Name: drosophila courtship conditioning as a measure of learning and memory
Uploaded: 2017-06-05T13:00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about Drosophila Courtship Conditioning As a Measure of Learning and Memory at JoVE.com

We acknowledge the Vienna Drosophila Resource Center for providing the Drosophila strains. Additionally, stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This research was supported in part by the European Union's FP7 large-scale integrated network Gencodys to K.K., H.v.B., and A.S. and by NSERC Discovery and CIHR Project Grants to J.M.K.

BEMÆRK: I protokollen skitseret nedenfor beskrives en replik af indsamling, træning og testning. For at teste reproducerbarheden af ​​resultaterne skal disse trin gentages parallelt, i flere dage og med separate grupper af fluer ( tabel 1) . Protokollen er baseret på en 10 dages livscyklus fra æg til voksen, hvilket er normalt ved opdræt af fluer under konstante forhold på 25 ° C, 70% fugtighed og en 12 h lys / mørk cyklus. Alle aspekter af denne protokol forudsætter, at betingelserne holdes konstante gennem hele analysen. Tiderne er angivet som timer, før lys tændes (BLO) eller efter lys tændes (ALO) i inkubatoren, da dette let kan indstilles afhængigt af forskerens foretrukne tidspunkt på dagen. Brug kun CO 2 gas til indledende indsamling af naive mannlige fluer og til indsamling af premierede kvinder. Denne protokol til domstolskendelse består af følgende trin: <ol><li> EEtablering af premierede kvindelige samlingskulturer </li><li> Etablering af kulturer til indsamling af mandlige forsøgspersoner </li><li> Forberedelse af boligblokke </li><li> Etablering af parringsflasker til fremstilling af standardiserede premierede hunner </li><li> Indsamling af mandlige forsøgspersoner </li><li> Uddannelse </li><li> Test </li><li> Video data analyse og statistik </li></ol> 1. Etablering af prædiverede kvindelige samlingskulturer <ol><li> Forbered strømforsyning 16 . Kogt 0,8% (vægt / volumen) agar, 8% (vægt / volumen) gær, 2% (vægt / volumen) gærekstrakt, 2% (vægt / volumen) pepton, 3% (vægt / volumen) saccharose, 6% Vægt / volumen) glucose, 0,05% (vægt / volumen) MgS04 og 0,05% (vægt / volumen) CaCl2 i vand i 15 minutter. Lad opløsningen afkøle til 70 ° C, inden der tilsættes 0,05% (w / v) methylparaben (FORSIGTIG: giftig) og 0,5% (vol / vol) propionsyre (FORSIGTIG: giftig). Bland godt ved omrøring under afkøling yderligere til 50 ° C for at opnå en homogen opløsning. </li><li> Før fødevaren såLokkes ved stuetemperatur, tilsæt ~ 50 ml strømforsyning til hvert 175 ml hætteglas af plast. Lad fødevaren afkøle yderligere. Luk hætteglasset med et stik. BEMÆRK: Powerfood er en specialiseret fødevareblanding formuleret specielt til produktion af et stort antal fluer, formodentlig ved at fremkalde æglægning. Powerfood bruges ikke til at producere mandlige fluer, der vil blive anvendt til adfærdsanalyse (trin 2), fordi den atypiske diæt og potentielle trængsel kan påvirke udviklingen. </li><li> På dag -11 ( Tabel 1 ) starter 5-20 vildtype kulturer med ca. 60-100 fluer (en blanding af hanner og kvinder) i powerfood hætteglas; Disse vil blive anvendt i trin 4 til fremstilling af standardiserede premierede kvinder 17 . Tilføj et filterpapir til hvert hætteglas for at øge det område, hvor larverne kan hvile Dette vil øge antallet af fluer, der kan lukke. </li><li> Gentag regelmæssigt trin 1.1-1.3 igennem eksperimentet for at opnå tilstrækkelige nyligt lukkede fluer som input til&quot;Etablering af parringsflasker til produktion af standardiserede premerede hunner&quot; (trin 4). </li></ol> 2. Etablering af kulturer til indsamling af testpersoner til mænd <ol><li> Forbered normal mad 16 , lavet med 0,5% (vægt / volumen) agar, 2,75% (vægt / volumen) gær, 5,2% (vægt / volumen) majsmel, 11% (vægt / volumen) sukker, 0,05% ) Methylparaben og 0,5% (vol / vol) propionsyre i vand som beskrevet i trin 1.1 og 1.2. Luk hætteglasene på 175 ml med et hætteglas med stikkeglas. </li><li> På dag -10 ( tabel 1 ) placeres ca. 10-20 mænd med ca. 30-75 jomfruhunner ( Materialer / Udstyrstabell ) i hvert 175 ml hætteglas indeholdende normal mad. Tilføj et filterpapir for at øge overfladearealet til pupering og for at maksimere produktiviteten. </li><li> Etablere tre til seks 175 ml hætteglas pr. Genotype for at opnå det krævede antal forsøgspersoner. BEMÆRK: Flere hætteglas kan være nødvendige, afhængigt af produktiviteten af ​​den ønskede genotype. </li></ol> 3. Fremstilling af boligblokke ( figur 2A ) <ol><li> Smelt ca. 50 ml strømforsyning pr. Boligblok i en mikrobølgeovn eller tilbered det frisk. </li><li> Tilsæt 500 μL strømforsyning til hver brønd i en 96-brønds flad bundblok ved brug af en multidispenser pipette. </li><li> Lad fødevaren størkne ved stuetemperatur. </li><li> Dæk blokken med PCR klæbende film og brug en nål til at lave mindst 4 huller pr. Brønd for at give frisk luft til fluerne. </li><li> For at kunne åbne hver brønd skal du bruge et barberblad til at skære klæbemidlet i længderetningen mellem hver række. Lad filmen være intakt i den ene ende af blokken. </li><li> Blokkene kan opbevares ved 4 ° C i op til 2 dage. BEMÆRK: Tillad blokken at genudligne til stuetemperatur før brug. </li></ol> 4. Etablering af parringsflasker til fremstilling af standardpensionerede kvinder <ol><li> På dag -1, fjern ogKassere alle voksne vildtype fluer fra premierede kvindelige samlingskulturer ved 2-5 timer BLO. </li><li> Indsamle fluer ved hjælp af aspiratoren ( figur 2B ) fra disse hætteglas i intervaller på 2- til 3 h ( f.eks. Ved 30 min, 2,5 h og 5 h ALO) og læg dem i et nyt hætteglas til powerfood suppleret med en lille mængde gær Indsæt og filtrer papir. </li><li> For at undgå trængsel og fremme en optimal parringsatmosfære, overfør ikke mere end 150-200 fluer til hvert nyt hætteglas. Sørg for parring af alle kvinder ved at give mindst 25% hanner. Sørg for, at der er tilstrækkelige hunder til stede i de parringsflasker, der passer til størrelsen af ​​eksperimentet. BEMÆRK: Da dette er et afgørende skridt i protokollen, skal du sørge for, at kun nyligt lukkede fluer og ingen gamle fluer, larver eller popper overføres til det nye parringsglas. </li><li> Inkubér disse &quot;parringshætteglas&quot; i fire dage for at give tilstrækkelig tid til at alle kvinder har parret. </li></ol> 5. ColLektion af mandlige testpersoner <ol><li> På dag 1 ( Tabel 1 ) i 2-3 timer BLO skal du bruge CO 2 til at fjerne alle voksne fluer fra hætteglassene til hankamling (trin 2), Men lad flere fluer lukke i løbet af de næste par timer. </li><li> I løbet af de næste 5-6 timer skal du fjerne nyligt lukkede fluer hver 20-30 minutter ved hjælp af CO 2 og sætte hver mand i en individuel brønd i husblokken (trin 3) ved hjælp af aspiratoren ( figur 2B ). </li><li> Forsegl brønden med den klæbende PCR film. BEMÆRK: Dette er et afgørende skridt i protokollen. Hannerne skal indsamles ofte. Samlede mænd skal isoleres i boligblokken tæt på tidspunktet for eclosion, når de viser bleg pigmentering og tilstedeværelsen af ​​meconium i den gennemskinnelige mave. BEMÆRK: Gentag brug af aspiratoren tillader overførsel af fluer; Uhensigtsmæssig brug vil imidlertid understrege fluerne og forårsage varians i analysen (se diskussionen). </li><li> Formålet med coLlect op til 48 mænd pr. Genotype. Dette giver et lille overskud til det maksimale antal mænd, der er brug for til analyse af både naive og uddannede betingelser, hvilket giver mulighed for noget tab under senere overførelsestrin. </li></ol> 6. Uddannelse <ol><li> Fjern fluerne fra parringsflasken (trin 4.2) ved hjælp af CO 2 og adskilt de premierede hunner fra hannerne. </li><li> Ved hjælp af aspiratoren skal der tilføjes en enkelt bedøvet, premieret kvinde til hver brønd i en række af en ny boligblok. </li><li> Brug aspiratoren og uden anæstesi, overfør en individuel naiv mand fra husblokken, der er opstillet i trin 5.2, til brønden, der indeholder en premieret kvinde. Efter at hanen er anbragt i brønden, skal man forsegle straks med klæbemiddelfilmen; Lad ikke hanen flygte. BEMÆRK: Overfør mandlige fluer fra aspiratoren til boligblokken ved at udnytte deres naturlige &quot;negative geotaxis&quot; -adfærd (se diskussionen). </li><li> Gentag trin6,2-6,3, indtil der er tilstrækkeligt mandlige kvindelige par. Ideelt set etablere 24 par, to fulde rækker af en boligblok, pr. Genotype. Efterlad de resterende naive mænd i den originale boligblok, der er oprettet i trin 5.2. </li><li> Forlad de mandlige kvindelige par uforstyrret i løbet af træningsperioden ( tabel 2 , figur 1B ). BEMÆRK: I løbet af denne tid vil hanen blive ret og blive afvist af den premierede kvinde. Til læring og STM er træningsperioden 1 time og for LTM er træningsperioden 7-9 timer. </li><li> Afslut træningen ( Tabel 2 , Figur 1B ) ved at bruge en aspirator til forsigtigt at adskille hanen fra den premierede hun; Brug ikke anæstesi. Placer den separerede han i en ny boligblok. </li><li> Brug aspiratoren til at overføre alle naive mænd forsigtigt og uden anæstesi fra husblokken i trin 5.2 til en ny boligblok. BEMÆRK: Dette trin er valgfrit for STM og LEARning, men det er meget vigtigt for LTM, fordi fluerne er anbragt i yderligere 24 timer for at teste LTM. </li><li> For STM og LTM, lad mændene hvile i henholdsvis 1 h og ~ 24 h ( tabel 2 , figur 1B ) før testning (trin 7). </li><li> Til læring skal du straks teste de uddannede og naive mænd (trin 7). </li></ol> 7. Testning <ol><li> Indsamle fluer fra parringsflasker (trin 4.2) ved hjælp af CO 2 og adskille de premierede kvinder fra hannerne. </li><li> Lad hunnene genvinde fra bedøvelsen i mindst 1 time i et hætteglas indeholdende normal mad. </li><li> Monter videooptageren på forhånd ( figur 2C ) for at få alt udstyr klar, før testen starter. </li><li> Start testen i henhold til de forskellige tidslinjer for læring, STM og LTM ( Tabel 2 , Figur 1B ). Udfør test straks efter træningTil læring, 1 time efter træning for STM og 24 timer efter træning for LTM. </li><li> Overfør forsigtigt en individuel mand fra den hvile boligblok eller fra træningshusblokken, hvis læringen testes (trin 6.7, trænet, trin 6.8, naivt) til den ene halvdel af en forankringsarena med divideret lukket ( figur 2D ; Se Fil S1 for en byggeplan). BEMÆRK: Brugen af ​​den naturlige &quot;negative geotaxis&quot; -adfærd bør være tilstrækkelig til at overføre de mandlige fluer fra aspiratoren til domstolenes arena (Diskussion). </li><li> Flyt forsigtigt indgangen til næste arena og gentag trin 7.5, indtil alle 18 arenaer indeholder en mand. </li><li> Ved hjælp af aspiratoren og uden CO2 tilsættes en premieret kvinde (samlet i trin 7.2) til den anden halvdel af alle 18 arenaer. </li><li> Placer forsigtigt kammerkammeret under kameraet, med åbningen af ​​brøndene nedad ( figur 2C </strong&gt;). </li><li> Fjern dividerne af arenaerne for at muliggøre direkte interaktion mellem hannerne og premierede kvinder. </li><li> Start straks optagelsen i mindst 10 minutter. BEMÆRK: Ved brug af en to-kamera opsætning kan paralleloptagelse af to skibsplader ske i overlappende tidsrammer for at maksimere effektiviteten. </li><li> Tøm domstolene ved hjælp af en håndholdt støvsuger og lad domstolskammeret ventilere før genanvendelse. </li><li> Gentag trin 7.4-7.11, indtil testen af ​​alle genotyper og betingelser ( dvs. naiv og trænet) er afsluttet. </li></ol> 8. Video data analyse og statistik <ol><li> Beregn domstolenes indeks (CI), defineret som den procentdel af tid, som mandlige domstole i løbet af de første 10 minutter af testperioden, for hver enkelt hanflyvning. BEMÆRK: Dette kan gøres manuelt ved at observere stereotyp frieriadfærd ( figur 1A ) eller af digSyng computer software til automatiseret kvantificering af domstolsadfærd (diskussion). BEMÆRK: Det anbefales at analysere 40-60 hanner pr. Tilstand i løbet af tre dage for at opnå tilstrækkelig statistisk effekt og dømme konsistensen af ​​CI-data. </li><li> Beregn læringsindekset (LI), defineret som procentvis reduktion i gennemsnitlig CI for uddannede mænd sammenlignet med naive mænd (LI = (CI naiv - CI uddannet ) / CI naiv ). Evaluer LI for hver testdag og sammenlign den med den kumulative LI beregnet fra alle testdage kombineret. </li><li> Lav en separat fladdatafil med to søjler med &quot;Genotype&quot; og &quot;CI&quot; som overskrifter. BEMÆRK: Disse overskrifter er store og små bogstaver. Navnet på genotypen for hvert CI skal bestå af en beskrivelse af genotypen efterfulgt af en understregning og træningsbetingelsen ( f.eks. Genotype_N og genotype_LTM osv., Hvor N = naiv og LTM = lang sigthukommelse; Se Supplerende fil S2 til et eksempel). Denne annotation er essentiel, da funktionen analearn vil identificere træne og naive fluer baseret på de tegn, der er til stede efter den første understregning i kolonnen &quot;Genotype&quot;. </li><li> Brug analearn R-scriptet (Supplemental File S3) til at udføre en randomiseringstest for at bedømme den statistiske betydning af forskelle mellem LI-værdierne fra forskellige genotyper. <ol><li> Kilde scriptet (Supplerende fil S3) i R 18 , som definerer en funktion kaldet &quot;analearn.&quot; BEMÆRK: Funktionsdefinitionen er: analearn &lt;- funktion (nboot = 10.000, naivelevel = &#39;N&#39;, refmutation = NA, datnavn = NA, header = TRUE, seed = NA, writeoutput = TRUE). </li><li> Start funktionen ved at indtaste &quot;analearn ()&quot; i kommandolinjen R og vælge den datafil, der skal analyseres (produceret i trin 8.3) via pop op-vinduet. </li><li> Vælg den referencemutation, somEr kontrolgenotypen ved at indtaste det tilsvarende tal og trykke på enter. BEMÆRK: Efter at have valgt referencegenotypen tager scriptet flere sekunder til at udføre 10.000 bootstrap-replikater. </li><li> Overhold udgangstabellen (tabel 3), som indeholder genotypen, indlæringsbetingelsen ( dvs. læring, STM eller LTM), gennemsnitlig CI naiv, gennemsnitlig CI uddannet, LI, forskellen mellem kontrollens LI i forhold til den eksperimentelle tilstand (LI dif), den nedre grænse (LL) og øvre grænse (UL) for 95% konfidensintervallet for LI dif og p- værdien, der indikerer sandsynligheden for, at der ikke er nogen signifikant forskel. BEMÆRK: analearn gemmer en output tekstfil i den mappe, hvor datafilen er placeret. Udgangstabellen vises dog også i R-Studio-konsollen. Standardnavnet er opbygget baseret på navnet på den givne datafil. </li><li> Der er flere argumenter i analearn-funktionen, som kan bruges til at ændre deFejlindstillinger af funktionen for at justere parametrene for bootstrapping. BEMÆRK: &quot;nboot&quot; definerer antallet af bootstrap-replikater og er som standard indstillet til 10.000. Denne værdi kan ændres til et helt tal, der er større end nul. Tabel 5 indeholder en række argumenter, der kan bruges til at ændre standardindstillingerne for funktionen. Det anbefales dog ikke at bruge data, der er produceret med et lavt antal bootstrap-replikater. </li></ol></li></ol>

<ol>
	<li>Quinn, W. G., Harris, W. A., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+behavior+in+Drosophila+melanogaster.">Conditioned behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. USA. 71, 708-712 (1974).</li><li>Livingstone, M. S., Sziber, P. P., Quinn, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+calcium/calmodulin+responsiveness+in+adenylate+cyclase+of+rutabaga,+a+Drosophila+learning+mutant.">Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant.</a> Cell. 37, 205-215 (1984).</li><li>Quinn, W. G., Sziber, P. P., Booker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+memory+mutant+amnesiac.">The Drosophila memory mutant amnesiac.</a> Nature. 277, 212-214 (1979).</li><li>Dudai, Y., Jan, Y. N., Byers, D., Quinn, W. G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dunce,+a+mutant+of+Drosophila+deficient+in+learning.">dunce, a mutant of Drosophila deficient in learning.</a> Proc. Natl. Acad. Sci. USA. 73, 1684-1688 (1976).</li><li>Dubnau, J., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+discovery+in+Drosophila:+new+insights+for+learning+and+memory.">Gene discovery in Drosophila: new insights for learning and memory.</a> Annu. Rev. Neurosci. 21, 407-444 (1998).</li><li>Siegel, R. W., 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=Conditioned+responses+in+courtship+behavior+of+normal+and+mutant+Drosophila.">Conditioned responses in courtship behavior of normal and mutant Drosophila.</a> Proc. Natl. Acad. Sci. USA. 76, 3430-3434 (1979).</li><li>Kamyshev, N. G., Iliadi, K. G., Bragina, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+conditioned+courtship:+two+ways+of+testing+memory.">Drosophila conditioned courtship: two ways of testing memory.</a> Learning &amp; memory. 6, 1-20 (1999).</li><li>McBride, S. 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=Mushroom+body+ablation+impairs+short-term+memory+and+long-term+memory+of+courtship+conditioning+in+Drosophila+melanogaster.">Mushroom body ablation impairs short-term memory and long-term memory of courtship conditioning in Drosophila melanogaster.</a> Neuron. 24, 967-977 (1999).</li><li>Keleman, K., Kruttner, S., Alenius, M., 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=Function+of+the+Drosophila+CPEB+protein+Orb2+in+long-term+courtship+memory.">Function of the Drosophila CPEB protein Orb2 in long-term courtship memory.</a> Nature Neurosci. 10, 1587-1593 (2007).</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, 1702-1714 (1994).</li><li>Keleman, K., 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=Dopamine+neurons+modulate+pheromone+responses+in+Drosophila+courtship+learning.">Dopamine neurons modulate pheromone responses in Drosophila courtship learning.</a> Nature. 489, 145-149 (2012).</li><li>Kramer, J. 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=Epigenetic+regulation+of+learning+and+memory+by+Drosophila+EHMT/G9a.">Epigenetic regulation of learning and memory by Drosophila EHMT/G9a.</a> PLoS Biol. 9. 9, e1000569 (2011).</li><li>Ishimoto, H., Sakai, T., Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+signaling+regulates+the+formation+of+long-term+courtship+memory+in+adult+Drosophila+melanogaster.">Ecdysone signaling regulates the formation of long-term courtship memory in adult Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. USA. 106, 6381-6386 (2009).</li><li>Kochinke, K., 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+Phenomics+Analysis+Deconvolutes+Genes+Mutated+in+Intellectual+Disability+into+Biologically+Coherent+Modules.">Systematic Phenomics Analysis Deconvolutes Genes Mutated in Intellectual Disability into Biologically Coherent Modules.</a> Am. J. Hum. Genet. 98, 149-164 (2016).</li><li>Buchert, 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+peroxisomal+disorder+of+severe+intellectual+disability,+epilepsy,+and+cataracts+due+to+fatty+acyl-CoA+reductase+1+deficiency.">A peroxisomal disorder of severe intellectual disability, epilepsy, and cataracts due to fatty acyl-CoA reductase 1 deficiency.</a> Am. J. Hum. Genet. 95, 602-610 (2014).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Biology+1:+yeast,+Drosophila+and+C.+elegans.+Drosophila+Maintenance.">Essentials of Biology 1: yeast, Drosophila and C. elegans. Drosophila Maintenance.</a> JoVE. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Biology+1:+yeast,+Drosophila+and+C.+elegans.+An+Introduction+to+Drosophila+melanogaster.">Essentials of Biology 1: yeast, Drosophila and C. elegans. An Introduction to Drosophila melanogaster.</a> JoVE. , (2017).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+Language+and+Environment+for+Statistical+Computing.">R: A Language and Environment for Statistical Computing.</a> R Foundation for Statistical Computing. , (2016).</li><li>Stepien, B. K., 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=RNA-binding+profiles+of+Drosophila+CPEB+proteins+Orb+and+Orb2).">RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2).</a> Proc. Natl. Acad. Sci. USA. 113, E7030-E7038 (2016).</li><li>Basu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomization+Analysis+of+Experimental+Data:+The+Fisher+Randomization+Test.">Randomization Analysis of Experimental Data: The Fisher Randomization Test.</a> J Am Stat Assoc. 75, 575-582 (1980).</li><li>Dankert, H., Wang, L., Hoopfer, E. D., Anderson, D. J., Perona, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+monitoring+and+analysis+of+social+behavior+in+Drosophila.">Automated monitoring and analysis of social behavior in Drosophila.</a> Nat Methods. 6, 297-303 (2009).</li><li>Reza, M. A., 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=Automated+analysis+of+courtship+suppression+learning+and+memory+in+Drosophila+melanogaster.">Automated analysis of courtship suppression learning and memory in Drosophila melanogaster.</a> Fly. 7, 105-111 (2013).</li><li>Schneider, J., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+identification+of+social+interaction+criteria+in+Drosophila+melanogaster.">Automated identification of social interaction criteria in Drosophila melanogaster.</a> Biol. Lett. 10, 20140749 (2014).</li></ol>

The authors have nothing to disclose.

The courtship conditioning assay is a classic paradigm for the analysis of learning and memory in Drosophila. The protocol presented here follows the general methodology described previously6,7,8,9 but includes unique aspects such as practical guidelines, specialized equipment, and a data analysis script9,12 for randomization tests. Using this protocol, it is possible to analyze large numbers of flies in parallel using 96-well flat-bottom blocks (Figure 2A) to collect and train males. The blocks are sealed with PCR adhesive film, which makes the flies easily accessible when required. Additionally, the unique courtship chambers described here allow for the simultaneous pairing of 18 male-female pairs in a nearly two-dimensional space that is optimal for video analysis. The custom-designed courtship chambers are easy to use, and a building plan is provided (File S1, Figure 2D). This protocol, from the establishment of the cultures used to collect test subjects to the acquisition of video data, takes approximately 20 days (Table 1). Additional time is required for the analysis of video data. In our experience, the STM assay is extremely robust. The LTM assay is also quite robust, but it is more sensitive to confounding environmental variables and therefore can be more difficult to master.
Animal behavior can be quite variable. Therefore, critical steps in the protocol must be performed with care to reduce this variance. First, gentle use of the aspirator (Figure 2B) will reduce the stress that can be imposed by rough handling or by blowing out too strongly. A suggested method of transferring individual flies out of the aspirator is by using negative geotaxis. As flies tend to walk up, one can simply point the tip of the aspirator up; just before the fly reaches the tip, a gentle blow is sufficient to let the fly out. Additionally, to let the males into the courtship chambers before testing, a blow is often not necessary.
Another important step is the collection and generation of male test subjects. All males must be collected when they are very young and socially na&#239;ve. This can be achieved by frequent collection during the peak periods of eclosion (step 5.2). If males are not collected in this tight timeframe, they can have early social interactions, which may result in poor learning or high variability in CI. Another factor of male test subjects that should be assessed is the genetic background. Different genetic backgrounds will exhibit different levels of na&#239;ve courtship and may also differ in general activity or locomotor ability. When comparing multiple genotypes, care should be taken with regard to genetic background in order to avoid these confounding factors that may influence LI scores. Additionally, the distribution of CI data should be carefully assessed. CI data can be both parametric and non-parametric, depending on the genotype or other environmental factors. In some cases, if the distribution of CI is dramatically skewed away from a normal distribution, it may be better to use the median CI rather than the mean for the calculation of LIs. However, in our experience, the use of median or mean CI does not make a difference in the statistical interpretation of the data, and the use of the mean CI is the common practice in the literature.
For successful courtship conditioning, the active rejection of male courtship attempts by premated females is crucial during the training period. It is important to ensure that the premated females used in this assay have been efficiently mated and are thus not allowing copulation. This premating is established in the mating vials prepared in step 4, where male and females flies are housed together for 4 days (Table 1). Subsequently, mating can be monitored by regular examination of testing videos and by observing male-female pairs during training. If mating does occur, there are several measures that can be taken during the preparation of premated females. First, premated females should be reared under optimal breeding conditions. Vials can be supplemented with yeast paste and a folded filter paper to increase potential mating surfaces. The incubation of flies under the conditions described here has produced robust premated females in the past, but this may vary in different labs and with the use of different genetic strains. Therefore, it may be necessary to optimize the generation of premated females by varying the incubation time and conditions.
Quantification of courtship behavior is another critical step in this protocol. This can be done manually or automatically using specialized software programs9. Automated quantification is fast and, in principle, unbiased. Several programs have been published21,22,23; however, they are not straightforward to use, often requiring specialized video formats and advanced computational skills. Manual quantification is easy and accurate, but it is highly labor intensive and subject to individual variability and bias. It is important to emphasize that this protocol does not address the requirements for video formatting that are potentially required for the automated quantification of CIs. For manual quantification, use any simple video recording device that has the potential to produce a video of sufficient quality to accurately observe courtship behavior. For automated quantification, there will likely be different requirements depending on the software used, and users should investigate this thoroughly if automated quantification is desired.
In combination with the extensive tools that are available for the genetic manipulation of flies, the courtship conditioning assay provides a robust readout that can be used to dissect molecular mechanisms and neuronal networks involved in learning and memory.

De molekylære mekanismer, der ligger til grund for læring og hukommelse, bevares gennem mange arter. Den banebrydende arbejde screening Drosophila melanogaster mutant linjer for defekter i olfaktorisk læring og hukommelse har givet nøgle molekylær indsigt i de processer, der ligger til grund for læring og hukommelse 1 . Disse undersøgelser identificerede nogle af de første gener involveret i læring og hukommelse , såsom rutabaga 2 , amnesiac 3 og dunce 4 , hvilket afslørede en kritisk rolle for signalering af cyclisk adenosinmonophosphat (cAMP) 5 . Tidlige genetiske skærme til hukommelsesmutanter blev primært udført under anvendelse af olfaktorisk konditionering. Flere metoder til måling af andre former for læring og hukommelse er dog opstået over tid. Et af de mest anvendte lærings- og hukommelsesparadigmer, og analysen beskrevet her, er kendt som cOurtship conditioning, som først blev beskrevet af Siegel og Hall 6 og senere blev raffineret af flere andre forskningsgrupper 7 , 8 , 9 . Courtship-konditionering er afhængig af tilstedeværelsen af ​​et specifikt feromon, cis- vaccenylacetat (cVA), på kvindens underliv, som aflejres af hanen under copulation. At mærke cVA på kvindens underliv reducerer naturligvis frieriadfærd, og når det kombineres med kvindens afvisning, er effekten af ​​cVA på mandlig kropsreduktion dramatisk forbedret. Reaktionen hos hanflyvninger i denne analyse kan let kvantificeres ved at observere deres særskilte retfærdighedsadfærd, som er kendetegnet ved orientering mod og efter kvinden, tapping, udstrækning og vibrerende vingen, slikker og forsøger kopiering 10 ( figur 1A ). Mandlige fluer lærer at distinguis<html> H mellem modtagelige, jomfruelige og ikke-modtagelige parede kvinder 11 og efter seksuel afvisning viser de nedsat frieri til ikke-modtagelige kvinder i op til 9 dage 8 . Denne naturlige opførsel kan bruges til at belyse de mekanismer, der ligger til grund for læring, kortvarig hukommelse (STM) og langvarig hukommelse (LTM) 8 , 9 , 12 . Læring er defineret som den øjeblikkelige reduktion i retssagens adfærd, der opstår i løbet af træningsperioden, og kaldes ofte som øjeblikkelig tilbagekaldshukommelse, målt 0 - 30 min efter udsættelse for en parret kvinde 6 , 8 . STM måles mellem 30 minutter og 1 time efter træning, mens LTM oftest måles 24 timer efter træning 8 ( figur 1B ). STM kan induceres ved hjælp af en 1-timers træningsperiode, men den varer kun i 2-3 timerS = &quot;xref&quot;&gt; 6 , 8 . I de fleste læringsparadigmer kan LTM kun fremkaldes ved adskillige forsøg på gentagen træning. Mcbride et al . (1999) 8 viste at tre adskilte, 1-timers træningssessioner var tilstrækkelige til at fremkalde hukommelseshukommelse, der varede i op til 9 dage, i modsætning til de 2-3 timer, der blev fremkaldt af en enkelt 1-timers træningssession. McBride et al . 8 viste også, at en enkelt 5-timers træningssession gav et lignende LTM respons i op til 9 dage. Fluer rømmer ikke konstant i løbet af denne 5-timers periode, og producerer faktisk deres egen indbyrdes træning for at fremkalde LTM i en enkelt træning. Dette er meget vigtigt fra et praktisk perspektiv, der øger den lethed, som denne analyse kan bruges til at undersøge LTM. Nuværende protokoller overvejende bruger en enkelt træningssession på 7-h for LTM 11 , 12 . Flere undersøgelser har undersøgt forskelligeMutante forhold, der har specifikke mangler i forskellige aspekter af courtship læring. For eksempel påvirker svampekroppens ablation STM og LTM, men ikke lærer 8 . Mutationer i amnesiacgenet , som først blev defineret som en specifik regulator af hukommelse ved hjælp af olfaktorisk konditionering 3 , påvirker STM, men ikke lærer 6 . Afbrydelse af oversættelsesregulatoren orb2 (oo18 RNA-bindende (orb) CPEB2-underfamilie) og ecdyson-signalering har udelukkende virkning på LTM 9 , 13 . Således er kriminalitetskonditionering et nyttigt paradigme til at opløse de mekanismer, der ligger til grund for de forskellige faser af læring og hukommelse. Dette arbejde demonstrerer en optimeret eksperimentel opsætning, der muliggør den relativt høje gennemløbstest af kriminalitetskonditionering. Desuden beskriver den et statistisk analyse script og diskuterer kritiske faktorer i analysen. Det er shEjer her, at Drosophila - gendihydroxyacetonphosphatacyltransferasen ( Dhap-at ) Er påkrævet i neuroner for LTM, men ikke for STM. Den menneskelige ortholog af dette gen, glycerolphosphat-O-acyltransferase ( GNPAT ), muteres i rhizomelisk chondrodysplasia punctata type 2 14 , en autosomal-recessiv lidelse karakteriseret ved alvorlig intellektuel invaliditet, anfald og flere andre kliniske egenskaber 15 . I denne sammenhæng kan kriminalitetskonditionering anvendes til funktionelt validering af rollen som menneskelige sygdomsgener i læring og hukommelse, der danner grundlag for mekanistiske undersøgelser.

<table border="0" cellpadding="0" cellspacing="0" width="1074">
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			<td height="21" style="height:21px;width:453px;">P{KK101437}VIE-260B</td>
			<td style="width:159px;">VDRC</td>
			<td style="width:119px;">101437</td>
			<td style="width:343px;">Dhat-at-RNAi in 60100 background&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">P{KK108109}VIE-260B</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;">-</td>
			<td>Control-RNAi in 60100 background (gift from K. Keleman)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">w+, UAS-dcr2/yhh;;elav-Gal4 (III)&#160;</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;">-</td>
			<td>panneuronal driver line</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Containers for plant tissue culture</td>
			<td style="width:159px;">VWR</td>
			<td style="width:119px;">960177</td>
			<td>175 mL plastic vials</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Folded filters</td>
			<td style="width:159px;">Whatman</td>
			<td style="width:119px;">10311643</td>
			<td>Filter paper to enlarge area flies can pupate on&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Flat-bottom blocks (96-wells)</td>
			<td style="width:159px;">Qiagen</td>
			<td style="width:119px;">19579</td>
			<td>Used for housing blocks</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">MicroAmp Clear Adhesive Film</td>
			<td style="width:159px;">Applied Biosystems</td>
			<td style="width:119px;">4306311</td>
			<td>PCR adhesive film as lid on flat-bottom blocks</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Razor blade</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;">-</td>
			<td>Any sharp will do</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Needle&#160;</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;">-</td>
			<td>0.8 mm diameter</td>
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		<tr height="168">
			<td height="168" style="height:168px;width:453px;">Aspirator</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:343px;">Cut a 1mL pipet tip with scissors in order to have two pieces. The narrow tip of the pipettip is placed as fly entrance in a ~80 cm flexible hose. To prevent a fly from getting in the hose, a normal piece of cotton or small mesh gaze is placed in between the tip and the hose. The other half of the pipettip can be used as mouth piece at the end of the hose.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Courtship chambers</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;">-</td>
			<td>file S1 can be opened with indicated CAD software&#160;</td>
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		<tr height="210">
			<td height="210" style="height:210px;width:453px;">Camcorder</td>
			<td style="width:159px;">Sony</td>
			<td style="width:119px;">-</td>
			<td style="width:343px;">camera specification: &#62;4M pixels, full HD. 
			For manual quantification, any simple video recording device has the potential to produce a video of sufficient quality to observe courtship behavior accurately. For automated quantification, there will likely be different requirements depending on the software to be used, and users should investigate this thoroughly if automated quantification is desired.</td>
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			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:159px;">Company</td>
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			<td>Comments</td>
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			<td height="21" style="height:21px;width:453px;">power food</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Agar</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">A7002</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Yeast</td>
			<td style="width:159px;">Bruggeman</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Yeast extract</td>
			<td style="width:159px;">MP biomedicals</td>
			<td>0210330391&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:453px;">Peptone</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P6838</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Sucrose</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">S9378</td>
			<td></td>
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			<td height="21" style="height:21px;width:453px;">Glucose</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">G7021</td>
			<td></td>
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			<td height="25" style="height:25px;width:453px;">MgSO4</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">M2643</td>
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			<td height="25" style="height:25px;width:453px;">CaCl2</td>
			<td style="width:159px;">Merck</td>
			<td>1023780500</td>
			<td></td>
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			<td height="21" style="height:21px;width:453px;">Methylparabene (CAUTION)</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">H5501</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Propionic acid (CAUTION)</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P1386</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Demineralized water</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Yeast paste</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;"></td>
			<td>yeast grains and water mixture in a 1:1 ratio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="21" style="height:21px;width:453px;">normal food</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Agar</td>
			<td style="width:159px;">MP biomedicals</td>
			<td>215017890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Yeast</td>
			<td style="width:159px;">bruggeman</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Corn flour</td>
			<td style="width:159px;">de Molen</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Sugar</td>
			<td style="width:159px;">de Molen</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Methylparabene (CAUTION)</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">H5501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Propionic acid (CAUTION)</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P1386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Demineralized water</td>
			<td style="width:159px;">-</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

drosophila courtship conditioning as a measure of learning and memory

Mange indblik i de molekylære mekanismer, der ligger til grund for læring og hukommelse, er blevet belyst ved brug af enkle adfærdsmæssige analyser i modelorganismer som frugtflyve , Drosophila melanogaster . Drosophila er nyttig til forståelse af de grundlæggende neurobiologiske underliggende kognitive underskud som følge af mutationer i gener forbundet med menneskelige kognitive lidelser, såsom intellektuel invaliditet (ID) og autisme. Dette værk beskriver en metode til afprøvning af læring og hukommelse ved hjælp af et klassisk paradigme i Drosophila kendt som kriminalitetskonditionering. Mand flyver domstol kvinder med et klart mønster af let genkendelige adfærd. Præsterede kvinder er ikke modtagelige for parring og vil afvise mannens copulationsforsøg. Som svar på denne afvisning reducerer mandlige fluer deres frieriadfærd. Denne lærte reduktion i retssagens adfærd måles over tid og tjener som indikator for læring og hukommelse. Den grundlæggende numéRical udgang af dette assay er domstolsindekset (CI), som defineres som den procentdel af tid, som en mand bruger hofning i løbet af et 10 min interval. Læringsindekset (LI) er den relative reduktion af CI i fluer, der har været udsat for en premieret kvinde sammenlignet med naive fluer uden tidligere sociale møder. Til statistisk sammenligning af LI&#39;er mellem genotyper anvendes en randomiseringstest med bootstrapping. For at illustrere hvordan analysen kan anvendes til at adressere rollen som et gen relateret til læring og hukommelse, blev den pan- neuronale nedbrydning af dihydroxyacetonphosphatacyltransferase ( Dhap-at ) karakteriseret her. Den menneskelige ortholog af Dhap-at , glyceronphosphat O-acyltransferase ( GNPT ) er involveret i rhizomelic chondrodysplasia punctata type 2, et autosomalt recessivt syndrom kendetegnet ved svær ID. Ved anvendelse af kriminalitetskonditioneringsassayet blev det fastslået, at Dhap-at er påkrævet til langsigtet hukommelse, men ikke forKortvarig hukommelse. Dette resultat tjener som grundlag for yderligere undersøgelse af de underliggende molekylære mekanismer.

The courtship conditioning assay can be used to measure learning and memory in Drosophila. In order to demonstrate this, the results presented here analyze STM and LTM in control flies compared to flies with the neuron-specific knockdown of Dhap-at. Control males express an RNA interference hairpin sequence targeting a Caenorhabditis elegans-specific gene, putative zinc finger protein C02F5.1219. This control strain ensures an equal number of upstream activating sequence (UAS) elements between the knockdown and control in the same genetic background, and the control RNAi hairpin accounts for any non-specific effects of the RNAi system. Control males (genotype: UAS-dcr2/+; P{KK108109}VIE-260B/+; elav-Gal4/+) show a significant reduction in CI in trained versus na&#239;ve for both STM (Figure 3A, p = 1.2 x 10-4, Mann-Whitney U-test) and LTM (Figure 3B, p = 1.2 x 10-4, Mann-Whitney U-test). This result reflects the normal capacity for learning and memory in these flies. Dhap-at knockdown flies (genotype: UAS-dcr2/+; P{KK101437}VIE-260B/+; elav-Gal4/+) also show a significant reduction of CI in trained versus na&#239;ve flies for STM (Figure 3A, p = 1.2 x 10-4, Mann-Whitney U-test). However, they do not show a significant reduction in CI after LTM training (Figure 3B, p = 0.33, Mann-Whitney U-test). The Mann-Whitney U-test was used to compare the mean CI of na&#239;ve and trained flies due to the non-parametric distribution of the CI data for some conditions (Figure 3A and 3B). The differences observed through the analysis of CIs are reflected by the LI for each genotype, which measures the percent reduction in CI in na&#239;ve versus trained flies (Figure 3C). There is no significant difference in LI between controls and Dhap-at knockdown flies for STM (p = 0.115, 10,000 bootstrap replicates, Figure 3C, Table 3), whereas the LI for LTM is significantly lower in Dhap-at knockdown flies (p = 0.0034, 10,000 bootstrap replicates, Figure 3C, Table 3). For the comparison of LI between genotypes, a randomization test20 adapted from a method first recommended by Kamyshev et al.7 was used. Since the LI is a single value derived from population data (i.e., mean CI-na&#239;ve and mean CI-trained), standard statistical methods that rely on experimentally derived distributions do not apply. The randomization test is distribution-free and uses bootstrapping (i.e., random sampling with replacement) to generate hypothetical data sets that are derived from the actual data. The analearn script (File S3) generates a set of hypothetical LIs for each genotype and calculates the difference between the control and the test genotype (LIdiff). This process is repeated 10,000 times, and the resulting values are used to determine the 95% confidence interval of LIdiff (Table 3). This data is used to generate a p-value indicating the probability that the LI of the two genotypes is different. The results shown here demonstrate that Dhap-at is required in neurons for LTM but not for STM.
In order to control for day-to-day variability, CIs and LIs are compared between replicate days (Table 4). Although some fluctuation in LI is observed from day to day, the results are generally reproducible. It is important to note that CI data can vary greatly depending on the control strain used and the environmental conditions of testing. The CI data shown here is typical for this control genotype, but other genotypes may exhibit a higher or lower mean CI and distribution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55808/55808fig1.jpg" /> 
Figure 1: Determination of the Courtship Index and Experimental Overview. (A) Images showing stereotypical male courtship behavior towards a female fly. Different stages of courtship behavior are shown: orientation (I), following (II), wing vibration (extension) and tapping (III), licking (IV), and attempted copulation (V). (B) Schematic overview of training and testing times relative to the incubator light cycle, marked in hours. Training times are indicated with bars, resting periods for STM and LTM are indicated with a dashed line, and the testing start point is indicated as an arrow. Note that the testing time for LTM is the day after after training. <a href="http://cloudflare.jove.com/files/ftp_upload/55808/55808fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55808/55808fig2.jpg" /> 
Figure 2: Equipment used for the Drosophila Courtship Conditioning Assay. (A) The housing block is a flat, bottom block with 500 &#181;L of powerfood per well. It is sealed with a qPCR adhesive film with at least 4 holes per well that were created using a syringe needle with a 0.8 mm diameter. Individual rows are cut lengthwise into strips using a razor blade to allow opening and closing. (B) The aspirator is required for the gentle transfer of male and female flies without the use of anesthesia. The inset shows the tip of the aspirator, closed with a piece of cotton to keep the flies within the tip. (C) Setup of a two-camera system for the simultaneous recording of two courtship chambers. (D) A courtship chamber with 18 arenas. Sliding entry holes are used to place the flies in the arenas. The white dividers can be simultaneously opened to initiate interaction between the males and females. <a href="http://cloudflare.jove.com/files/ftp_upload/55808/55808fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55808/55808fig3.jpg" /> 
Figure 3: Analysis of STM and LTM in Control and Dhap-at Knockdown Flies. (A-B) Boxplots showing the distribution of CI values for na&#239;ve (N) and trained (T) flies of the control (gray) and Dhap-at knockdown (white) genotypes tested for STM (A) and LTM (B). (C) Corresponding LI values for control and Dhap-at knockdown flies tested for STM and LTM. Differences in LI between control and knockdown genotypes were compared using a randomization test (10,000 bootstrap replicates). Error bars indicate the standard error of the mean derived from the LI values calculated on different test days. The genotypes are: w+, UAS-dcr2/+; P{KK108109}VIE-260B/+; and elav-Gal4/+ (Control) and w+, UAS-dcr2/+;P{KK101437}VIE-260B/+; and elav-Gal4/+ (Dhap-at-RNAi). <a href="http://cloudflare.jove.com/files/ftp_upload/55808/55808fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>General</td>
			<td>Collect</td>
			<td>Train</td>
			<td>Test</td>
		</tr>
		<tr>
			<td>day -11</td>
			<td>Start premated female collection cultures (step 1.3)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>day -10</td>
			<td>Start cultures for the collection of male test subjects (step 2.2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>day 1</td>
			<td></td>
			<td>rep. 1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>day 2</td>
			<td></td>
			<td>rep. 2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>day 3</td>
			<td></td>
			<td>rep. 3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>day 4</td>
			<td></td>
			<td>rep. 4</td>
			<td>rep. 1</td>
			<td></td>
		</tr>
		<tr>
			<td>day 5</td>
			<td></td>
			<td></td>
			<td>rep. 2</td>
			<td>rep. 1</td>
		</tr>
		<tr>
			<td>day 6</td>
			<td></td>
			<td></td>
			<td>rep. 3</td>
			<td>rep. 2</td>
		</tr>
		<tr>
			<td>day 7</td>
			<td></td>
			<td></td>
			<td>rep. 4</td>
			<td>rep. 3</td>
		</tr>
		<tr>
			<td>day 8</td>
			<td></td>
			<td></td>
			<td></td>
			<td>rep. 4</td>
		</tr>
		<tr>
			<td>day 9</td>
			<td>Video data analysis and statistics (step 8)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">rep = repeat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Example Timeline for Testing LTM over Three Replicates on Individual Days.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Learning</td>
			<td>STM</td>
			<td>LTM</td>
		</tr>
		<tr>
			<td>Training time</td>
			<td>1 h.</td>
			<td>1 h.</td>
			<td>8 h.</td>
		</tr>
		<tr>
			<td>Resting time</td>
			<td>0 h.</td>
			<td>1 h.&#160;</td>
			<td>~ 24 h.</td>
		</tr>
		<tr>
			<td>start training</td>
			<td>0 h. ALO</td>
			<td>0 h. ALO</td>
			<td>4 h. BLO</td>
		</tr>
		<tr>
			<td>stop training</td>
			<td>1 h. ALO</td>
			<td>1 h. ALO</td>
			<td>4 h. ALO</td>
		</tr>
		<tr>
			<td>start test&#160;</td>
			<td>1 h. ALO</td>
			<td>2 h. ALO&#160;</td>
			<td>0 h. ALO (next day)</td>
		</tr>
		<tr>
			<td colspan="4">ALO = after lights turn on, BLO = before lights turn on, STM = short term memory, LTM = long term memory&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: Training Duration, Training Times, and Testing Times for Learning, STM, and LTM.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genotype</td>
			<td>Learning 
			condition</td>
			<td>CI 
			naive</td>
			<td>CI 
			trained</td>
			<td>LI</td>
			<td>LI 
			difference</td>
			<td>Lower limit 
			(95% conficence interval)</td>
			<td>Upper limit 
			(95% conficence interval)</td>
			<td>p-value</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>STM</td>
			<td>0.467</td>
			<td>0.116</td>
			<td>0.752</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Dhap-at-RNAi</td>
			<td>STM</td>
			<td>0.699</td>
			<td>0.257</td>
			<td>0.633</td>
			<td>0.119</td>
			<td>-0.030</td>
			<td>0.265</td>
			<td>0.116</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>LTM</td>
			<td>0.590</td>
			<td>0.384</td>
			<td>0.348</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Dhap-at-RNAi</td>
			<td>LTM</td>
			<td>0.697</td>
			<td>0.650</td>
			<td>0.068</td>
			<td>0.280</td>
			<td>0.103</td>
			<td>0.446</td>
			<td>0.003</td>
		</tr>
	</tbody>
</table>
Table 3: Statistical Data Produced from the Analearn Script. 
Statistical data produced from the analearn script. The output file of the bootstrapping R-script containing the genotype, learning condition (i.e., learning, STM, or LTM), mean CI na&#239;ve, mean CI trained, LI, difference between LI of the control compared to experimental condition (LI dif), the lower limit (LL) and upper limit (UL) of the 95% confidence interval of LI dif, and the p-value indicating the probability that there is no significant difference.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Control</td>
			<td></td>
			<td></td>
			<td>Dhap-at-RNAi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Average CI na&#239;ve</td>
			<td>Average CI trained</td>
			<td>LI</td>
			<td>Average CI na&#239;ve</td>
			<td>Average CI trained</td>
			<td>LI</td>
		</tr>
		<tr>
			<td>STM</td>
			<td>Day 1</td>
			<td>0.300</td>
			<td>0.125</td>
			<td>0.584</td>
			<td>0.679</td>
			<td>0.239</td>
			<td>0.648</td>
		</tr>
		<tr>
			<td></td>
			<td>Day 2</td>
			<td>0.634</td>
			<td>0.107</td>
			<td>0.831</td>
			<td>0.720</td>
			<td>0.276</td>
			<td>0.617</td>
		</tr>
		<tr>
			<td></td>
			<td>All Days</td>
			<td>0.467</td>
			<td>0.116</td>
			<td>0.752</td>
			<td>0.699</td>
			<td>0.257</td>
			<td>0.633</td>
		</tr>
		<tr>
			<td>LTM</td>
			<td>Day 1</td>
			<td>0.590</td>
			<td>0.441</td>
			<td>0.252</td>
			<td>0.630</td>
			<td>0.646</td>
			<td>-0.027</td>
		</tr>
		<tr>
			<td></td>
			<td>Day 2</td>
			<td>0.640</td>
			<td>0.363</td>
			<td>0.432</td>
			<td>0.709</td>
			<td>0.710</td>
			<td>-0.002</td>
		</tr>
		<tr>
			<td></td>
			<td>Day 3</td>
			<td>0.547</td>
			<td>0.349</td>
			<td>0.363</td>
			<td>0.738</td>
			<td>0.598</td>
			<td>0.190</td>
		</tr>
		<tr>
			<td></td>
			<td>All Days</td>
			<td>0.590</td>
			<td>0.384</td>
			<td>0.348</td>
			<td>0.697</td>
			<td>0.650</td>
			<td>0.068</td>
		</tr>
	</tbody>
</table>
Table 4: CI and LI Values Obtained on Separate Testing Days.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#8220;naivelevel&#8221; determines the text that will identify na&#239;ve values for each genotype. The default is &#8220;N,&#8221; but this can be changed into any other alphanumerical text.</td>
		</tr>
		<tr>
			<td>&#8220;refmutation&#8221; is set to &#8220;NA&#8221; (not applicable) by default, but can be changed to the name of the control or the genotype in order to perform statistical comparisons.&#160; 
			This will cause the script to automatically select the control genotype.</td>
		</tr>
		<tr>
			<td>&#34;datname&#8221; refers to the name of the data file and can be specified in this argument instead of the default file selection.&#160;</td>
		</tr>
		<tr>
			<td>&#34;header&#8221; can be used to indicate whether or not the data file contains column headers.&#160; 
			The default is &#8220;TRUE,&#8221; but a file with no headers can be used when this argument is changed to &#8220;FALSE.&#8221;&#160;</td>
		</tr>
		<tr>
			<td>&#34;seed&#8221; initializes the random number generator. This is set by default to &#8220;NA&#8221; and ensures a random number each time the script is used.&#160; 
			By design, a bootstrap analysis will give slightly different results each time it is run, even when using the same data file.&#160; 
			When the seed is specified by any integer number larger than zero, the same set of random bootstrap samples is obtained.&#160;</td>
		</tr>
		<tr>
			<td>&#34;writeoutput&#8221; can be set to &#8220;TRUE&#8221; or &#8220;FALSE&#8221; in order to determine whether an output file will be generated. The default is &#8220;TRUE.&#8221;</td>
		</tr>
	</tbody>
</table>
Table 5: Arguments used in the Analearn Function that Can Alter the Default Settings of the Function to Adjust the Parameters of the Bootstrapping
Supplemental File S1: Building plan of a courtship chamber. The file can be opened with any application that allows .stp extensions (CAD-files). <a href="http://cloudflare.jove.com/files/ftp_upload/55808/FileS1.zip" target="_blank">Please click here to download this file.</a>
Supplemental File S2: Example of an Input File for the Analearn Script. <a href="http://cloudflare.jove.com/files/ftp_upload/55808/FileS2.zip" target="_blank">Please click here to download this file.</a>
Supplemental File S3: The Analearn.R Acript. The file can be opened with R-studio18. <a href="http://cloudflare.jove.com/files/ftp_upload/55808/FileS3.zip" target="_blank">Please click here to download this file.</a>

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