Nous tenons à remercier Amsale Belay pour son aide dans le tournage de cet article vidéo. Ce travail a été financé par les IRSC, le CRSNG et les subventions à la CRC JDL.

Partie 1. Filet de préparation de l&#39;abdomen adulte chez la drosophile. La procédure décrite ci-dessous immédiate prépare l&#39;abdomen pour l&#39;élimination ultérieure de l&#39;oenocytes (voir Part2 du protocole). La même procédure peut également être utilisé pour la préparation des autres tissus attachés à la surface interne de la cuticule, y compris le vaisseau dorsal (coeur par exemple) et de graisse corporelle. Notez que dans l&#39;article la vidéo de la technique de dissection oenocyte est démontrée sur un adulte de type sauvage (Canton-S) mouche mâle, six jours d&#39;âge. Techniques identiques peuvent être utilisés pour enlever les oenocytes des mouches femelles. La préparation de filet de l&#39;abdomen prend environ 5 minutes à effectuer. Avant de commencer il est important d&#39;avoir sous la main plusieurs épingles de dissection fine (voir ci-dessous et partie 2) et une aiguille de dissection (voir partie 2). Broches de dissection disponibles dans le commerce et les aiguilles sont souvent trop grands pour ce protocole, nous avons pris à la fabrication de notre propre laboratoire. Pour créer l&#39;aiguille de dissection fil de tungstène, (0,005 &quot;) est d&#39;abord enfilé à travers l&#39;arbre de l&#39;aiguille hypodermique (27G, moyeu aluminium). Enfiler le fil à travers la pointe de l&#39;aiguille hypodermique afin qu&#39;elle émerge du moyeu. Sertir le fil en saillie du moyeu;. ce qui empêche le fil d&#39;être tiré hors de l&#39;aiguille hypodermique Ensuite, couper le fil à l&#39;extrémité opposée de l&#39;exemple de sertissage que plusieurs millimètres (~ 3-4mm) de fil de saillie de la pointe de l&#39;aiguille hypodermique Passant. la pointe de tungstène exposés à travers une flamme va aiguiser le fil d&#39;une pointe très fine. Une pipette jetables en plastique quand 5ml coincé dans le moyeu de l&#39;aiguille hypodermique agit comme poignée pratique pour cet outil de dissection. Les broches de dissection fines sont faites dans un même chemin. Après l&#39;étape de l&#39;affûtage, saisissez la pointe du fil fermement avec une pince pour l&#39;empêcher de bouger et de faire une coupe d&#39;environ 3mm de la pointe pour libérer la goupille de dissection petits. Utilisez soin d&#39;éviter d&#39;être jeté broches de l&#39;emprise du la pince. Pour préparer les filets de l&#39;abdomen adulte drosophile: <ol><li> Fixez la volée à la plaque de dissection (Sylgard; Dow-Corning) en plaçant une épingle de dissection fines que le thorax. </li><li> Enlevez les pattes et les ailes de la mouche. Lors du retrait de l&#39;ensemble le plus postérieure de jambes, de créer une ouverture dans la cuticule au point juste en avant du thorax, où se réunit l&#39;abdomen. Cette ouverture est utilisée plus tard comme un point d&#39;accès pour faire une incision le long de la surface ventrale de l&#39;abdomen (voir étape 5) </li><li> Fixez l&#39;abdomen de la mouche en place en plaçant une épingle de dissection seconde à travers le segment génital. </li><li> Couvrir la volée avec Shields réfrigérés et Sang insectes milieu M3 (Sigma). Retirer les bulles d&#39;air en le dessinant avec une pipette Pasteur. </li><li> En utilisant une pince ouvrir l&#39;abdomen en faisant une incision le long de la ligne médiane ventrale de l&#39;ouverture de la cuticule créé par la suppression des jambes vers le segment génital. Retirer les tripes et les gonades de l&#39;abdomen. </li><li> Sever la cuticule et trachées reliant l&#39;abdomen au thorax. Le tissu ne reste reliant l&#39;abdomen au thorax devrait être le cœur. Ceci fournit la cuticule abdominale avec une certaine stabilité, et l&#39;empêche de tourner ou de torsion lors de la prochaine étape. </li><li> Pin plat antérieur plupart des coins de la cuticule abdominale. Retirez le thorax. Tirer la cuticule tendue, broches plates coins postérieure de la cuticule. La broche dans le segment génital peut être nécessaire de repositionner pour faire la cuticule à plat. </li></ol> Partie 2. Dissection Oenocyte La préparation de filet de l&#39;abdomen expose la surface interne de la cuticule abdominale et les tissus attachés, y compris le cœur, le corps gras, trachées, muscles de la paroi du corps, l&#39;épiderme, et les oenocytes. Le coeur se trouve le long de la ligne médiane dorsale. De graisse corporelle (tissu opaque) couvre la plupart de la surface interne cuticulaire. Trachées (blanc structures tubulaires) s&#39;étendent à partir des ouvertures latérales spiracle, et faire des branches étendues qui s&#39;infiltrent dans ces tissus. Situé au pied de ces tissus, la oenocytes pigmentées (couleur ambrée cellules) rayonnent à partir de la ligne médiane au bord latéral de la cuticule dans la région postérieure de chaque segment. Le oenocytes se trouvent directement en dessous des régions brunies par le soleil de chaque tergite (dorsale plaques cuticulaires) et peuvent être difficiles à identifier, sans connaissance préalable de leur emplacement. Une autre population de oenocytes ventralement situé est associée à la sternites (ventrale plaques cuticulaires); bien qu&#39;il soit possible d&#39;isoler ces cellules, ce protocole vidéo sera seulement démontrer la dissection de la oenocytes dorsale. Dans chaque segment abdominal, une feuille de premier plan du tissu de graisse corporelle est antérieur à la oenocytes; une petite feuille de moins évident de graisse corporelle est postérieure à la oenocytes. La feuille d&#39;éminents du tissu de graisse du corps couvre souvent le oenocytes du segment qui le précède. Le vaisseau dorsal et de la graisse corporelle doit être retiré en premier afin d&#39;exposer le oenocytes. Il est important de noter que la pigmentation de l&#39;ambre oenocytes, d&#39;où le nom de leur dérive (oeno - de oinos grec, «vin») 1, qui distingue clairement ces cellules des tissus environnants; l&#39;intensité de la pigmentation augmente avec l&#39;âge de la mouche. Par ailleurs, une lame basale ensheathes l&#39;2,3 oenocytes, apportant un soutien à l&#39;amas de cellules et d&#39;empêcher les cellules de devenir dissocié lors de la dissection. La lame basale contribue également à l&#39;élimination des tissus extraneously respectées, comme le tissu de graisse du corps, en créant un point faible, où ces adhérences peut facilement être perturbé. La procédure décrite ci-dessous fournit des instructions détaillées pour l&#39;élimination de la oenocytes du tégument abdominal. Le oenocytes peut être facilement retiré de segments abdominaux 2-5 (mâle) et 2-6 (femelle). Le oenocytes de la première et la dernière segments abdominaux (segments 1, et 6 ou 7 dans les mâles chez les femelles) sont souvent perturbés par les broches de dissection tenant les coins de la cuticule et sont donc évités. La suppression de la oenocytes prend environ 10 minutes à effectuer. Pour isoler l&#39;oenocytes adulte: <ol><li> Sever les troncs trachéale émanant des stigmates à l&#39;aide d&#39;une aiguille de dissection. </li><li> Retirez la graisse du corps et le cœur en coupant les connexions à la surface interne de la cuticule avec une aiguille de dissection. Pour ce faire, placez le point de l&#39;aiguille de dissection entre le corps gras et la oenocytes, et le faire glisser à partir du bord latéral de la cuticule, grâce à la ligne médiane, se déplaçant au-dessous du cœur, et en continuant à côté. Progressant de l&#39;arrière en avant, retirez le tissu de graisse du corps et le cœur de la cuticule de chaque segment successifs. Si bien exécuté l&#39;oenocytes restent attachés à la cuticule, et la graisse du corps et du coeur peut être retiré entier. </li><li> Paroi du corps muscles dragonne l&#39;oenocytes à la surface intérieure de la cuticule. Utilisation de l&#39;aiguille de dissection couper les muscles longitudinaux paroi du corps de chaque segment. Pour ce faire, faites glisser l&#39;aiguille de dissection le long de la face postérieure du brin oenocyte, délicatement les soulever de la surface de la cuticule. Les brins en forme de ruban oenocyte doit se détacher de la cuticule et peut être posé sur le côté tandis que le reste oenocytes sont supprimés. </li><li> Une fois retiré de la cuticule et couché sur la plaque de la dissection, il peut être nécessaire de nettoyer toute respectées tissus de graisse du corps de la oenocytes. Ceci est fait en plaçant simplement l&#39;aiguille de dissection entre le tissu de graisse corporelle et le oenocytes, et la rupture de la connexion en poussant l&#39;aiguille vers le bas et contre la plaque de dissection. </li><li> Rassemblez les brins oenocyte simplement en les poussant avec le bout de l&#39;aiguille de dissection. Une fois collé à l&#39;extrémité de l&#39;aiguille de dissection du oenocytes peut être tiré lentement sur la surface du milieu de culture. Le oenocytes disséqué peuvent être placés directement dans le tampon de lyse cellulaire approprié pour ARN ou d&#39;extraction de protéines. Notez que le oenocytes (segments abdominaux 2-5) à partir d&#39;une mouche mâle unique rendements généralement 15-20ng d&#39;ARN total. </li></ol> Figure Légende: <img src="/files/ftp_upload/2242/2242fig1.jpg" alt="Figure 1" /> Figure 1. L&#39;expression des gènes désaturase, desat1 et desatF, dans oenocytes mâles et femelles de D. melanogaster. transcription inverse-amplification par PCR des desat1 et desatF d&#39;ADNc dérivé de l&#39;adulte oenocytes masculins et féminins. Le gène est exprimé dans desat1 deux oenocytes mâle et femelle, l&#39;expression desatF est un dimorphisme sexuel, étant exprimée exclusivement dans oenocytes femelle. Correspondant à ces différences dans l&#39;expression des gènes, desat1 est nécessaire pour la production de composés d&#39;hydrocarbures communs aux hommes et aux femmes âgées de 9 et desatF est impliqué dans la production de composés d&#39;hydrocarbures spécifiques aux femmes 10,11. DesatF a été montré pour être exprimé spécifiquement au féminin par oenocytes dans 12 hybridation in situ. Notez que, en moyenne 15 20ng d&#39;ARN total est obtenu à partir de l&#39;oenocytes disséqués à partir d&#39;un avion individuel. L&#39;ARN a été isolé en utilisant le micro RNeasy (Qiagen); ADNc a été produite en utilisant les qScript synthèse cDND kit (Quanta Biosciences)

<ol>
	<li>Wielowiejski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ueber+das+blutgewebe+der+insekten.">Ueber das blutgewebe der insekten.</a> Zeit. Wiss. Zool. 43, 512-536 .</li><li>Krupp, J. J., Kent, C., Billeter, J. C., Azanchi, R., So, A. K., Schonfeld, J. A., Smith, B. P., Lucas, C., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+experience+modifies+pheromone+expression+and+mating+behavior+in+male+Drosophila+melanogaster.">Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster.</a> Curr. Biol. 18, 1373-1383 (2008).</li><li>Locke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+membranes,+Golgi+complexes,+and+vacuolar+systems.">Surface membranes, Golgi complexes, and vacuolar systems.</a> Annu. Rev. Entomol. 48, 1-27 (2003).</li><li>Billeter, J. C., Atallah, J., Krupp, J. J., Millar, J. G., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+cells+tag+sexual+and+species+identity+in+Drosophila+melanogaster.">Specialized cells tag sexual and species identity in Drosophila melanogaster.</a> Nature. 461, 987-991 (2009).</li><li>Gould, A. P., Elstob, P. R., Brodu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+oenocytes:+a+model+system+for+studying+cell-fate+specification+by+Hox+genes.">Insect oenocytes: a model system for studying cell-fate specification by Hox genes.</a> J. Anat. 199, 25-33 (2001).</li><li>Lawrence, P. A., Johnston, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+the+Drosophila+abdomen:+the+epidermis,+oenocytes+and+ventral+muscles.">Cell lineage of the Drosophila abdomen: the epidermis, oenocytes and ventral muscles.</a> J. Embryol. Exp. Morphol. 72, 197-208 (1982).</li><li>Johnson, M. B., Butterworth, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+aging+of+adult+fat+body+and+oenocytes+in+Drosophila+as+revealed+by+light+microscopic+morphometry.">Maturation and aging of adult fat body and oenocytes in Drosophila as revealed by light microscopic morphometry.</a> J. Morphol. 184, 51-59 (1985).</li><li>Gutierrez, E., Wiggins, D., Fielding, B., Gould, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+hepatocyte-like+cells+regulate+Drosophila+lipid+metabolism.">Specialized hepatocyte-like cells regulate Drosophila lipid metabolism.</a> Nature. 445, 275-280 (2007).</li><li>Marcillac, F., Bousquet, F., Alabouvette, J., Savarit, F., 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=A+mutation+with+major+effects+on+Drosophila+melanogaster+sex+pheromones.">A mutation with major effects on Drosophila melanogaster sex pheromones.</a> Genetics. 171, 1617-1628 (2005).</li><li>Chertemps, T., Duportets, L., Labeur, C., Ueyama, M., Wicker-Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+female-specific+desaturase+gene+responsible+for+diene+hydrocarbon+biosynthesis+and+courtship+behaviour+in+Drosophila+melanogaster.">A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster.</a> Insect Mol. Biol. 15, 465-473 (2006).</li><li>Legendre, A., Miao, X. X., Da Lage, J. L., Wicker-Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+a+desaturase+involved+in+female+pheromonal+cuticular+hydrocarbon+biosynthesis+and+courtship+behavior+in+Drosophila.">Evolution of a desaturase involved in female pheromonal cuticular hydrocarbon biosynthesis and courtship behavior in Drosophila.</a> Insect. Biochem. Mol. Biol. 38, 244-255 (2008).</li><li>Shirangi, T. R., Dufour, H. D., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+sex+pheromone-producing+enzyme+expression+in+Drosophila.">Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.</a> PLoS Biol. 7, e1000168-e1000168 (2009).</li></ol>

Dans cet article nous présentons la vidéo d&#39;un protocole de dissection détaillées pour la préparation de la oenocytes de l&#39;adulte D. melanogaster d&#39;une manière appropriée pour l&#39;analyse moléculaire. Une base de texte abrégé compte de la méthode de dissection a été décrit ailleurs 2, et la préparation obtenue oenocyte a été démontré pour être appropriés pour l&#39;extraction de l&#39;ARN et la protéine 2. Utiliser cette préparation, il peut également ...

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<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tungsten Wire</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>27-11</td>
<td>0.005&#x201D;/20&#x2019;</td>
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<td>Forceps</td>
<td>&nbsp;</td>
<td>Dumont</td>
<td>11252-30</td>
<td>#5</td>
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<td>Shields and Sang M3 Insect Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
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<td>RNeasy Micro Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74004</td>
<td>&nbsp;</td>
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<td>qScript cDNA Synthesis Kit</td>
<td>&nbsp;</td>
<td>Quanta Biosciences</td>
<td>95047-100</td>
<td>&nbsp;</td>
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<td>Monoject Hypodermic Needle</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
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dissection of oenocytes from adult drosophila melanogaster

Dans<em&gt; Drosophila melanogaster</em&gt;, Comme dans d&#39;autres insectes, une couche cireuse sur la surface externe de la cuticule, composée principalement de composés d&#39;hydrocarbures, offre une protection contre la dessiccation et autres défis environnementaux. Plusieurs composés de ces hydrocarbures cuticulaires (CHC) fonctionnent aussi comme des signaux sémiochimiques, et en tant que telle médiation communication phéromonale entre les membres de la même espèce, ou, dans certains cas entre les différentes espèces, et influencer les comportements. Des cellules spécialisées appelées oenocytes sont considérés comme le principal site de synthèse du CCH. Cependant, on sait relativement peu sur l&#39;implication de l&#39;oenocytes dans la régulation de l&#39;biosynthèse, le transport, et les voies de dépôt de contribuer à la sortie du CCH. Étant donné le rôle significatif que jouent les CSC dans plusieurs aspects de la biologie des insectes, y compris la communication chimique, la résistance dessiccation, et l&#39;immunité, il est important d&#39;acquérir une meilleure compréhension de la régulation moléculaire et génétique de la production du CCH au sein de ces cellules spécialisées. Le oenocytes adulte<em&gt; D. melanogaster</em&gt; Sont situés dans le tégument abdominal, et sont disposés en metamerically ruban comme les grappes de rayonnement le long de la surface intérieure de la cuticule chaque segment abdominal. Dans cet article, vidéo, nous démontrer une technique de dissection utilisé pour la préparation de oenocytes de l&#39;adulte<em&gt; D. melanogaster</em&gt;. Plus précisément, nous fournissons une analyse détaillée étape par étape, la démonstration de (1) comment préparer les filets d&#39;un adulte<em&gt; Drosophile</em&gt; Abdomen, (2) comment identifier les oenocytes et les distinguer des autres tissus, et (3) comment faire pour supprimer les grappes intactes oenocyte du tégument abdominal. Une brève illustration expérimentale de la façon dont cette préparation peut être utilisée pour examiner l&#39;expression de gènes impliqués dans la synthèse d&#39;hydrocarbures est inclus. La préparation disséqué démontrée ici permettra l&#39;analyse détaillée moléculaires et génétiques de la fonction oenocyte chez la drosophile adulte.

Watch this Scientific Journal Video about Dissection of Oenocytes from Adult Drosophila melanogaster at JoVE.com

Dissection of Oenocytes from Adult Drosophila melanogaster

Chez les insectes, les oenocytes produire des composés d&#39;hydrocarbures cuticulaires. Ces composés se protéger contre le dessèchement et faciliter la communication chimique. Ici nous démontrons une technique de dissection utilisée pour isoler les oenocytes de l&#39;adulte<em&gt; Drosophila melanogaster</em&gt;, Et d&#39;illustrer comment cette préparation peut être utilisée pour étudier les gènes impliqués dans la synthèse d&#39;hydrocarbures.

Dissection de Oenocytes d&#39;adultes Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

dissection-of-oenocytes-from-adult-drosophila-melanogaster

Research

JoVE Journal

Biology

Dissection of Oenocytes from Adult Drosophila melanogaster

20.3K vues.  University of Toronto. Chez les insectes, les oenocytes produire des composés d'hydrocarbures cuticulaires. Ces composés se protéger contre le dessèchement et faciliter la communication chimique. Ici nous démontrons une technique de dissection utilisée pour isoler les oenocytes de l'adulte Drosophila melanogaster, Et d'illustrer comment cette préparation peut être utilisée pour étudier les gènes impliqués dans la synthèse d'hydrocarbures.

Vidéo: Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection de SNC larvaire chez Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Recherche

Biologie

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissection de disques imaginaux de la 3ème Instar larves de drosophile

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

Enregistrements à partir de cellules entières du cerveau d&#39;adulte drosophile

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Suivi vidéo haute résolution de la locomotion chez les adultes Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissection de l&#39;hippocampe gyrus denté de souris adulte

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissection d&#39;organes sur le poisson zèbre pour adultes

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolement des Drosophila melanogaster Testicules

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Injection d&#39;insuline et d&#39;extraction hémolymphe pour mesurer la sensibilité d&#39;insuline dans les adultes Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Mesure de la durée de vie en<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Dissection et Immunocoloration Imaginal disques de<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...