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...

<ol>
	<li>Sturtevant, A. H., Bridges, C. B., Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+relations+of+genes.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Embryonic Development of Drosophila melanogaster. , (1985).</li><li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+Approaches+to+Biological+Rhythms+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+neuronal+morphogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Dscam+Is+an+Axon+Guidance+Receptor+Exhibiting+Extraordinary+Molecular+Diversity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLH+proteins,+fly+neurogenesis,+and+vertebrate+myogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuitry+that+governs+Drosophila+male+courtship+behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Organization+of+the+Neural+Circuit+that+Drives+Drosophila+Courtship+Behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Brain+Neurons+Expressing+doublesex+Regulate+Female+Receptivity+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+sexual+differentiation+and+behavior+by+the+doublesex+gene+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-specific+fruitless+specifies+the+neural+substrates+of+Drosophila+courtship+behaviour.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruitless+specifies+sexually+dimorphic+neural+circuitry+in+the+Drosophila+brain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+the+fly+brain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+Chemosensory+Circuits+Controlling+Male+Courtship+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation+and+inhibition+onto+central+courtship+neurons+biases+Drosophila+mate+choice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+control+of+Drosophila+courtship+song.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+neural+circuitry+mediating+courtship+song+perception+in+male+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+contact+activates+male-specific+interneurons+that+trigger+stereotypic+courtship+behavior+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+induced+initiation+of+Drosophila+innate+courtship-like+following+pursuit+is+mediated+by+central+excitatory+state.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+neural+mechanisms+that+inhibit+Drosophila+from+mating+with+other+species.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+class+of+olfactory+neurons+mediates+behavioural+responses+to+a+Drosophila+sex+pheromone.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalization+of+Courtship+Learning+in+Drosophila+Is+Mediated+by+cis-Vaccenyl+Acetate.">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., Kr&#252;ttner, S., Yu, J. Y., Kurtovic-Kozaric, A., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neurons+modulate+pheromone+responses+in+Drosophila+courtship+learning.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Identification+and+Separation+of+Innate+and+Experience-Dependent+Courtship+Behaviors+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+Circuitry+Underlying+Mating+Drive.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+behavior+during+L-dopa+treatment+for+Parkinsonism.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Awakenings. , (1999).</li><li>Dietzl, G., Chen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+dopaminergic+and+GABAergic+neurons+control+the+duration+and+persistence+of+copulation+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradual+Release+of+Sperm+Bound+Sex-Peptide+Controls+Female+Postmating+Behavior+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+receptor+that+mediates+the+post-mating+switch+in+Drosophila+reproductive+behaviour.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Sensillum+Recordings+in+the+Insects+Drosophila+melanogaster+and+Anopheles+gambiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Attractiveness+to+males+of+female+Drosophila+melanogaster:+effects+of+mating,+age+and+diet.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Motivational Systems (Problems in the Behavioural Sciences). , (1986).</li><li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mating+of+a+fly.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+manipulating+neural+circuits+in+the+fly+brain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Manipulation+of+Genes+and+Cells+in+the+Nervous+System+of+the+Fruit.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+optical+excitation+of+distinct+neural+populations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BDGP+gene+disruption+project:+single+transposon+insertions+associated+with+40%+of+Drosophila+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Berkeley+Drosophila+Genome+Project+gene+disruption+project:+Single+P-element+insertions+mutating+25%+of+vital+Drosophila+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+generation+of+high-resolution+deletion+coverage+of+the+Drosophila+melanogaster+genome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+resources+for+Drosophila:+the+expanding+universe.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+resource+of+transgenic+RNAi+lines+for+neurogenetics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-scale+shRNA+resource+for+transgenic+RNAi+in+Drosophila.">A genome-scale shRNA resource for transgenic RNAi in Drosophila.</a> Nature. 8 (5), 405-407 (2011).</li></ol>

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.

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			<td height="21" style="height:21px;width:216px;">1/16 inch clear acrylic</td>
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			<td style="width:119px;">8589K12</td>
			<td style="width:743px;">Used to make arenas; see Supplemental Material 1 for designs.</td>
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			<td height="21" style="height:21px;">1/8 inch clear acrylic</td>
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			<td height="21" style="height:21px;">3/16 inch clear acrylic</td>
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			<td height="21" style="height:21px;">1/32 inch black delrin</td>
			<td style="width:119px;">McMaster-Carr</td>
			<td>8575K132</td>
			<td>Used to make arenas; see Supplemental Material 1 for designs.</td>
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			<td height="21" style="height:21px;">Hex screws, 1 inch long (50x)</td>
			<td style="width:119px;">McMaster-Carr</td>
			<td>92314A115&#160;</td>
			<td>Used to make arenas. Can be replaced by 3/4 inch screws (92314A113, McMaster-Carr) for 32-chamber arenas.</td>
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			<td height="21" style="height:21px;">Camcorder</td>
			<td style="width:119px;">Canon</td>
			<td>Vixia HF R700</td>
			<td>Can be replaced by any consumer comcorder.</td>
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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 &#177;0.3 (mean &#177;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 throug...

Watch this Scientific Journal Video about Measuring and Altering Mating Drive in Male Drosophila melanogaster at JoVE.com

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.

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, ...

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.

measuring-and-altering-mating-drive-in-male-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

10.9K vistas.  Boston Children’s Hospital. 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 too...