, M., Yamamoto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Female+contact+activates+male-specific+interneurons+that+trigger+stereotypic+courtship+behavior+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Female contact activates male-specific interneurons that trigger stereotypic courtship behavior in Drosophila.</a> Neuron. 69 (3), 498-508 (2011).</li>
<li>Kohatsu, S., Yamamoto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visually+induced+initiation+of+Drosophila+innate+courtship-like+following+pursuit+is+mediated+by+central+excitatory+state%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Visually induced initiation of Drosophila innate courtship-like following pursuit is mediated by central excitatory state.</a> Nature Communications. 6, 6457(2015).</li>
<li>Fan, P., Manoli, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+and+neural+mechanisms+that+inhibit+Drosophila+from+mating+with+other+species%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genetic and neural mechanisms that inhibit Drosophila from mating with other species.</a> Cell. 154 (1), 89-102 (2013).</li>
<li>Kurtovic, A., Widmer, A., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+class+of+olfactory+neurons+mediates+behavioural+responses+to+a+Drosophila+sex+pheromone%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone.</a> Nature. 446 (7135), 542-546 (2007).</li>
<li>Ejima, A., Smith, B. P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generalization+of+Courtship+Learning+in+Drosophila+Is+Mediated+by+cis-Vaccenyl+Acetate%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Generalization of Courtship Learning in Drosophila Is Mediated by cis-Vaccenyl Acetate.</a> Current Biology. 17, 599-605 (2007).</li>
<li>Keleman, K., Vrontou, E., Krüttner, S., Yu, J. Y., Kurtovic-Kozaric, A., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+neurons+modulate+pheromone+responses+in+Drosophila+courtship+learning%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Dopamine neurons modulate pheromone responses in Drosophila courtship learning.</a> Nature. 489 (7414), 145-149 (2012).</li>
<li>Pan, Y., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+Identification+and+Separation+of+Innate+and+Experience-Dependent+Courtship+Behaviors+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genetic Identification and Separation of Innate and Experience-Dependent Courtship Behaviors in Drosophila.</a> Cell. 156 (1-2), 236-248 (2014).</li>
<li>Zhang, S. X., Rogulja, D., Crickmore, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopaminergic+Circuitry+Underlying+Mating+Drive%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Dopaminergic Circuitry Underlying Mating Drive.</a> Neuron. 91 (1), 168-181 (2016).</li>
<li>Bowers, M. B., Van Woert, M., Davis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+behavior+during+L-dopa+treatment+for+Parkinsonism%5BTitle%5D)+AND+%22The+American+journal+of+psychiatry%22%5BJournal%5D)">Sexual behavior during L-dopa treatment for Parkinsonism.</a> The American journal of psychiatry. 127 (12), 1691-1693 (1971).</li>
<li>Sacks, O. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSacks%2C+OW+%22Awakenings%22">Awakenings</a>. , Vintage Books. New York. (1999).</li>
<li>Dietzl, G., Chen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448 (7150), 151-156 (2007).</li>
<li>Crickmore, M. A., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Opposing+dopaminergic+and+GABAergic+neurons+control+the+duration+and+persistence+of+copulation+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila.</a> Cell. 155 (4), 881-893 (2013).</li>
<li>Peng, J., Chen, S., Busser, S., Liu, H., Honegger, T., Kubli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gradual+Release+of+Sperm+Bound+Sex-Peptide+Controls+Female+Postmating+Behavior+in+Drosophila%5BTitle%5D)+AND+%22Current+biology%22%5BJournal%5D)">Gradual Release of Sperm Bound Sex-Peptide Controls Female Postmating Behavior in Drosophila.</a> Current biology. 15 (3), 207-213 (2005).</li>
<li>Yapici, N., Kim, Y. J., Ribeiro, C., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+receptor+that+mediates+the+post-mating+switch+in+Drosophila+reproductive+behaviour%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A receptor that mediates the post-mating switch in Drosophila reproductive behaviour.</a> Nature. 451 (7174), 33-37 (2008).</li>
<li>Pellegrino, M., Nakagawa, T., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Sensillum+Recordings+in+the+Insects+Drosophila+melanogaster+and+Anopheles+gambiae%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae.</a> Journal of Visualized Experiments. 36 (36), 1-5 (2010).</li>
<li>Cook, R., Cook, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Attractiveness+to+males+of+female+Drosophila+melanogaster%3A+effects+of+mating%2C+age+and+diet%5BTitle%5D)+AND+%22Animal+behaviour%22%5BJournal%5D)">The Attractiveness to males of female Drosophila melanogaster: effects of mating, age and diet.</a> Animal behaviour. 23, 521-526 (1975).</li>
<li>Toates, F. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aToates%2C+FM+%22Motivational+Systems+%28Problems+in+the+Behavioural+Sciences%29%22">Motivational Systems (Problems in the Behavioural Sciences)</a>. , (1986).</li>
<li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mating+of+a+fly%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The mating of a fly.</a> Science. 264 (5166), 1702-1714 (1994).</li>
<li>Simpson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+and+manipulating+neural+circuits+in+the+fly+brain%5BTitle%5D)+AND+%22Advances+in+genetics%22%5BJournal%5D)">Mapping and manipulating neural circuits in the fly brain.</a> Advances in genetics. 65 (9), 79-143 (2009).</li>
<li>Venken, K. J. T., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+Manipulation+of+Genes+and+Cells+in+the+Nervous+System+of+the+Fruit%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit.</a> Neuron. 72 (2), 202-230 (2011).</li>
<li>Klapoetke, N. C., Murata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Independent+optical+excitation+of+distinct+neural+populations%5BTitle%5D)+AND+%22Nature+methods%22%5BJournal%5D)">Independent optical excitation of distinct neural populations.</a> Nature methods. 11 (3), 338-346 (2014).</li>
<li>Bellen, H. J., Levis, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+BDGP+gene+disruption+project%3A+single+transposon+insertions+associated+with+40%25+of+Drosophila+genes%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.</a> Genetics. 167 (2), 761-781 (2004).</li>
<li>Spradling, A. C., Stern, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Berkeley+Drosophila+Genome+Project+gene+disruption+project%3A+Single+P-element+insertions+mutating+25%25+of+vital+Drosophila+genes%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.</a> Genetics. 153 (1), 135-177 (1999).</li>
<li>Parks, A. L., Cook, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+generation+of+high-resolution+deletion+coverage+of+the+Drosophila+melanogaster+genome%5BTitle%5D)+AND+%22Nature+genetics%22%5BJournal%5D)">Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.</a> Nature genetics. 36 (3), 288-292 (2004).</li>
<li>Matthews, K. A., Kaufman, T. C., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Research+resources+for+Drosophila%3A+the+expanding+universe%5BTitle%5D)+AND+%22Nature+reviews.+Genetics%22%5BJournal%5D)">Research resources for Drosophila: the expanding universe.</a> Nature reviews. Genetics. 6 (3), 179-193 (2005).</li>
<li>Ni, J. Q., Liu, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Drosophila+resource+of+transgenic+RNAi+lines+for+neurogenetics%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">A Drosophila resource of transgenic RNAi lines for neurogenetics.</a> Genetics. 182 (4), 1089-1100 (2009).</li>
<li>Ni, J. Q., Zhou, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genome-scale+shRNA+resource+for+transgenic+RNAi+in+Drosophila%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genome-scale shRNA resource for transgenic RNAi in Drosophila.</a> Nature. 8 (5), 405-407 (2011).</li>
</ol>

The authors have nothing to disclose.

Motivational states can be satiated, maintained, and recovered34. We present a 2-D satiety assay that quickly and robustly measures all of these aspects of mating drive in the fly. This assay opens up the possibility of using advanced fly genetic manipulations to study the molecular and circuit components of a motivated behavior.
The satiety assay relies on the male&#39;s ability to successfully court and copulate, and to terminate copulations at the appropriate time. T...

Work in Drosophila has provided deep and pioneering insight into many biological phenomena, including the nature of the gene1, principles of embryonic development2, circadian rhythms3, and the development and wiring of the nervous system4,5,6. Motivation remains far less well understood than these phenomena, perhaps because of the limitations on the systems that have been studied thus far. Motivation in the fly is primarily studied in the context of hunger, which presents many challenges due to their vanishingly small food intake per feeding bout and exoskeleton which precludes overt signs of fat deposition. Consequently, there is a need to expand the systems used to study motivation in the fly.
We describe a behavioral framework for the study of mating drive in Drosophila. This system takes advantage of the neurogenetic tools in the fly as well as the accessibility7,8,9,10,11,12 and the putative connectome of its sexually dimorphic circuitry8,13. In addition, much of the innate14,15,16,17,18,19,20,21 and learned22,23,24 sensory-motor circuitry controlling courtship has been worked out in detail, providing a rare opportunity to locate the exact circuit node upon which motivation impinges. We recently reported that, in the fly, as in humans, dopamine levels are central to mating drive25,26,27. We have gained genetic access to the relevant dopamine-producing and receiving neurons in the fly, facilitating detailed molecular- and circuit-level analyses of this conserved phenomenon using the assays we describe here25.
We add to the behavioral assays in Zhang et al.25 a new flat behavioral arena that allows video scoring, which we call a 2-dimensional (2-D) satiety assay, an important improvement over previous methods. Consequently, the new assay is more scalable and quantifiable, and therefore more suitable for genetic screens of genes and neurons involved in motivation. We use this new assay, together with courtship assays and neurogenetic manipulations, to demonstrate how to measure and alter mating drive in the fly.

<table><tbody><tr><td>1/16 inch clear acrylic</td><td>McMaster-Carr</td><td>8589K12</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>1/8 inch clear acrylic</td><td>McMaster-Carr</td><td>8589K42</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>3/16 inch clear acrylic</td><td>McMaster-Carr</td><td>8560K219</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>1/32 inch black delrin</td><td>McMaster-Carr</td><td>8575K132</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>Hex screws, 1 inch long (50x)</td><td>McMaster-Carr</td><td>92314A115&amp;nbsp;</td><td>Used to make arenas. Can be replaced by 3/4 inch screws (92314A113, McMaster-Carr) for 32-chamber arenas.</td></tr><tr><td>Thumb nuts (25x)</td><td>McMaster-Carr</td><td>92741A100</td><td>Used to make arenas. Can be replaced by regular hex nuts (90480A005, McMaster-Carr).</td></tr><tr><td>Camcorder</td><td>Canon</td><td>Vixia HF R700</td><td>Can be replaced by any consumer comcorder.</td></tr></tbody></table>

measuring and altering mating drive in male drosophila melanogaster

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.

To characterize Drosophila mating drive, 3-day-old, WT Canton-S males were tested in a 2-D satiety assay. Over the course of the assay (4.5 h), males mate an average of 4.8 ±0.3 (mean ±standard error of mean, SEM) times. Matings initiate mostly in the first 2 h (78%) (Figure 6A, 6B) and become less frequent as the assay progresses (Figure 6A, 6B). This decrease is not due to the lack of mating partners (74% females remain unmated throughout the a...

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, ...

The overall goal of this assay is to measure and manipulate the male Drosophila mating drive in order to examine motivation in a reductionist system. This method can help answer key questions in the study of motivation like what genes and neurons control mating drive potentially revealing the logic behind the motivational circuitry. The main advantage of this method is that it provides a high throughput assay for measuring mating drive that can be used in concert with the powerful genetic tools available in the fly.At least three days before a 2-D satiety assay prepare female flies from a bottle of white 11 18 over heat shock hid fly stock. Once 20 to 50%of the pupae have eclosed and there are at least five males to propagate the stock, flip flies in to a new bottle. Next heat shock the original bottle in a 37 degree Celsius hot water bath for one hour to sufficiently activate heat shock hid to kill male larvae and pupae.After the heat shock only females will eclose from these bottles. To prepare the arena for experiments partially assemble the 2-D satiety arena and tighten it with thumb nuts and hex screws. Next place fresh fly food in to a clean, microwave safe bottle and add just enough water to cover the bottom surface.Microwave the food on high for 30 to 45 seconds. Visually check the progress and stir the food every 15 seconds until it is melted. Once the food is melted use a blunted 1000 microliter pipette tip to slowly transfer about 1 milliliter of melted food in to each chamber.Allow the food to re-solidify at four degrees Celsius for 10 minutes before completely assembling the arena. The completed arena can be used for experiments. Store the leftover food at four degrees Celsius for reuse.Before starting the 2-D satiety assay, set the incubator to 23 degrees Celsius for standard room temperature experiments and make sure the humidity is greater than 30%Leave a cup of water in the incubator if the incubator does not have humidity control. Place the 2-D satiety assay arena in the incubator for 30 minutes to equilibrate the temperature and prevent condensation in the chambers during the course of the experiment. Keep the rotating doors in the open position.Next use a fly aspirator to aspirate 15 to 20 virgin females in to each chamber of the arena. After the female flies are transferred, aspirate one male in to each chamber. Then place the loaded arena in to incubator under a standard consumer camcorder and film the flies for 4.5 hours.After filming use CO2 or cold temperature to anesthetize the flies before removing them from the arena. Score videos by noting when each male fly starts and ends each mating. Use the provided code to generate an ethogram.After logging all mating times, sum the number of matings for each fly. Aspirate one male in to each of the 32 chambers in a courtship arena. Then aspirate one female in to each chamber.Film the flies for twenty minutes using a standard consumer camcorder. Score the courtship index to quantify courtship over a five minute window. If a male fly does not start courtship in the first fifteen minutes, the courtship index is zero.Naive wild type flies should show a courtship index greater than 0.9 Whereas a fully satiated fly should have a courtship index below 0.2. After performing a 2-D satiety assay, aspirate the male flies from the chambers in to food vials. Isolate the male flies from the females at 23 degrees Celsius for the desired number of days.Then perform a 2-D satiety assay with the recovered males and score their matings over the course of just one hour. To characterize the Drosophila mating drive, three day old wild type Canton-S males were tested in a 2-D satiety assay. Over 4.5 hours males mate an average of 4.8 times.78%of matings initiate in the first two hours. Matings become less frequent as the assay progresses. This decline in mating behavior is also seen when the males are tested in courtship assays with new females and is recovered after the males have been isolated from females for three days.At the end of 2-D satiety assays dopaminergic stimulation increased both courtship and copulation. The reversal effects are not observed with parental controlled genotypes. Once mastered, this assay can be prepared in 30 minutes and scored in an hour and a half.This assay can be performed in concert with genetic and neuronal manipulations such as RNAi knockdown and optogenetics to determine which genes and neurons control mating drive.

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Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Assessing Differences in Sperm Competitive Ability in Drosophila

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High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

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A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

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Drosophila Courtship Conditioning As a Measure of Learning and Memory

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Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Methods to Test Endocrine Disruption in Drosophila melanogaster

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

Learn to measure and manipulate male Drosophila melanogaster mating drive using a high-throughput behavioral assay. This method reveals genetic and neural mechanisms underlying motivation in a reductionist system.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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