Name: quantification of drosophila grooming behavior
Uploaded: 2017-07-19T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Drosophila Grooming Behavior at JoVE.com

Vi ønsker å takke Brian Shepherd, Tat Udomritthiruj, Aaron Willey, Ruby Froom, Elise Pitmon og Rose Hedreen for tidlig arbeid i testing og etablering av denne metodikken og kammerdesign. Vi takker Kelly Tellez og Graham Buchan for å lese og redigere manuskriptet. Vi takker Andrew Seeds og Julie Simpson for deres banebrytende arbeid og deres råd og støtte i å foreslå bruken av Brilliant Yellow Dye (Sigma). Dette arbeidet støttes delvis av Mary E. Groff Kirurgisk og medisinsk forskning og utdanning Charitable Trust, Bronfman Science Center, og Hellman Fellows Program.

1. Fremstilling <ol><li> Klargjør en aspirator for å flytte levende Drosophila fra et kulturflaske til pleiekammeret. Aspiratorer tillater overføring av bevisste dyr til adferdskamrene for å sikre at anestesi ikke påvirker etterfølgende atferdsobservasjon. <ol><li> Bruk saks kutt 1,5 fot tygon tubing ID ⅛ &quot;, OD ¼&quot;. Ta minst 1 tommers av spissen av en 1 ml engangs mikropipettespiss. Hold et 1 cm kvadratmaske (åpning 0,196 tommer) over kuttespissen. MERK: De fleste 1m...

<ol>
	<li>Szebenyi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+Behaviour+in+Drosophila-Melanogaster.">Cleaning Behaviour in Drosophila-Melanogaster.</a> Animal. Behaviour. 17, (1969).</li><li>Corfas, G., Dudai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+and+dishabituation+of+a+cleaning+reflex+in+normal+and+mutant+Drosophila.">Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila.</a> J Neurosci. 9 (1), 56-62 (1989).</li><li>Yanagawa, A., Guigue, A. M. A., Marion-Poll, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hygienic+grooming+is+induced+by+contact+chemicals+in+Drosophila+melanogaster.">Hygienic grooming is induced by contact chemicals in Drosophila melanogaster.</a> Frontiers in Behavioral Neuroscience. 8, (2014).</li><li>Dawkins, R., Dawkins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+Organization+and+Postural+Facilitation+-+Rules+for+Grooming+in+Flies.">Hierarchical Organization and Postural Facilitation - Rules for Grooming in Flies.</a> Animal Behaviour. 24 (Nov), 739-755 (1976).</li><li>Seeds, A. M., 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+suppression+hierarchy+among+competing+motor+programs+drives+sequential+grooming+in+Drosophila.">A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila.</a> Elife. 3, e02951 (2014).</li><li>King, L. B., 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=Neurofibromin+Loss+of+Function+Drives+Excessive+Grooming+in+Drosophila.">Neurofibromin Loss of Function Drives Excessive Grooming in Drosophila.</a> G3-Genes Genomes Genetics. 6 (4), 1083-1093 (2016).</li><li>Tauber, J. M., Vanlandingham, P. A., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+Levels+of+the+Vesicular+Monoamine+Transporter+and+a+Novel+Repetitive+Behavior+in+the+Drosophila+Model+of+Fragile+X+Syndrome.">Elevated Levels of the Vesicular Monoamine Transporter and a Novel Repetitive Behavior in the Drosophila Model of Fragile X Syndrome.</a> Plos One. 6 (11), e27100 (2011).</li><li>Kaur, K., Simon, A. F., Chauhan, V., Chauhan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bisphenol+A+on+Drosophila+melanogaster+behavior--a+new+model+for+the+studies+on+neurodevelopmental+disorders.">Effect of bisphenol A on Drosophila melanogaster behavior--a new model for the studies on neurodevelopmental disorders.</a> Behav Brain Res. 284, 77-84 (2015).</li><li>Chang, H. 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=Overexpression+of+the+Drosophila+vesicular+monoamine+transporter+increases+motor+activity+and+courtship+but+decreases+the+behavioral+response+to+cocaine.">Overexpression of the Drosophila vesicular monoamine transporter increases motor activity and courtship but decreases the behavioral response to cocaine.</a> Molecular Psychiatry. 11 (1), 99-113 (2006).</li><li>Yellman, C., Tao, H., He, B., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+sexually+dimorphic+behavioral+responses+to+biogenic+amines+in+decapitated+Drosophila.">Conserved and sexually dimorphic behavioral responses to biogenic amines in decapitated Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (8), 4131-4136 (1997).</li><li>Feng, G. 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=Cloning+and+functional+characterization+of+a+novel+dopamine+receptor+from+Drosophila+melanogaster.">Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster.</a> Journal of Neuroscience. 16 (12), 3925-3933 (1996).</li><li>Gotzes, F., Balfanz, S., Baumann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Structure+and+Functional-Characterization+of+a+Drosophila+Dopamine-Receptor+with+High+Homology+to+Human+D(1/5).">Primary Structure and Functional-Characterization of a Drosophila Dopamine-Receptor with High Homology to Human D(1/5).</a> Receptors. Receptors &amp; Channels. 2 (2), 131-141 (1994).</li><li>Han, K. A., Millar, N. S., Grotewiel, M. S., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAMB,+a+novel+dopamine+receptor+expressed+specifically+in+Drosophila+mushroom+bodies.">DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies.</a> Neuron. 16 (6), 1127-1135 (1996).</li><li>Sugamori, K. S., Demchyshyn, L. L., Mcconkey, F., Forte, M. A., Niznik, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primordial+Dopamine+D1-Like+Adenylyl+Cyclase-Linked+Receptor+from+Drosophila-Melanogaster+Displaying+Poor+Affinity+for+Benzazepines.">A Primordial Dopamine D1-Like Adenylyl Cyclase-Linked Receptor from Drosophila-Melanogaster Displaying Poor Affinity for Benzazepines.</a> Febs Letters. 362 (2), 131-138 (1995).</li><li>Pitmon, E., 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+D1+family+dopamine+receptor,+DopR,+potentiates+hind+leg+grooming+behavior+in+Drosophila.">The D1 family dopamine receptor, DopR, potentiates hind leg grooming behavior in Drosophila.</a> Genes Brain and Behavior. 15 (3), 327-334 (2016).</li><li>Phillis, 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=Isolation+of+mutations+affecting+neural+circuitry+required+for+grooming+behavior+in+Drosophila+melanogaster.">Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster.</a> Genetics. 133 (3), 581-592 (1993).</li><li>Hampel, S., Franconville, R., Simpson, J. H., Seeds, A. 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+neural+command+circuit+for+grooming+movement+control.">A neural command circuit for grooming movement control.</a> Elife. 4, e08758 (2015).</li><li>Kays, I., Cvetkovska, V., Chen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+analysis+of+single+neurons+to+correlate+synaptic+connectivity+with+grooming+behavior.">Structural and functional analysis of single neurons to correlate synaptic connectivity with grooming behavior.</a> Nature Protocols. 9 (1), 1-10 (2014).</li></ol>

Grooming-analysen er relativt enkel, men vi vil forsiktighetseksperimenter være spesielt oppmerksomme på følgende problemer. Opprettholde en tett forsegling ved å stramme skruene på topp- og bunnplaten etter å ha innført fluer og fargestoff er viktig for reproducerbare resultater. Brilliant Yellow-fargestoffet er veldig fint, og løse ledd vil tillate tap av fargestoffer fra kammerets kanter. Uregelmessigheten i fargestoffinnholdet for hver brønn kan lett kaste av groomkvantifisering, da ikke-jevn støving vil ?...

Grooming i Drosophila melanogaster ( D. melanogaster ) er en robust medfødt atferd som involverer koordinering av flere uavhengige motorprogrammer 1 . Fruktfluer renser deres støv, mikrober og andre patogener som kan hemme normal fysiologisk funksjon som syn og fly, eller føre til betydelige immunforsvar. Ved å føle og reagere på både mekanisk 2 og immun aktivering 3 , flyr fluene gjentatt på bena sammen eller på en målrettet kroppsregion til den er tilstrekkelig ren og grooming utvikler seg til en annen del av kroppen. Fluer utfører bevegelsesbevegelser i forskjellige begivenheter som stort sett forekommer i stereotype mønstre 1 , 4 . Et oppførselshierarki blir tydelig som groomsignaler prioriteres. Kretskort og aktivitetsmønster er identifisert i støtte oFa modell som grooming programmer øverst i hierarkiet oppstår først og undertrykker parallelle signaler fra områder av kroppen som blir preparert senere 5 . Høyeste prioritet er gitt til hodet, deretter magen, vingene og til slutt thoraxen 5 . Lekeprogrammet i D. melanogaster er et ideelt system for å studere nevrale kretser, modulerende molekylære signaler og nevrotransmittere. For eksempel, kompromittering av nevrofibrominfunksjon 6 , tap av Drosophila fragile X mentalt retardasjonsprotein ( dfmr1 ) 7 og eksponering for bisfenol A (BPA) 8 forårsaker altfor groom og annen oppførsel som er analog med diskrete menneskelige symptomer på nevrofibromatose, skjøre X Syndrom og aspekter av autismespektrumforstyrrelser og henholdsvis ADHD (Attention-Deficit Hyperactivity Disorder). Grooming atferd kan også være habituaTed differensielt over mutantstammer 2 , utlån denne motorprogrammet til studier av atferdsmessig plastisitet. Bredden av nevrologiske fenomener som kan modelleres av Drosophila krever en ny komparativ tilnærming for å måle flygens evne til å pleie seg selv. Den kombinerte virkningen av vesikulære monoamintransportører og den relative overflaten av dopamin og andre biogene aminer i kroppen har vist seg å formidle fruktluftspleieadferd 9 , 10 . Octopamin og dopamin stimulerer sammenlignbar hindleg grooming aktivitet i decapitated fluer, mens tyramin, forløperen av oktopamin, også utløser grooming i mindre grad 7 . Fire dopaminreseptorer er identifisert i D. melanogaster 11 , 12 , 13 , 14 </sup&gt;. Ved å bruke groomanalysemetoden beskrevet i denne protokollen, bestemte vi en rolle for type I-familien Dopamin Receptor DopR ( DopR, dDA1, dum ) i hindleg grooming behavior 15 . Grooming kan indirekte kvantifiseres ved å se på omfanget av renslighet som et dyr kan fullstendig grooms etter dusting hele kroppen med et markørfarger eller fluorescerende støv 5 , 16 . Resten av støv igjen på kroppen kan brukes som en relativ markør for den generelle oppførelsen. Dusty flyr etter å ha fått tilstrekkelig tid til å gifte seg, kan vise seg et bestemt underskudd i grooming behavior. Som grooming undersøkelser har blitt mer omfattende, har protokoller innarbeidet slik praksis som halshugning for å legge farmakologiske behandlinger på nakkebindende nerver 10 , taktil stimulering av børster for å fremkalle grooming respons 2 ,Og videoopptak av oppførsel 15 . Direkte observasjon av grooming kan enkelt studeres ved hjelp av visuell observasjon og manuell opptak av frekvens og varighet av bestemte grooming hendelser 4 . Vi har designet et femten brønnkammer som kan bygges med en 3D-skriver eller laserkutter, og tegningene er tilgjengelige for reproduksjon 15 . Designet bruker to sammenføyde sentralplater med åpninger som er tilpasset og adskilt av mesh og to ytterligere glidende topp- og bunnplater, hvorfra fluer og / eller fargestoffer er lastet. Etter at støvfluene har tid til å pleie, legger vi dem i etanol for å oppløseliggjøre fargestoffet og måle absorbansen av denne løsningen ved bølgelengden av fargestoffet. En plate leser kan brukes til flere parallelle prøver eller et enkeltlest spektrofotometer kan brukes til individuelle prøver. Denne metoden minimerer feilen fremkalt ved håndtering og alLows for grooming analyser skal kjøres på en mindre, kostnadseffektiv skala. Denne metoden er avledet og modifisert fra metodene pioneret av Julie Simpson og Andrew Seeds, som bruker større pleiekamre med varmeelementer for temperaturfølsomme kretsmanipulasjoner 5 . Følgende protokoll viser kvantifiseringen av grooming av hele kroppen, samt viser alternative metoder for kvantifisering av fargestoffer på individuelle kroppsdeler. Vi presenterer også prøve-sammenligningsdata mellom WT- og DopR- mutanter, samt metoder for beregning av en enkel ytelsesindeks for pleieadferd.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High-Flex Tygon PVC Clear Tubing</td>
			<td>McMaster-Carr</td>
			<td>5229K54</td>
			<td>ID 1/8&#34;, OD 1/4&#34;, used with micropipettor tips and mesh to construct mouth aspirators</td>
		</tr>
		<tr>
			<td>Micropipette tips (1ml and 200ul)</td>
			<td>Genesee Scientific</td>
			<td>24-165, 24-150R</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Screen, 2&#34; x 2.6&#34;</td>
			<td>McMaster-Carr</td>
			<td>9318T44</td>
			<td>Used to construct grooming chamber and mouth aspirators</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Yellow Dye</td>
			<td>Sigma-Aldrich</td>
			<td>201375-25G</td>
			<td>we recommend use of nitrile gloves while handling this product</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td>set to &#34;touch&#34;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carolina Biological Supply</td>
			<td>86-1282</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR International</td>
			<td>10025-726</td>
			<td></td>
		</tr>
		<tr>
			<td>0.65 ml microcentrifuge tubes</td>
			<td>VWR International</td>
			<td>20170-293</td>
			<td>tubes can be reused with successive assays</td>
		</tr>
		<tr>
			<td>UV 96 well plate</td>
			<td>Corning</td>
			<td>26014017</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HTX Platereader</td>
			<td>BioTek</td>
			<td>need to download catalog to access product number</td>
			<td>http://www.biotek.com/products/microplate_detection/synergy_htx_multimode_microplate_reader.html?tab=overview</td>
		</tr>
		<tr>
			<td>Gen5 Microplate Reader and Imager Software</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>https://www.microsoftstore.com/store/msusa/en_US/pdp/Excel-2016/productID.323021400?tduid=(65d098c0e83b86c952bdff5b0719c83f)(256380)(2459594)(SRi0yYDlqd0-LI..ql4M2LoZBEhcBljvIA)()</td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Tritech</td>
			<td>DT2-CIRC-TK</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34; acrylic plastic</td>
			<td>McMaster-Carr</td>
			<td>8473K341</td>
			<td></td>
		</tr>
		<tr>
			<td>8-32 nuts</td>
			<td>McMaster-Carr</td>
			<td>90257A009</td>
			<td></td>
		</tr>
		<tr>
			<td>8-32 x 1&#34; hex cap screws</td>
			<td>McMaster-Carr</td>
			<td>92185A199</td>
			<td>the bottom plate needs to be tapped for this size screw</td>
		</tr>
		<tr>
			<td>8-32 x 1/2&#34; hex cap screws</td>
			<td>McMaster-Carr</td>
			<td>92185A194</td>
			<td>the second plate from the top needs to be tapped</td>
		</tr>
		<tr>
			<td>2-56 3/8&#34; flat head phillips machine screws</td>
			<td>McMaster-Carr</td>
			<td>91500A088</td>
			<td>these hold the two middle plates together</td>
		</tr>
		<tr>
			<td>0.175&#34; ID, 1/4&#34; OD, 0.34&#34; aluminum pipe</td>
			<td>McMaster-Carr</td>
			<td>92510A044</td>
			<td>Manufactured in-house; product listed is approximately the same dimensions and should work for size 8 screws.&#160; These act as sheaths for the 1&#34; screws and set the hex cap up slightly from the surface of the top plate</td>
		</tr>
	</tbody>
</table>

quantification of drosophila grooming behavior

Drosophila grooming behavior er et komplekst multi-trinns lokomotorisk program som krever koordinert bevegelse av både forben og bakben. Her presenterer vi en grooming assay protokoll og ny kammer design som er kostnadseffektiv og skalerbar for enten små eller store studier av Drosophila grooming. Fluer er støvet over hele kroppen med Brilliant Yellow farge og gitt tid til å fjerne fargestoffet fra kroppene sine i kammeret. Fluer blir deretter avsatt i et sett volum etanol for å oppløse fargestoffet. Den relative spektralabsorbansen av fargestoff-etanolprøver for preparerte versus ungromede dyr måles og registreres. Protokollen gir kvantitative data om fargestoffsakkumulering for individuelle fluer, som lett kan beregnes og sammenlignes på tvers av prøver. Dette tillater eksperimentelle design å enkelt vurdere grooming evne for mutant dyre studier eller krets manipulasjoner. Denne effektive prosedyren er både allsidig og skalerbar. Vi viser wOrk-flow av protokollen og sammenlignende data mellom WT dyr og mutant dyr for Drosophila type I Dopamine Receptor ( DopR ).

Groom-analysen gir kvantitative data for å vurdere atferdsprestasjon basert på den relative resten av akkumulert fargestoff som er igjen på flygellegemene etter en bestemt måltid for grooming (30 min). Prøvebilder av glidekrympekammerdesignen og hovedtrinnene i analysen er fremhevet i figur 1 . Flyver aggregerer en betydelig mengde fargestoff fra umiddelbar støvdannelse ved vortexing i nærvær av fargestoff ( Figur 2d, 2e ). Dusted fluer kan behol...

Watch this Scientific Journal Video about Quantification of Drosophila Grooming Behavior at JoVE.com

Quantification of Drosophila Grooming Behavior

Denne protokollen beskriver en skalerbar individuell grooming assay teknikk i Drosophila som gir robust, kvantitativ data for å måle grooming oppførsel. Metoden er basert på å sammenligne forskjellen i opphopning av fargestoffer på kroppene til ukjente dyr i forhold til preparerte dyr over en bestemt tidsperiode.

Kvantifisering av Drosophila Grooming Behavior

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

The overall goal of this method is to measure the grooming behavior of Drosophila by quantifying and comparing the remainder of colored brilliant yellow dye on the body of the animals in groomed and ungroomed conditions. This method can help answer key questions in the field of neurobiology and behavioral genetics such as the coordination of fine locomotive skills and the associated molecular regulators and circuits. The main advantage is as of this technique, are that it's rapid, robust and easy to perform by many experimentalists and students at a variety of skill levels.Demonstrating this procedure will be Frankie Barradale, a student in my laboratory. To begin the experiment, prepare an aspirator to move live Drosophila from a culture vial to the grooming chamber. Using scissors, cut 1.5 feet of Tygon tubing.Cut at least one inch of the tip of a one milliliter disposable micro-pipette tip and hold the one centimeter square piece of mesh over the 196 inch opening of the cut tip. Snugly place a fresh, one milliliter micro-pipette tip over the mesh and cut tip to create a holding chamber for flies. Cut the extreme tip of the new micro-pipette tip to widen the opening enough to allow the passage of a single fly.The hole will roughly match the opening of the grooming chamber. Snugly fit the nested tips onto the end of the Tygon tubing. Push the tips into the tube to ensure a tight fit to allow for vacuum pressure.On the other end of the tubing, cut the tip of a 200 milliliter micro-pipette tip and fit the narrow cut end into the tubing to form the mouth side of the aspirator. To prepare dust aliquot tubes, weigh five milligrams of brilliant yellow dye on weighing paper. Pour the dust into a 8 milliliter micro-centrifuge tube and tightly close the cap.Next, to prepare ethanol tubes, pipette one milliliter of 100%ethanol into 15 milliliter micro-centrifuge tubes. Label the tubes, cap and save the ethanol tubes for completion of the grooming assay. Also include at least one blank sample with only one milliliter of ethanol without a fly for negative controls.Assemble each grooming chamber by screwing the sliding top plate with the smaller holes, but leaving the bottom plate with the larger holes removed for the addition of the dust. Place each necessary grooming chamber flat on a table with the top face-down. Load five milligrams of brilliant yellow dye aliquot into each chamber, by tapping the tube against the surface of the chamber above the desired well.Tap the chamber against the table to ensure that all the dust falls through the mesh to the top of the plate. Then screw the bottom plate onto each chamber, so the wider ends of the conical openings face outward and the holes do not rest over the wells. Secure the bottom plate tightly, so that it does not slide and release dye.Flip the chamber over and knock it flat against the table a few times so that the dust falls through the mesh to rest on the bottom plate. Slide the top plate of the chamber into the open positions that the small holes align with the 15 wells, allowing the introduction of flies into the individual wells. Load one fly into the mouth aspirator by gently sucking through one end of the aspirator as if using a straw.Deposit the flies by lightly blowing and directing the opening toward the target. After loading a well, slide the top plate so that the fly is trapped in the chamber and place tape over the opening of the well during the loading of the hole chamber, to prevent the fly's escape while loading the other wells. If multiple flies are aspirated into a single well, leave that opening uncovered until only one fly remains.After all of the necessary wells are filled with flies, flip the chamber so that the bottom plate is upward and the dust has the potential to coat the flies by falling through the mesh. Tightly secure the top and bottom plates by tightening the screws and vortex the chamber to dust the flies for four seconds. Knock the chamber, top plate face-down against the table twice, so that the dye falls through the other side.Then, flip the chamber over, top plate face-up and knock the chamber twice, to ensure dye settles on the floor of the bottom chamber, away from the fly, held in the top chamber. For control flies, which are not allowed to groom, immediately proceed to sample preparation. Rest the chamber flat on the table or countertop, with the top plate upward for 30 minutes to allow the flies to groom.Place the chamber in a noise vibration free space, to keep your environment constant for all grooming experiments. Keeping the chamber level with the top plate upward, gently unscrew the bottom plate and remove it from the chamber, taking care not to lose any dye. Place the chamber on a CO2 pad with low air flow until the flies are anesthetized.Unscrew the top plate from the chamber, carefully use forceps to grab a leg and remove one dusted fly from each chamber and deposit it in a 1.5 milliliter micro-centrifuge tube containing ethanol. Once each micro-centrifuge tube contains one fly, close the cap and invert the tubes three times to agitate and mix the solution of dye and ethanol. Incubate the tubes containing ethanol and flies for five hours at room temperature to ensure full removal of the dye from the fly into solution.After five hours, briefly vortex each tube for two seconds to ensure clearance of the dyes from the flies. Aliquot 50 microliters of the experimental 1.5 milliliter tube into a well of a 96 well plate, taking note of the location of each sample on the plate. Add 200 microliters of ethanol to dilute the sample five times to avoid sealing effects of heavily dusted flies or large grooming defects.Use a plate reader to analyze each sample at 397 nanometers. Record the sample through the plate reader and save the spreadsheet with the recording samples on the plate. We used this method to study DopR by comparing the grooming performance of a strong hypomorph and to null DopR mutants to a wild type strain.The DopR mutants retained significantly more dye than their wild type of rescued counterparts, indicating that the DopR mutants were less efficient at grooming behavior. In a parallel experiment of Rutabaga flies, mutants display the higher dye accumulation levels than DopR mutants, suggesting a significant role of DopR in grooming behavior. After watching this video, you should have a good understanding of how to handle flies for behavioral assays and also measure the grooming behavior.

Research

JoVE Journal

Neuroscience

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

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 ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

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Methods to Assay Drosophila Behavior

Methods to Assay Drosophila Behavior

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Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

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A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

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Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

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

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

Høyoppløselig Kvantifisering av Lukt styrt Atferd<em&gt; Drosophila melanogaster</em&gt; Bruke<em&gt; Flywalk</em&gt; Paradigm

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Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

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Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...