Nous tenons à remercier Astha Maltare pour générer les données crawling larvaires. Nous tenons à remercier le Dr Nicholas Lanson Jr. pour donner ses commentaires sur le manuscrit. Ce travail a été soutenu par le Centre Robert Packard pour la SLA à l&#39;Université Johns Hopkins (à l&#39;UBP) et le Sclérose latérale amyotrophique Association (UBP), et R01MH083689 de la National Institutes of Mental Health (CDN).

Essai A. Crawling larvaire 1. Collection larves <ol><li> Mettre en place une bouteille de 8 oz de mouches (10-15 mâles + femelles 10-15). </li><li> Laissez-mouches pondent leurs œufs pendant 24 heures, puis de bouteilles claire de mouches. (Transfert des adultes dans un nouveau flacon et répéter au besoin). </li><li> Incuber bouteille pendant 3-4 jours, ou jusqu&#39;au troisième stade larvaire sont visibles. </li><li> Ajouter 50 à 100 ml de saccharose à 20% à la bouteille avec les larves et les laisser reposer pendant 20 minutes. Les larves flottent à la surface. </li><li> Recueillir les larves à l&#39;aide d&#39;une pipette de 25 ml sérologique avec la pointe coupée, et le placer dans un panier en treillis. </li><li> Laver les larves dans le panier à maille deux fois avec désionisée H 2 O. Les larves sont maintenant prêts pour les expériences. </li></ol> 2. Pour traiter les larves à la drogue <ol><li> Utilisation d&#39;une brosse pour transporter le nombre souhaité de larves à un bécher contenant 5 ml de saccharose une solution à 5% + médicament. </li><li> Laissez les larves se nourrissent pendant au moins 15 minutes.</li><li> Verser la drogue traitée larves dans un panier en treillis et rincer. Ils sont maintenant prêts à utiliser. </li></ol> 3. Essai locomoteur (mesure de la distance totale parcourue ou des contractions paroi du corps) <ol><li> Utilisez une brosse pour le transport de larve à un individu: <ol><li> 15 cm boîte de Pétri contenant 2% d&#39;agarose (précédemment versé et on laisse durcir) sur du papier millimétré avec une épaisseur de 0,2 cm 2 de grille. <ol><li> Compter le nombre de lignes de la grille traversé en 1 minute. </li></ol></li><li> Eh bien d&#39;un plat de dissection verre contenant une solution de levure diluée pâte. <ol><li> Comptez le péristaltisme (contractions antérieure sur le mouvement de contraction postérieure = 1) en une minute tout en observant sous un microscope à dissection. </li></ol></li></ol></li><li> Répétez jusqu&#39;à ce que les chiffres désirés de larves ont été comptés. </li></ol> B. itératif rapide géotaxie négative (RING) Protocole Cet essai a été à l&#39;origine deprescrite par Gargano et al 5. <ol><li> Recueillir nouvellement écloses mouches mâles adultes de moins de CO 2 anesthésie légère et le placer dans un flacon standard contenant des aliments (ou de la nourriture + médicament). </li><li> Maintenir les mouches à la température ambiante (sur la paillasse. ~ 22 ° C) pendant 2-3 jours pour permettre la récupération du CO 2 (et l&#39;accumulation des concentrations de médicament l&#39;état d&#39;équilibre, le cas échéant). </li><li> Transférer environ 25 mouches sans anesthésie dans des flacons en polystyrène préparés. </li><li> Assemblez les flacons avec des mouches dans l&#39;appareil RING (Figure 1). </li><li> Permettez-mouches pour s&#39;acclimater à l&#39;environnement, à l&#39;abri, pendant 15-20 minutes. </li><li> Au cours de cet appareil photo numérique du temps lieu ~ 1 m en avant de l&#39;appareil (sur une plate-forme si nécessaire pour aligner le centre de la lentille à mi-hauteur de la flacons), mise au point et zoom de la caméra sur l&#39;appareil, et régler une minuterie pour 3,0 secondes. </li><li> Soigneusement s&#39;emparer de l&#39;appareil sonner avec votre main gauche afin de ne pas disturb les mouches, et tiennent la minuterie avec votre main droite. </li><li> Fortement appuyez sur l&#39;appareil vers le bas sur la surface du banc à trois reprises, en s&#39;assurant que l&#39;eau du robinet est déjà assez difficile de faire tomber toutes les mouches au fond des flacons. </li><li> Parallèlement à l&#39;achèvement de la troisième prise, démarrez le compte à rebours de 3 secondes. </li><li> A trois secondes de prendre une photo. </li><li> Réinitialiser la minuterie pendant 1 minute et commencer. Pendant ce temps, réinitialiser la caméra et se concentrer sur l&#39;appareil, et fixer un autre canal de la minuterie pendant trois secondes. </li><li> Après 1 minute, répétez les étapes 1.7 à 1.10 </li><li> Après un total de 5-6 essais, télécharger des images sur un ordinateur et d&#39;utiliser votre visionneuse d&#39;image préférée pour ouvrir, et marquer la hauteur moyenne a grimpé pour chaque flacon. </li><li> Effectuer une analyse statistique sur vos différents groupes en comparant la hauteur moyenne a grimpé. </li></ol> Courtship C. et dosage d&#39;accouplement <ol><li> La première chose le matin, claires et producteurs de bouteilles de flies à être utilisé. </li><li> Au cours de la journée (toutes les 3-4 heures), de recueillir nouvellement écloses mâles sexuellement naïfs et les femmes: <ol><li> Placez les hommes individuellement dans des flacons ou des tubes avec le milieu. </li><li> Placez 5-6 femelles ensemble, par flacon / tube. </li><li> Isoler les mouches recueillies à 25 ° C de moins de 12 heures de lumière / obscurité pendant 5 jours. </li><li> Transfert d&#39;une femelle dans la chambre d&#39;une roue d&#39;accouplement. </li><li> Transférer un mâle dans la chambre d&#39;une roue d&#39;accouplement. </li><li> Observez paire sous un microscope à dissection pour les comportements suivants: <ol><li> Orientation (les mâles oriente vers la femelle) </li><li> Tapping (le mâle tape la femme) </li><li> Chanson Wing (le mâle s&#39;étend et vibre d&#39;une aile) </li><li> Lèche (les mâles lèche les organes génitaux féminins) </li><li> Curling (le mâle se recourbe son abdomen sous lui-même) </li><li> Tentative de copulation (curling activité tout en essayant de monter sur la femelle) </li></ol></li></ol></li><li> Observer pour 10 minutes ou jusqu&#39;à ce que la copulation, en notant l&#39;heure à laquelle chaque problème se produit (latence), le temps total engagé dans le comportement de cour jusqu&#39;à ce que la copulation (pour calculer l&#39;indice de cour), ainsi que le nombre de paires qui ont réussi à adopter un comportement donné ( fréquence). 100% des paires de type sauvage est généralement s&#39;accoupler dans les 5 minutes. </li><li> Calculer un indice de cour (CI) en divisant le temps passé à faire la cour, divisé par le temps total jusqu&#39;à la copulation. Pour les couples de type sauvage de cette oscille habituellement entre 0.6-0.8. </li></ol> D. des résultats représentatifs Crawling test Normale larve de type sauvage va errer ~ 3 cm / minute, et d&#39;exposition de ~ 40-50 contractions paroi du corps en une minute. Nous avons récemment développé un modèle de drosophile du FUS / TLS liée sclérose latérale amyotrophique qui montre des larves rampant défaut, la durée de vie réduite et des adultes d&#39;escalade dépréciation 6. Nous avons ciblé l&#39;expression of de type sauvage et des formes mutantes de FUS / TLS afin de les neurones moteurs (OK-371-gal4 pilote) et a effectué un test exploration des larves. Comme indiqué ci-dessous, les larves de type sauvage ramper jusqu&#39;à 12 cm, alors que l&#39;expression du FUS de type sauvage a diminué la capacité des larves rampant à environ 6 cm. Animaux exprimant la mutation R521C SLA causant dans FUS / TLS démontrer une déficience très sévère dans leur mouvement rampant (Figure 1), ramper seulement d&#39;environ 1 cm par minute. Négatif dosage RING géotaxie Les jeunes mouches adultes de type sauvage doit avoir une hauteur moyenne d&#39;escalade de ~ 4-5 cm dans un délai de 3 secondes (le temps peut être réglé de 3 secondes pour accueillir différentes souches ou des niveaux d&#39;activité pour définir une hauteur moyenne donnée pour une donnée la souche / traitement). Mouches qui restent à la partie inférieure est attribuée une valeur de 0. Il n&#39;est pas conseillé d&#39;utiliser plus de 25 mouches par flacon, car il devient alors difficile de déterminer l&#39;emplacement de chaque individu à la mouche pour mesurer la hauteur. Ndésensibilisation o a été observée à la hauteur des 6 essais consécutifs espacés de 1 minute d&#39;intervalle que nous avons employée. Il est essentiel de ne pas réutiliser les flacons d&#39;essai en polystyrène dans cet essai, après les premières poses de données sont recueillies en raison de nouvelles mouches placées dans des flacons utilisés ne veut pas monter dans la même mesure que dans des flacons frais. <img alt="Figure 1" src="/files/ftp_upload/3795/3795fig1.jpg" /> Figure 1. Configuration pour le test RING. L&#39;appareil photo numérique est placé ~ 1 m en avant de l&#39;appareil contenant les mouches dans les tubes en polystyrène; mise au point et zoom de la caméra sur l&#39;appareil et régler une minuterie pour 3,0 secondes. <img alt="Figure 2" src="/files/ftp_upload/3795/3795fig2.jpg" /> Les données représentatives Figure 2. De l&#39;essai ramper larves à l&#39;aide mouches ectopique exprimer UAS-FUS WT, et UAS-FUS R521C sous le contrôle d&#39;un conducteur du neurone moteur (OK371-Gal4). <pclass = &quot;jove_content&quot;&gt;

<ol>
	<li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacol. Rev. 63 (2), 411-436 (2011).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+Parkinson's+disease.">A Drosophila model of Parkinson's disease.</a> Nature Mar. 23 (6776), 394-398 (2000).</li><li>Auluck, P. K., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+prevention+of+Parkinson+disease+in+Drosophila.">Pharmacological prevention of Parkinson disease in Drosophila.</a> Nat. Med. 8 (11), 1185-1186 (2000).</li><li>Whitworth, A. J., Theodore, D. A., Greene, J. C., Benes, H., Wes, P. D., Pallanck, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutathione+Stransferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione Stransferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (22), 8024-8029 (2005).</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> Exp. Gerontol. 40 (5), 386-395 (2005).</li><li>Lanson, N. A., Maltare, A., King, H., Smith, R., Kim, J. H., Taylor, J. P., Lloyd, T. E., 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=A+Drosophila+model+of+FUS-related+neurodegeneration+reveals+genetic+interaction+between+FUS+and+TDP-43.">A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43.</a> Hum. Mol. Genet. 20 (13), 2510-2523 (2011).</li><li>Batlevi, Y., Martin, D. N., Pandey, U. B., Simon, C. R., Powers, C. M., Taylor, J. P., Baehrecke, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+light+chain+1+is+required+for+autophagy,+protein+clearance,+and+cell+death+in+Drosophila.">Dynein light chain 1 is required for autophagy, protein clearance, and cell death in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (2), 742-747 (2010).</li><li>Sang, T. K., Chang, H. Y., Lawless, G. M., Ratnaparkhi, A., Mee, L., Ackerson, L. C., Maidment, N. T., Krantz, D. E., Jackson, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+mutant+human+parkin-induced+toxicity+demonstrates+selective+loss+of+dopaminergic+neurons+and+dependence+on+cellular+dopamine.">A Drosophila model of mutant human parkin-induced toxicity demonstrates selective loss of dopaminergic neurons and dependence on cellular dopamine.</a> J. Neurosci. 27 (5), 981-992 (2007).</li><li>Stacey, S. M., Muraro, N. I., Peco, E., Labb&#233;, A., Thomas, G. B., Baines, R. A., van Meyel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+glial+glutamate+transporter+Eaat1+is+regulated+by+fringe-mediated+notch+signaling+and+is+essential+for+larval+locomotion.">Drosophila glial glutamate transporter Eaat1 is regulated by fringe-mediated notch signaling and is essential for larval locomotion.</a> J. Neurosci. 30 (43), 14446-14457 (2010).</li><li>Repnikova, E., Koles, K., Nakamura, M., Pitts, J., Li, H., Ambavane, A., Zoran, M. J., Panin, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialyltransferase+regulates+nervous+system+function+in+Drosophila.">Sialyltransferase regulates nervous system function in Drosophila.</a> J. Neurosci. 30 (18), 6466-6476 (2010).</li><li>Repnikova, E., Koles, K., Nakamura, M., Pitts, J., Li, H., Ambavane, A., Zoran, M. J., Panin, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialyltransferase+regulates+nervous+system+function+in+Drosophila.">Sialyltransferase regulates nervous system function in Drosophila.</a> J. Neurosci. 30 (18), 6466-6476 (2010).</li><li>Nedelsky, N. B., Pennuto, M., Smith, R. B., Palazzolo, I., Moore, J., Nie, Z., Neale, G., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+functions+of+the+androgen+receptor+are+essential+to+pathogenesis+in+a+Drosophila+model+of+spinobulbar+muscular+atrophy.">Native functions of the androgen receptor are essential to pathogenesis in a Drosophila model of spinobulbar muscular atrophy.</a> Neuron. 67 (6), 936-952 (2010).</li><li>Lorenzo, D. N., Li, M. G., Mische, S. E., Armbrust, K. R., Ranum, L. P., Hays, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrin+mutations+that+cause+spinocerebellar+ataxia+type+5+impair+axonal+transport+and+induce+neurodegeneration+in+Drosophila.">Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila.</a> J. Cell Biol. 189 (1), 143-158 (2010).</li><li>Wang, J. W., Brent, J. R., Tomlinson, A., Shneider, N. A., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ALS-associated+proteins+FUS+and+TDP-43+function+together+to+affect+Drosophila+locomotion+and+life+span.">The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.</a> J. Clin. Invest. , (2011).</li><li>Choi, J. K., Jeon, Y. C., Lee, D. W., Oh, J. M., Lee, H. P., Jeong, B. H., Carp, R. I., Koh, Y. H., Kim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+GSS+syndrome+suggests+defects+in+active+zones+are+responsible+for+pathogenesis+of+GSS+syndrome.">A Drosophila model of GSS syndrome suggests defects in active zones are responsible for pathogenesis of GSS syndrome.</a> Hum. Mol. Genet. 19 (22), 4474-4489 (2010).</li><li>Ruan, H., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+interaction-mediated+lifespan+extension+of+Drosophila+Cu/Zn+superoxide+dismutase+mutants.">Social interaction-mediated lifespan extension of Drosophila Cu/Zn superoxide dismutase mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (21), (2008).</li><li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+video+tracking+of+locomotion+in+adult+Drosophila+melanogaster.">High-resolution video tracking of locomotion in adult Drosophila melanogaster.</a> J. Vis. Exp. (24), (2009).</li><li>Becnel, J., Johnson, O., Luo, J., N&#228;ssel, D. R., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serotonin+5-HT7+Dro+receptor+is+expressed+in+the+brain+of+Drosophila,+and+is+essential+for+normal+courtship+and+mating.">The serotonin 5-HT7 Dro receptor is expressed in the brain of Drosophila, and is essential for normal courtship and mating.</a> PLoS One. 6 (6), e20800 (2011).</li><li>Johnson, O., Becnel, J., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+5-HT(2)+and+5-HT(1A)-like+receptors+differentially+modulate+aggressive+behaviors+in+Drosophila+melanoga-+ster.">Serotonin 5-HT(2) and 5-HT(1A)-like receptors differentially modulate aggressive behaviors in Drosophila melanoga- ster.</a> Neuroscience. 158 (2), 1292-1300 (2009).</li><li>Bastock, M., Manning, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Courtship+of+Drosophila+Melanogaster.">The Courtship of Drosophila Melanogaster.</a> Behaviour. , 85-111 (1955).</li><li>Greenspan, R. J., Ferveur, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Courtship+in+Drosophila.">Courtship in Drosophila.</a> Annu. Rev. Genet. 34, 205-232 (2000).</li><li>Villella, A., 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=Neurogenetics+of+courtship+and+mating+in+Drosophila.">Neurogenetics of courtship and mating in Drosophila.</a> Adv. Genet. 62, 67-184 (2008).</li></ol>

Nous n&#39;avons rien à communiquer.

Comportement chez la drosophile est étroitement régulée par des facteurs génétiques et environnementaux. Nous, et d&#39;autres, ont déjà utilisé les tests décrits ici pour recueillir des données afin d&#39;examiner les gènes liés à voler et à des comportements humains les maladies neurodégénératives modélisés chez la drosophile 5-19. Pour le dosage de l&#39;exploration, la sélection minutieuse de 3 e stade larvaire est une étape critique. Si le traitement avec...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Saccharose </td><td> Fisher Scientific </td><td> S5-500 </td></tr><tr><td> Agarose </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> 6 oz Drosophila bouteille </td><td> Genesee scientifique </td><td> 32-130 </td></tr><tr><td> Paint Brush (# 1) </td><td> Ted Pella, Inc </td><td> 11859 </td></tr><tr><td> Fly composants alimentaires </td><td></td><td></td></tr><tr><td> Semoule de maïs </td><td> Fisher Scientific </td><td> NC9109741 </td></tr><tr><td> Gélose </td><td> Genesee scientifique </td><td> 66-104 </td></tr><tr><td> Mélasse </td><td> Fisher Scientific </td><td> NC9349176 </td></tr><tr><td> L&#39;acide propionique </td><td> Acros </td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td> Apex </td><td> 20-258 </td></tr><tr><td> L&#39;éthanol </td><td> PêcheurScientifique </td><td> BP2818-4 </td></tr><tr><td> Levure </td><td> Genesee scientifique </td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

Drosophila melanogaster, la mouche des fruits, a été utilisé pour étudier les mécanismes moléculaires d&#39;un large éventail de maladies humaines comme le cancer, les maladies cardiovasculaires et de diverses maladies neurologiques 1. Nous avons optimisé simples et robustes tests comportementaux pour déterminer locomotion de la larve, la capacité d&#39;escalade adulte (dosage RING), et les comportements de séduction de la drosophile. Ces tests comportementaux sont largement applicables pour étudier le rôle des facteurs génétiques et environnementaux sur le comportement à la mouche. La capacité de ramper larvaire peut être utilisé de manière fiable pour déterminer les changements à un stade précoce dans les capacités rampants de larves de drosophile et aussi pour examiner l&#39;effet de médicaments ou de gènes de maladies humaines (des mouches transgéniques) sur leur locomotion. Le dosage des larves rampant devient plus applicable si l&#39;expression ou la suppression d&#39;un gène provoque une létalité à un stade nymphal ou adulte, que ces mouches ne survivent pas à l&#39;âge adulte où ils pourraient par ailleurs être évalué. Cette base, unssay peut également être utilisé en conjonction avec la lumière vive ou le stress d&#39;examiner d&#39;autres réponses comportementales chez les larves de Drosophila. Comportement de cour a été largement utilisée pour étudier les bases génétiques du comportement sexuel, et peut également être utilisé pour examiner l&#39;activité et de la coordination, ainsi que l&#39;apprentissage et la mémoire. Comportement de cour chez la drosophile implique l&#39;échange de divers stimuli sensoriels, y compris visuelles, auditives, et les signaux chimiosensoriels entre les hommes et les femmes qui mènent à une série complexe de comportements moteurs bien caractérisés ont abouti à la copulation avec succès. Dosages adultes traditionnels d&#39;escalade (géotaxie négatifs) sont fastidieux, de main-d&#39;œuvre, et prend du temps, avec une variation significative entre les différents essais 2-4. Le itératif rapide géotaxie négative (RING) test 5 a de nombreux avantages sur des protocoles plus largement utilisés, en fournissant une approche débit reproductible, sensible, et de haute de quantifier locomotrice des adultes et négatifs géotaxie behaviors. Dans le test RING, plusieurs génotypes ou de traitements médicamenteux peuvent être testés simultanément en utilisant grand nombre d&#39;animaux, à l&#39;approche à haut débit rend plus propice pour des expériences de dépistage.

Watch this Scientific Journal Video about Methods to Assay Drosophila Behavior at JoVE.com

Methods to Assay Drosophila Behavior

Drosophila melanogaster est un système modèle génétiquement et comportemental souple qui a été utilisée pour comprendre la base moléculaire et cellulaire de nombreux processus biologiques importants pour plus d&#39;un siècle 1. Drosophile a été bien exploitée afin de mieux comprendre la base génétique du comportement à la mouche.

Méthodes de dosage à Drosophila Comportement

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

42.6K vues.  Louisiana State University Health Sciences Center. Drosophila melanogaster est un système modèle génétiquement et comportemental souple qui a été utilisée pour comprendre la base moléculaire et cellulaire de nombreux processus biologiques importants pour plus d'un siècle 1. Drosophile a été bien exploitée afin de mieux comprendre la base génétique du comportement à la mouche.

Vidéo: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Enregistrements Sensillum simple chez les insectes Drosophila melanogaster Et Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Recherche

Neurosciences

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Stimulation optogenetic de comportement de fuite en<em&gt; Drosophila melanogaster</em

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

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

Une simple mouche Essai de comportement alimentaire dans<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilisation Agarose linéaire Canaux pour étudier<em&gt; Drosophila</em&gt; Larvaire Ramper Comportement

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

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Un moyen simple pour mesurer Transformations en récompense le comportement de recherche Utilisation<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Quantification du comportement de toilettage de la drosophile

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

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Analyse de haut-débit du comportement locomoteur dans le dosage de l’île de drosophile

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Analyser le comportement larves de drosophile en réponse à une Stimulation des neurones olfactifs Optogenetic

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster In Vivo

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Test mécanique d’évitement des conflits pour mesurer le comportement de la douleur chez la souris

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...