Name: a simple technique to assay locomotor activity in drosophila
Uploaded: 2023-02-24T14:00:00
Description: Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

Dette arbeidet ble støttet av et spesielt lanseringsfond fra Soochow University og National Science Foundation of China (NSFC) (82171414). Vi takker professor Chunfeng Lius laboratoriemedlemmer for deres diskusjon og kommentarer.

W1118 voksne fluer og tredje instar larver ble brukt til denne studien.
1. Eksperimentell forberedelse
MERK: En åpen arena for Drosophila bevegelsessporing er laget medacolorless og luktfri silikagel.
<ol><li>Bland reagens A og reagens B i forholdet 1:10, i henhold til produsentens instruksjoner for silikasettet (se materialfortegnelse). Sørg for at natriumbikarbonat tilsettes til blandingen ved omr?...

<ol>
	<li>Ham, S. J., 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=Loss+of+UCHL1+rescues+the+defects+related+to+Parkinson's+disease+by+suppressing+glycolysis.">Loss of UCHL1 rescues the defects related to Parkinson's disease by suppressing glycolysis.</a> Science Advances. 7 (28), (2021).</li><li>Algarve, T. D., Assmann, C. E., Aigaki, T., da Cruz, I. B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parental+and+preimaginal+exposure+to+methylmercury+disrupts+locomotor+activity+and+circadian+rhythm+of+adult+Drosophila+melanogaster.">Parental and preimaginal exposure to methylmercury disrupts locomotor activity and circadian rhythm of adult Drosophila melanogaster.</a> Drug and Chemical Toxicology. 43 (3), 255-265 (2020).</li><li>Jones, M. A., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+age-related+impairment+in+locomotor+and+other+behaviors.">Drosophila as a model for age-related impairment in locomotor and other behaviors.</a> Experimental Gerontology. 46 (5), 320-325 (2011).</li><li>Yamaguchi, M., Yoshida, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+organism.">Drosophila as a model organism.</a> Advances in Experimental Medicine and Biology. 1076, 1-10 (2018).</li><li>Rothenfluh, A., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs,+files,+and+videotape:+the+effects+of+ethanol+and+cocaine+on+Drosophila+locomotion.">Drugs, files, and videotape: the effects of ethanol and cocaine on Drosophila locomotion.</a> Current Opinion in Neurobiology. 12 (6), 639-645 (2002).</li><li>Tsuda, L., Lim, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+model+system+using+Drosophila.">Alzheimer's disease model system using Drosophila.</a> Advances in Experimental Medicine and Biology. 1076, 25-40 (2018).</li><li>Dung, V. M., Thao, D. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+model.">Parkinson's disease model.</a> Advances in Experimental Medicine and Biology. 1076, 41-61 (2018).</li><li>Liguori, F., Amadio, S., Volonte, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fly+for+ALS:+Drosophila+modeling+on+the+route+to+amyotrophic+lateral+sclerosis+modifiers.">Fly for ALS: Drosophila modeling on the route to amyotrophic lateral sclerosis modifiers.</a> Cellular and Molecular Life Sciences. 78 (17-18), 6143-6160 (2021).</li><li>Cao, 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=An+automated+rapid+iterative+negative+geotaxis+assay+for+analyzing+adult+climbing+behavior+in+a+Drosophila+model+of+neurodegeneration.">An automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a Drosophila model of neurodegeneration.</a> Journal of Visualized Experiments. (127), 56507 (2017).</li><li>Eidhof, I., 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=High-throughput+analysis+of+locomotor+behavior+in+the+Drosophila+island+assay.">High-throughput analysis of locomotor behavior in the Drosophila island assay.</a> Journal of Visualized Experiments. (129), 55892 (2017).</li><li>Scaplen, K. 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=Automated+real-time+quantification+of+group+locomotor+activity+in+Drosophila+melanogaster.">Automated real-time quantification of group locomotor activity in Drosophila melanogaster.</a> Scientific Reports. 9 (1), 4427 (2019).</li><li>Werkhoven, Z., Rohrsen, C., Qin, C., Brembs, B., de Bivort, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MARGO+(Massively+Automated+Real-time+GUI+for+Object-tracking),+a+platform+for+high-throughput+ethology.">MARGO (Massively Automated Real-time GUI for Object-tracking), a platform for high-throughput ethology.</a> PLoS One. 14 (11), e0224243 (2019).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental Gerontology. 40 (5), 386-395 (2005).</li><li>Cichewicz, K., 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=ShinyR-DAM:+a+program+analyzing+Drosophila+activity,+sleep+and+circadian+rhythms.">ShinyR-DAM: a program analyzing Drosophila activity, sleep and circadian rhythms.</a> Communications Biology. 1, 25 (2018).</li><li>McParland, A. L., Follansbee, T. L., Ganter, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+larval+activity+in+the+Drosophila+activity+monitor.">Measurement of larval activity in the Drosophila activity monitor.</a> Journal of Visualized Experiments. 98, e52684 (2015).</li><li>Walter, T., Couzin, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRex,+a+fast+multi-animal+tracking+system+with+markerless+identification,+and+2D+estimation+of+posture+and+visual+fields.">TRex, a fast multi-animal tracking system with markerless identification, and 2D estimation of posture and visual fields.</a> eLife. 10, (2021).</li><li>Maia Chagas, A., Prieto-Godino, L. L., Arrenberg, A. B., Baden, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#8364;100+lab:+A+3D-printable+open-source+platform+for+fluorescence+microscopy,+optogenetics,+and+accurate+temperature+control+during+behaviour+of+zebrafish,+Drosophila,+and+Caenorhabditis+elegans.">The &#8364;100 lab: A 3D-printable open-source platform for fluorescence microscopy, optogenetics, and accurate temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis elegans.</a> PLoS Biology. 15 (7), e2002702 (2017).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> Journal of Visualized Experiments. (61), (2012).</li><li>Xiao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+the+behavior+of+Drosophila+melanogaster+for+toxicity+study.">Methods to assay the behavior of Drosophila melanogaster for toxicity study.</a> Methods in Molecular Biology. 2326, 47-54 (2021).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Johnson, M. E., Bobrovskaya, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+rotenone+models+of+Parkinson's+disease:+their+ability+to+reproduce+the+features+of+clinical+disease+and+model+gene-environment+interactions.">An update on the rotenone models of Parkinson's disease: their ability to reproduce the features of clinical disease and model gene-environment interactions.</a> Neurotoxicology. 46, 101-116 (2015).</li><li>Coulom, H., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+rotenone+models+sporadic+Parkinson's+disease+in+Drosophila+melanogaster.">Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster.</a> The Journal of Neuroscience. 24 (48), 10993-10998 (2004).</li><li>Kumar, P. P., Bawani, S. S., Anandhi, D. U., Prashanth, K. V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotenone+mediated+developmental+toxicity+in+Drosophila+melanogaster.">Rotenone mediated developmental toxicity in Drosophila melanogaster.</a> Environmental Toxicology and Pharmacology. 93, 103892 (2022).</li><li>Gulyas, M., Bencsik, N., Pusztai, S., Liliom, H., Schlett, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AnimalTracker:+an+ImageJ-based+tracking+API+to+create+a+customized+behaviour+analyser+program.">AnimalTracker: an ImageJ-based tracking API to create a customized behaviour analyser program.</a> Neuroinformatics. 14 (4), 479-481 (2016).</li><li>Qu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EasyFlyTracker:+a+simple+video+tracking+Python+package+for+analyzing+adult+Drosophila+locomotor+and+sleep+activity+to+facilitate+revealing+the+effect+of+psychiatric+drugs.">EasyFlyTracker: a simple video tracking Python package for analyzing adult Drosophila locomotor and sleep activity to facilitate revealing the effect of psychiatric drugs.</a> Frontiers in Behavioral Neuroscience. 15, 809665 (2022).</li><li>Yarwais, Z. H., Najmalddin, H. O., Omar, Z. J., Mohammed, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+data+collection+of+Drosophila+movement+behaviour+assays+using+computer+vision+in+Python.">Automated data collection of Drosophila movement behaviour assays using computer vision in Python.</a> International Journal of Innovative Approaches in Science Research. 4 (1), 15-22 (2020).</li></ol>

Vi har utviklet en metode, basert på open source-materialet AnimalTracker API som er kompatibelt med Fijis bildebehandlingsprogram, som kan gjøre det mulig for forskere å systematisk evaluere bevegelsesaktivitet ved å spore både voksne og individuelle larvefluer. AnimalTracke er et verktøy skrevet i Java som enkelt kan integreres i eksisterende databaser eller andre verktøy for å lette analysen av applikasjonsdesignet dyresporingsadferd24. Ved en ramme-for-ramme-analyse v...

Drosophila melanogaster gir en utmerket modellorganisme for å undersøke cellulære og molekylære funksjoner i nevronale sykdomsmodeller opprettet av genmodifisering1, medikamentell behandling2 og senescens3. Den høye bevaringen av biologiske veier, fysiske egenskaper og sykdomsassosierte homologgener mellom mennesker og Drosophila gjør fruktfluen til en ideell etterligning fra molekylært til atferdsnivå4. I mange sykdomsmodeller er atferdsmangel en viktig indeks, og gir en nyttig modell for ulike menneskelige nevropatier 5,6. Drosophila brukes nå til å studere flere menneskelige sykdommer, nevroutvikling og nevrodegenerative sykdommer som Parkinsons sykdom og amyotrofisk lateralsklerose 7,8. Påvisning av sykdomsmodellenes motoriske evne er avgjørende for å forstå den patogene utviklingen og kan gi en fenotypisk korrelasjon til de molekylære mekanismene som ligger til grunn for sykdomsprosessen.
Nylig har kommersielt tilgjengelige programvareverktøy og kostnadseffektive programmer blitt utviklet for Drosophila lokomotoriske deteksjonsstrategier, for eksempel testing med høy gjennomstrømning i grupperte fluer9,10 og måling av bevegelse i sanntid11,12. En slik konvensjonell tilnærming er rapid interactive negative geotaxis (RING), også kalt klatreanalysen, som inkluderer flere kanaler som gjør det mulig å inneholde en stor fluepopulasjon med samme kjønn og alder, noe som reduserer variasjonen mens data samlerinn 9,13. En annen pre-testing metode for å analysere lokomotorisk oppførsel er TriKinetics Drosophila aktivitet monitor (DAM), en enhet som bruker flere stråler for å oppdage fly aktivitet bevegelse i et tynt glassrør 14. Enheten registrerer posisjon kontinuerlig, noe som representerer automatisert bevegelse ved å beregne stråleovergangene for å studere aktiviteten og døgnrytmen til fluer over en lengre periode15. Selv om disse metodene har blitt mye brukt i å analysere atferdsdefekter i fruktfluer for å bestemme endringer i atferdsbevegelse, krever de alltid spesielt testutstyr eller komplekse analyseprosesser, og begrenser deres anvendelse i noen modeller med en begrenset, enkel enhet. Gruppebaserte strategier for testing av den voksne Drosophila, som FlyGrAM11 og Drosophila-øyanalysen 10, implementerer sosial rekruttering og individuell sporing i et forhåndsdefinert område. Likevel kan sosial individuell begrensning i trossede områder ha en negativ effekt på identifikasjoner i bildene, forårsaket av kollisjon eller overlapping av fluer. Selv om noen åpen kildekode-materialbaserte metoder, som TRex16, MARGO 12 og FlyPi17, har en nødsituasjon, kan de spore fluene raskt med fleksibel bruk i atferdstesting. Disse testmetodene er knyttet til forseggjorte eksperimentelle apparatinstallasjoner, spesielle programvarekrav eller profesjonelle dataspråk. For larver er måling av total tilbakelagt avstand over antall gittergrenselinjer per tidsenhet18, eller grovtelling av kroppsveggkontraksjonene for individer manuelt19, de dominerende metodene for å vurdere deres lokomotoriske evne. På grunn av mangel på presisjon i utstyr eller enheter og analysemetoder, kan noen atferdsbevegelse av larver unnslippe deteksjon, noe som gjør det vanskelig å nøyaktig vurdere atferdsbevegelse, spesielt fin bevegelse15.
Den nåværende utviklede metoden benytter AnimalTracker applikasjonsprogrammeringsgrensesnitt (API), kompatibelt med Fiji (ImageJ) bildebehandlingsprogram, for systematisk å evaluere den lokomotoriske aktiviteten til både voksne og larvefluer ved å analysere deres sporingsadferd fra HD-videoer (HD). Fiji er en åpen kildekode-programvare ImageJ-distribusjon som kan kombinere robuste programvarebiblioteker med mange skriptspråk, noe som resulterer i rask prototyping av bildebehandlingsalgoritmer, noe som gjør den populær blant biologer for sine bildeanalysemuligheter20. I den nåværende tilnærmingen utnyttes Fijis integrering i AnimalTracker API til å utvikle en unik Drosophila-atferdsanalyse med personlig algoritmeinnsetting, og gir et nyttig trinn for detaljert dokumentasjon og opplæringsprogrammer for å støtte robuste analytiske evner for lokomotorisk oppførsel (figur 1). For å omgå komplikasjonen av objektive identifikasjoner i bildene forårsaket av kollisjon eller overlapping av fluer, er hver arena begrenset til å være vert for bare en flue. Ved vurdering av sporingspresisjonen til tilnærmingen ble det implementert for å spore og kvantifisere de lokomotoriske bevegelsene til Drosophila som ble administrert med det giftige stoffet rotenon, som vanligvis brukes til dyremodeller av Parkinsons sykdom, og til slutt oppdaget bevegelsessvikt i narkotikabehandlingen21. Denne metoden, som benytter åpen kildekode og fri programvare, krever ikke høykostinstrumentering, og kan nøyaktig og reproduserbart analysere Drosophila atferdsbevegelse.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal tracker</td>
			<td>Hungarian Brain Research Program</td>
			<td>version: 1.7</td>
			<td>pfficial website: http://animaltracker.elte.hu/main/downloads</td>
		</tr>
		<tr>
			<td>Camera software</td>
			<td>Microsoft</td>
			<td>version: 2021.105.10.0</td>
			<td>built-in windows 10 system</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>DELL</td>
			<td>Vostro-14-5480</td>
			<td>a comupter running win 10 system is available</td>
		</tr>
		<tr>
			<td>Drosophila carbon dioxide anesthesia workstation</td>
			<td>Wu han Yihong technology</td>
			<td>#YHDFPCO2-018</td>
			<td>official website: http://www.yhkjwh.com/</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Fiji team</td>
			<td>version: 1.53v</td>
			<td>official website: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Format factory software</td>
			<td>Pcfreetime</td>
			<td>version: X64 5.4.5</td>
			<td>official website: http://www.pcfreetime.com/formatfactory/CN/index.html</td>
		</tr>
		<tr>
			<td>Graph pad prism</td>
			<td>GraphPad Software</td>
			<td>version: 8.0.2</td>
			<td>official website: https://www.graphpad-prism.cn</td>
		</tr>
		<tr>
			<td>Hight definition camera</td>
			<td>TTQ</td>
			<td>Jingwang2 (HD1080P F1.6 6-60mm)</td>
			<td>official website: http://www.ttq100.com/product_show.php?id=35</td>
		</tr>
		<tr>
			<td>Office software</td>
			<td>Microsoft</td>
			<td>version: office 2019</td>
			<td>official website: https://www.microsoftstore.com.cn/software/office</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Bkman</td>
			<td>110301003</td>
			<td>size: 60 mm</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>DOW</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Mix well according to the instructions</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Macklin</td>
			<td>#144-55-8</td>
			<td>Mix well with silica gel</td>
		</tr>
	</tbody>
</table>

a simple technique to assay locomotor activity in drosophila

Drosophila melanogaster er en ideell modellorganisme for å studere ulike sykdommer på grunn av sin overflod av avanserte genetiske manipulasjonsteknikker og ulike atferdsegenskaper. Identifisering av atferdsmangel i dyremodeller er et avgjørende mål på sykdommens alvorlighetsgrad, for eksempel i nevrodegenerative sykdommer der pasienter ofte opplever nedsatt motorisk funksjon. Men med tilgjengeligheten av ulike systemer for å spore og vurdere motoriske underskudd i fluemodeller, for eksempel narkotikabehandlede eller transgene individer, mangler det fortsatt et økonomisk og brukervennlig system for presis evaluering fra flere vinkler. En metode basert på AnimalTracker applikasjonsprogrammeringsgrensesnitt (API) er utviklet her, som er kompatibel med Fiji bildebehandlingsprogram, for systematisk å evaluere bevegelsesaktivitetene til både voksne og larveindivider fra innspilt video, og dermed muliggjøre analyse av deres sporingsadferd. Denne metoden krever bare et HD-kamera og en perifer maskinvareintegrasjon for å registrere og analysere atferd, noe som gjør det til en rimelig og effektiv tilnærming for screening av fluemodeller med transgene eller miljømessige atferdsmangler. Eksempler på atferdstester ved bruk av farmakologisk behandlede fluer er gitt for å vise hvordan teknikkene kan oppdage atferdsendringer hos både voksne fluer og larver på en svært repeterbar måte.

I denne studien ble lokomotoriske underskudd hos voksne fluer og tredje instar larver behandlet med rotenon undersøkt og sammenlignet i motorisk aktivitet med en kontrollflue matet med medikamentet løsningsmidlet dimetylsulfoksid (DMSO). Behandling med rotenon i Drosophila har vist seg å forårsake dopaminergt nevrontap i hjernen22 og føre til betydelige lokomotoriske utfall23. Som vist i figur 11 og figur 12 har voksne fluer og tr...

Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

A Simple Technique to Assay Locomotor Activity in Drosophila

Den nåværende protokollen vurderer den lokomotoriske aktiviteten til Drosophila ved å spore og analysere bevegelsen av fluer i en håndlaget arena ved hjelp av åpen kildekode-programvare Fiji, kompatibel med plugins for å segmentere piksler i hver ramme basert på HD-videoopptak for å beregne parametere for hastighet, avstand, etc.

En enkel teknikk for å analysere lokomotorisk aktivitet i Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Metoden er basert på åpen kildekode-programvare for systematisk å evaluere de lokale motoraktivitetene for sporingsadferd hos voksne og larvene flyr fra innspilt video. Denne metoden krever lavpris enhetsintegrasjon for å registrere og analysere atferd, noe som gjør det til en rimelig og effektiv tilnærming for screening av fluebevegelse. Denne teknologien brukes til å identifisere atferdsmodifikasjon i en fluemodell av menneskelig sykdom med lokal bevegelseshemming, men den er ikke egnet for behandling av sykdom, funksjonshemninger eller vanskeligheter.Det er viktig å fjerne det aktuelle videovinduet under analyseprosessen, nemlig originalen og behandlingsvideovinduet. For å begynne, bland reagens A og reagens B av silikasettet i forholdet en til 10, i henhold til produsentens instruksjoner, for å forberede friluftsarenaen for drosophila bevegelsessporing. Sett deretter HD-kameraet på et stativ, juster det slik at kameralinsen er vinkelrett på overflaten av silikaarenaen.Åpne deretter videoen med Fiji. Dra fremdriftsindikatoren til startbildet, og godkjenn stilltiende. Velg hele kroppen av fluen ved hjelp av frihåndsmarkeringsverktøyet.Klikk på bilde, juster og lysstyrke og kontrast for å justere hvitbalansen til den grå verdien i det valgte området nærmer seg den brede bakgrunnen. Overfør den bedøvede fluen til friluftsarenaen. Når flua kommer seg etter bedøvelsen, legger du arenafatet med flua under kameraet og rister det raskt fra side til side for å sikre at flua er i bevegelse når opptaket begynner.Trykk på opptaksknappen på kameraapplikasjonen for å starte videoopptak. Når opptaket er fullført, trykker du på stoppknappen for å avslutte videoopptaket. Konverter de innspilte videoene til AVI-format med MJ hedensk koding slik at de kan åpnes og analyseres ved hjelp av Fiji.Sett også rammene per sekund av videoen til 15 fps for voksne fluer og 12 fps for larver. For å åpne videoen med Fiji, velg de to alternativene, bruk virtuell stabel og konverter til gråtoner, i popup-vinduet. Få et behandlingsvindu ved å bruke det angitte aktive bildeverktøyet til dyresporingsprogrammet, og opprett et sporingsområde som sirkler arenaen i det originale videovinduet, ved hjelp av det ovale verktøyet.Angi filtrene og parametrene for de to filtrene for den første tomme rammen i behandlingsvinduet. Velg deretter neste ramme i det opprinnelige videovinduet, og velg den filtrerte overflaten til behandlingsvinduet. Når et filtrert behandlingsvindu er valgt, bruker du det angitte terskelverktøyet til å snu kanalfluen med en rød profil, dekket i behandlingsvinduet.Bruk deretter den innstilte blob-detektoren for å la datamaskinen gjenkjenne fluen med en rød profil, dekket i behandlingsvinduet. Sett den siste rammen for den voksne fluen eller larvene, som beskrevet i tekstmanuskriptet. Bruk verktøyet Vis blobber til å presentere et sporingsrektangel i det opprinnelige videovinduet.Start sporingen og eksporter sporingsfilen etter at overvåkingen er fullført. Last inn spor- og sonefilene ved hjelp av plugin-modulen for sporing av dyr og sporingsanalysator. Velg ønsket indeks ved hjelp av soneinnstillinger, og endre parameterinnstillingene.Beregn tiden for rammeintervallet ved hjelp av bildefrekvensen. Produser de kvantitative analysediagrammene ved hjelp av regnearkprogramvare og grafputeprisme, etter å ha blitt analysert i sporingsanalysatoren. Åpne sporfilen.Kopier alle koordinatene til Microsoft Office Excel, og del cellene ved hjelp av mellomromstasten. For å beregne immobilitetstiden per rammeintervall, velg alle beregnede resultater og sett inn et kolonnediagram for visuelt å vise immobilitetstiden med margen på hele kolonnediagrammet. Hvis du vil illustrere endringene i retningsvinkelen, merker du alle beregnede resultater og setter inn et punktdiagram.Voksne fluer og tredje Instar-larver, behandlet med rotenon, hadde signifikante lokomotoriske underskudd sammenlignet med kontrollfluer, matet med løsemiddel DMSO. Kvantitativ analyse av rotenonbehandling hos voksne fluer viste en signifikant reduksjon i tilbakelagt distanse og gjennomsnittlig hastighet. Og en betydelig økning i immobilitetstiden.Representative grafer viste færre topper for bevegelseshastigheten per rammeintervall i fluer matet med rotenon sammenlignet med de i kontrollen, noe som indikerer alvorlighetsgraden av det lokomotoriske aktivitetsunderskuddet. Den institusjonistiske immobilitetskolonnen med bevegelige piksler per ramme viste også betydelig mindre bevegelse innen ett minutt for rotenonfôrede fluer, sammenlignet med kontrollfluene. Representative grafer som illustrerer den bevegelige vinkelen på retningsendringer i rotenonfôret og kontrolldyr avslørte også endringer i retningen valgt av fluer.Lignende resultater ble observert for den tredje Instar larver. Resultatene viste at atferdsbevegelsen til sporingslarver matet med rotenon var betydelig svekket sammenlignet med kontrollen. Det mest avgjørende trinnet i analyseprosessen er å danne den første av blank ramme, da det viser om programvaren kan sjekke effektivt.Denne teknologien kan tillate forskere å dykke dypere inn i den underliggende mekanismen for flere defekter, for eksempel nerve- eller muskelskade.

a-simple-technique-to-assay-locomotor-activity-in-drosophila

Research

JoVE Journal

Behavior

A Simple Technique to Assay Locomotor Activity in Drosophila

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Mus Kort-og langsiktige Locomotor aktivitet Analyzed by Video Tracking Software

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

Atferds-og bevegelses målinger ved hjelp av et åpent felt Activity Monitoring System for skjelettmuskelsykdommer

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Bruk av det åpne feltet Maze å måle Locomotor og angst-lignende oppførsel i Mus

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Den tredemølle Fatigue Test: En enkel, High-throughput analyse av Fatigue-lignende oppførsel for Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. However, studying human locomotor learning can be challenging. During infancy and childhood, the neuromuscular system is quite immature, and it is unlikely that locomotor learning during early stages of development is governed by the same mechanisms as in adulthood. By the time humans reach maturity, they are so proficient at walking that it is difficult to come up with a sufficiently novel task to study de novo locomotor learning. The split-belt treadmill, which has two belts that can drive each leg at a different speed, enables the study of both short- (i.e., immediate) and long-term (i.e., over minutes-days; a form of motor learning) gait modifications in response to a novel change in the walking environment. Individuals can easily be screened for previous exposure to the split-belt treadmill, thus ensuring that all experimental participants have no (or equivalent) prior experience. This paper describes a typical split-belt treadmill adaptation protocol that incorporates testing methods to quantify locomotor learning and generalization of this learning to other walking contexts. A discussion of important considerations for designing split-belt treadmill experiments follows, including factors like treadmill belt speeds, rest breaks, and distractors. Additionally, potential but understudied confounding variables (e.g., arm movements, prior experience) are considered in the discussion.

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

Bruke en Split-beltet tredemølle evaluere generalisering av menneskelig Locomotor tilpasning

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

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

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

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

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

En metode for å teste effekten av miljøvennlige påkjenningshendelser i<em&gt; Drosophila melanogaster</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

En datametode å kvantifisere Fly Circadian aktivitet

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Anvendelse av flerfarget FlpOut teknikk høyoppløselig enkeltcelle Morphologies og celle interaksjoner av Glia i Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

En automatisert rask iterativ Negative Geotaxis analysen for å analysere voksen klatring atferd i Drosophila modell av Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

En enkel og rimelig analysen for å måle Ambulation i musen modeller av muskeldystrofi

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...