Les auteurs remercient le RIKEN pour le financement. Nous remercions également Cagri Yalçin, Caroline Delandre, et Jay Parrish pour les discussions sur les protocoles génétiques et immunohistochimie.

1.Préparation de réactifs <ol><li> Préparer Ca + + libre HL3.1 salée 28. <ol><li> En mm: 70 NaCl, KCl 5, 20 MgCl2, 10 NaHCO 3, HEPES 5, 115 le saccharose, le tréhalose et 5; pH 7,2. Filtre stériliser et conserver à 4 ° C. Note: Ca + solution + libre empêche la contraction des muscles lors de la dissection. </li></ol></li><li> Faire de poly-L-lysine (PLL) lamelles. <ol><li> Dissoudre 100 mg de PLL dans l&#39;eau 4,2 ml et de faire des aliquots 300μl dans des tubes Eppendorf et congeler à -20 ° C. </li><li> Avant de lamelles de revêtement, premier dégel d&#39;une partie aliquote, le mettre à 10ml dans le trou DDH 2 O et d&#39;ajouter 20 pi de Kodak Photo-Flo, la concentration finale de PLL est de 0,7 mg / ml. </li><li> Plonger des lamelles pour les 30mins dans une solution de PLL, enlever et sécher, puis rincer rapidement à l&#39;eau et à sec. Répéter deux fois. Lamelles traitées durer environ un mois. </li></ol></li></ol> 2. Croisements génétiques <ol><li> To générer des clones MARCM. Voici par exemple une croix en utilisant un pilote pan-DA 11,27: FRT2A x hsFLP; Gal4 109 (2) 80, SAMU-mCD8:: GFP; baignoire-GAL80 FRT2A/SM5-TM6B </li><li> Pour générer Flp-out clones. Voici une croix par exemple en utilisant une classe IV-pilote spécifique 8: PPK-Gal4 x yw, hsFLP; SAMU-FRT-CD2, y +-stop-FRT-mCD8:: GFP </li></ol> 3. Collecte des embryons <ol><li> Gardez drosophile croise dans une bouteille de collection à 25 ° C et de recueillir des embryons sur un jus de pomme gélose 29 répandit avec une fine couche de pâte de 30,31 levure. </li></ol> 4. Traitement par choc thermique des embryons <ol><li> Placer une assiette vide en opposition à la plaque de gélose de pomme jus de sur lequel les embryons ont été posées. Seal autour de ces deux plaques avec du parafilm (Fig. 2). </li><li> Lors de choc thermique ème submerger,Plaque e dans le bain d&#39;eau et la maintenir enfoncée en utilisant un poids de métal. </li><li> Pour les embryons MARCM <ol><li> Recueillir des embryons pour 2h et incuber dans un plat de 10cm de Pétri entouré par des tissus humidifié à 25 ° C pour une 2h supplémentaires. Remarque: Régler le protocole de choc thermique modifie la fréquence à laquelle les clones sont générés. </li><li> Pour un plus petit nombre de clones, visant simples neurones isolés, submerger et de choc thermique dans le bain-marie pendant 1h à 38 ° C. </li><li> Pour un plus grand nombre de clones, de choc thermique pour les 45mins à 38 ° C, de récupérer à la température ambiante 30 minutes, puis un choc thermique à nouveau pour une 30mins supplémentaires. </li></ol></li><li> Recueillir Flp-out embryons pour 24h, et le choc thermique pour 1h à 38 ° C. </li></ol> 5. Le dépistage de clones <ol><li> Après un choc thermique, enlevez le couvercle de l&#39;arrangement étanche à l&#39;eau et placez la plaque de gélose de pomme jus dans une boîte de Pétri 10cm entourées de tissus humidifiés. Culture des embryons et des larves subséquentes à 25 ° C jusqu&#39;à ce que se promenerING 3 ème stade. Remarque: les conditions de culture, en particulier la nutrition, ont été montré de modification DA morphologie dendritique tonnelle 32,33. Assurez-vous que les larves de plus en plus avoir accès à la pâte de levure à tout moment, de suivre et reconstituer la levure coller comme requis. </li><li> De ce point dans le protocole de partir, manipuler délicatement avec une pince les larves d&#39;insectes. </li><li> Brièvement et rincer doucement les larves dans l&#39;eau du robinet, puis placez-les sur une plaque de gélose. Remarque: Cette étape permet de réduire de fond auto-fluorescence de la nourriture ou de saleté sur la surface des larves. </li><li> Examiner les larves sous un microscope à fluorescence puissante dissection. Identifier les larves avec GFP-positives neurones / cellules de la paroi du corps. </li></ol> 6. Imagerie in vivo dans des dendrites <ol><li> Placer la larve dans un verre à glisser la dépression avec une petite goutte de glycérine à 80%. Placer une lamelle sur la lame d&#39;immobiliser la larve. Assurez-vous que l&#39;air ne reste entre la larve et l&#39;coverslip. </li><li> Poussez doucement la lamelle à rouler la larve pour permettre la visualisation du neurone d&#39;intérêt. L&#39;image de la mCD8:: GFP-étiquetée tonnelle dendritiques par microscopie confocale. </li></ol> 7. Dissection des larves Notez avant de commencer: DA dendrites neuronales se dégrader rapidement après le début de la dissection. Disséquer chaque larve individu dans moins de 5min à assurer la morphologie des dendrites bon. <ol><li> Disséquer errance 3 e stade larvaire sur une plaque Sylgard comme décrit précédemment 34 avec les modifications suivantes: <ol><li> Pour l&#39;imagerie des neurones DA Termini axone, s&#39;assurer que le système nerveux central est intact et les nerfs segmentaires ne sont pas brisés. Après l&#39;ouverture de la larve, d&#39;abord soigneusement couper les connexions trachéale à la paroi du corps puis retirez délicatement le tube digestif entier. </li><li> Si l&#39;imagerie des dendrites seul, enlever la SNC pour permettre à un plat de montage. </li></ol></li></ol> 8. Fixation et le blocage des filets larvaires <ol><li> Avec la larve encore épinglé à la plaque Sylgard, le fixer dans PFA 4% dans du PBS pendant 20 minutes à température ambiante sur une légère rotation shaker. </li><li> Supprimer les traces des tissus larvaires (la graisse corporelle, la trachée, etc) après fixation. </li><li> Laver dans du PBST (PBS avec 0,1% de Triton X-100) 10mins 3x sur la plaque Sylgard et agitateur. Si l&#39;examen de Termini axone, garder le filet sur la plaque Sylgard. (Si dendrites coloration, il est possible de transférer les filets des larves dans un tube de 0,5 ml à cette étape 34) </li><li> Bloquer les larves pendant 20 minutes à température ambiante dans 5% des taux sériques normaux âne (NDS) dans PBST sur un agitateur. </li></ol> 9. Coloration des filets larvaires <ol><li> Retirer la solution de blocage et incuber en anticorps primaire (dans 5% NDS / PBST) nuit à 4 ° C dans un petit récipient Tupperware entouré par humidifié tissus. </li><li> Retirer les tubes de 4 ° C et incuber une heure supplémentaire à température ambiante. </li><li> Lavez 10mins 6x dans le PBST. Ajouter le ANTIB secondairesOdy (dans 5% NDS / PBST), et le couvercle pour éviter la photo-blanchiment fluorophore 34. </li><li> Incuber soit à température ambiante pendant deux heures ou une nuit à 4 ° C suivie d&#39;une heure à température ambiante. </li><li> Laver les larves 6x 10 minutes dans du PBST et procéder à un montage. </li></ol> 10. Montage des filets de larves d&#39;examen de la tonnelle dendrite <ol><li> Mont chaque filet des larves aussi plate que possible en utilisant des ciseaux de dissection ou un scalpel pour couper la tête (y compris les crochets bouche), et la partie arrière (y compris les stigmates) 34. </li><li> Mettez le filet des larves sur la diapositive cuticule vers le bas, montez dans le glycérol 80%, et sceller les bords de la lamelle avec du vernis à ongles pour une «rapide» de montage 34. </li><li> Pour un montage permanent et une image plus claire <ol><li> Placer le filet de muscles disséqués larves vers le bas sur une goutte de PBS sur une lamelle de PLL, il sera rapidement adhérer à la lamelle. </li><li> Retirez autant de liquide que possible après le montage, uter ne lui permettent pas de sécher complètement. </li><li> Prenez la lamelle (avec filet de larves jointe) grâce à une série d&#39;éthanol: l&#39;éthanol 35%, suivie par 50%, 70%, 95%, et enfin 2x éthanol à 100% pour chacun des 10 minutes (le 2ème solution 100% EtOH doit être changé fréquemment ), et enfin, laver 2-3x 10 minutes dans le xylène. </li><li> Mettre une goutte de milieu de montage DPX (Xylène Plastifiant Distyrene) sur une lame propre et soigneusement préparer le filet de la lamelle à l&#39;envers sur le dessus. Gardez à l&#39;obscurité, et d&#39;attendre une journée pour le DPX de mettre en avant l&#39;imagerie. </li></ol></li></ol> 11. Montage des filets de larves d&#39;examen des terminus axone <ol><li> Gardez les nerfs segmentaires courant entre le PNS et le SNC intacte lors de la dissection, la fixation et coloration afin de permettre le traçage des axones des neurones périphériques du corps de cellules sensorielles de la VNC. </li><li> Montez chaque filet de la cuticule des larves vers le bas dans 80% de glycérine dans une diapositive dépression. Trace les axones du corps cellulaire DA auVNC sous le microscope confocal. Remarque: PNS neurones dans chaque projet segment du corps mur pour le segment apparenté de la VNC. </li></ol> 12. Les résultats représentatifs: Les résultats représentatifs sont présentés dans les figures 3-5. Fig. 3 montre la tonnelle entière d&#39;un neurone de classe IV da, capturé in vivo sous le microscope confocal. Fig. 4 montre un gros plan d&#39;une partie de la tonnelle d&#39;un dendrite immunohistochimiquement étiquetés neurone de classe III qui a été correctement fixé afin de préserver la morphologie. L&#39;encart montre la dégradation associée qui peut survenir après une dissection infructueuses et la fixation (deux colorés avec des anticorps anti-GFP). Fig. 5 montre une seule classe IV axone terminal dans le SNC (anti-GFP, vert); tous les terminus de classe IV sont co-colorés avec anti-CD2 (magenta). <img alt="Figure 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> Figure 1 Aperçu Protocole. a) Recueillir et ensuite de choc thermique embryons. b)<html> Sélectionnez une larve avec un clone GFP-positives DA neurone, puis l&#39;image de la GFP-étiquetée dendrite tonnelle dans la larve vit. c) Disséquer la larve, puis immunohistochimiquement tacher le filet. d) Monter le filet taché, puis l&#39;image de la tonnelle dendritiques et projections axonales des neurones DA les clones. <img alt="Figure 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> Figure 2: préparation de la plaque de gélose de pomme jus pour choc thermique. Prenez la plaque (flèche blanche, AB), ajouter un second plat de Pétri sur le dessus (flèche rouge b) et joint d&#39;étanchéité autour de Parafilm (flèche bleue, b). <img alt="Figure 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> Figure 3 en image in vivo de la tonnelle d&#39;un dendrite mCD8:: GFP-étiquetée clone MARCM (génotype comme en 2.1), représentant une classe IV (v&#39;ada) neurone d&#39;une larve de 3 e stade larvaire. La barre d&#39;échelle est 50 microns. d/3111/3111fig4.jpg &quot;/&gt; Figure 4 mCD8:: GFP-étiquetée clone MARCM d&#39;un neurone de classe III (ddaA) colorés avec des anticorps anti-GFP, montrant une bonne morphologie après dissection réussie et la fixation (en vert). L&#39;encart montre la dégradation des dendrites (flèches blanches) survenant après la dissection infructueuses et la fixation (magenta). La barre d&#39;échelle est 25 um. <img alt="Figure 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> Figure 5 La 3 e stade larvaire VNC. Un clone Flp-out d&#39;un terminus de classe VI seul axone (VDAB) (génotype comme en 2.2) est détecté à l&#39;aide d&#39;anticorps anti-GFP (vert). Anti-CD2 étiquettes tous les terminus de classe IV (magenta). La barre d&#39;échelle est 25 um.

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J., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Embryonic+Nervous+System+in+Whole-mount+Drosophila+Embryos.">Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos.</a> J. Vis. Exp. (46), e2150-e2150 (2010).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> J Vis Exp. , (2009).</li><li>Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., 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=Drosophila+Larval+NMJ+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

La Drosophile larvaire DA neurone modèle fournit un excellent système pour étudier les mécanismes génétiques que la morphologie des neurones de contrôle et de la formation des circuits. MARCM est généralement utilisé pour l&#39;étiquetage et pour générer des clones mutants DA neurone. Pour MARCM nous utilisons soit un pan-neural (p. ex Gal4 C155) ou spécifique des neurones DA pilote. L&#39;utilisation d&#39;un pilote pan-neuronal, il est possible d&#39;utiliser directem...

<table border="1"><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> SZX16 microscope à dissection de fluorescence (avec GFPHQ filtre) </td><td> Olympus </td><td> SZX16 </td><td></td></tr><tr><td> Pince en direct insectes </td><td> TSF </td><td> 26030-10 </td><td></td></tr><tr><td> 26mm x 76mm en verre glisser la dépression </td><td> Toshinriko Co. </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (ou Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poly-L-lysine </td><td> Sigma </td><td> P-1524 </td><td> Ce produit a prouvé plus efficace </td></tr><tr><td> DPX milieu de montage </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> Lapin anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Dilution 1:500 </td></tr><tr><td> Rat anti-CD8 </td><td> Caltag </td><td> 5h10 </td><td> Dilution 1:200 </td></tr><tr><td> Souris anti-CD2 </td><td> AbD serotec </td><td> MCA443R </td><td> Dilution 1:700 </td></tr><tr><td> Souris anti-Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td> Dilution 1:10 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

Développement du système nerveux requiert la spécification correcte de la position du neurone et de l&#39;identité, suivie par le développement des neurones dendritiques précise classe spécifique et le câblage axonal. Récemment, le dendritiques arborisation (DA) des neurones sensoriels de l&#39;Drosophile larvaire du système nerveux périphérique (SNP) sont devenus puissants modèles génétiques dans lesquels d&#39;élucider les mécanismes à la fois générales et spécifiques à chaque classe de la différenciation neuronale. Il ya quatre principales classes de neurones DA (I-IV) 1. Ils sont nommés dans l&#39;ordre croissant de complexité des dendrites tonnelle, et ont de la classe des différences spécifiques dans le contrôle génétique de leur différenciation 2-10. Le DA système sensoriel est un modèle pratique pour étudier les mécanismes moléculaires derrière le contrôle de la morphologie dendritique 11-13 parce que: 1) il peut profiter de l&#39;puissants outils génétiques disponibles chez la drosophile, 2) le neurone DA dendrite tonnelle se propage en seulement deux dimensions sous un article optiquementar des larves cuticule rendant facile à visualiser en haute résolution in vivo, 3) la diversité des classes spécifiques de morphologie dendritique facilite une analyse comparative de trouver des éléments clés contrôlant la formation de simples vs très ramifiées arbres dendritiques, et 4) dendritiques tonnelle stéréotypées formes de neurones DA différents de faciliter les analyses statistiques morphométriques. DA neurone modifie l&#39;activité de la sortie d&#39;un générateur de pattern de locomotion des larves centrale 14-16. Les différentes classes de neurones DA ont des modalités sensorielles distinctes, et leur activation provoque différentes réactions comportementales 14,16-20. Par ailleurs des classes différentes envoyer des projections axonales stéréotypée dans le système central de la drosophile larvaires nerveux dans la corde nerveuse ventrale (VNC) 21. Ces projections se terminent par des représentations topographiques de deux neurones DA modalité sensorielle et la position dans la paroi du corps du champ dendritique 7,22, 23. Ainsi l&#39;examen des projections axonales DA peut être utilisée pour élucider les mécanismes qui sous-tendent la cartographie topographique 7,22,23, ainsi que le câblage d&#39;un circuit simple de locomotion des larves modulant 14-17. Nous présentons ici un guide pratique pour générer et analyser les mosaïques génétiques marquant 24 neurones DA via MARCM (Analyse mosaïque avec un marqueur cellulaire répressible) 1,10,25 et FLP-out 22,26,27 techniques (résumées dans la Fig. 1).

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Les neurones sensoriels de l&#39;arborisation dendritique des<em&gt; Drosophile</em&gt; Larvaires système nerveux périphérique sont des modèles utiles pour élucider la fois générales et spécifiques à chaque classe neurone mécanismes de la différenciation neuronale. Nous présentons un guide pratique pour générer et analyser dendritiques arborisation neurone mosaïques génétique.

Analyse morphologique des Drosophile Larvaires périphérique dendrites et des axones des neurones sensoriels utilisant mosaïques génétiques

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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JoVE Journal

Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

15.2K vues.  RIKEN Brain Science Institute. Les neurones sensoriels de l'arborisation dendritique des Drosophile Larvaires système nerveux périphérique sont des modèles utiles pour élucider la fois générales et spécifiques à chaque classe neurone mécanismes de la différenciation neuronale. Nous présentons un guide pratique pour générer et analyser dendritiques arborisation neurone mosaïques génétique.

Vidéo: Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Visualisation in vivo des vésicules synaptiques Dans Drosophila larves axones segmentaire

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

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Neurosciences

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Étiquetage immunohistologique des microtubules dans dendrites des neurones sensoriels, trachées et les muscles dans la Drosophile Mur Corps Larve

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisation et la manipulation génétique des dendrites et des épines dans le cortex cérébral et l&#39;hippocampe de souris à l&#39;aide<em&gt; In utero</em&gt; Électroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Enregistrement colorant sensible à la tension d&#39;axones, des dendrites et des épines dendritiques des neurones individuels dans des tranches de cerveau

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Analyse fonctionnelle du circuit d&#39;alimentation des larves dans Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tout le mont immunomarquage des récepteurs olfactifs neurones dans le<em&gt; Drosophila</em&gt; Antenne

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

La production et l&#39;isolement des axones de neurones sensoriels de l&#39;analyse biochimique Utilisation des filtres poreux

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Enlèvement de<em&gt; Drosophila</em&gt; Muscle Tissue de Fillets larvaires pour immunofluorescence Analyse des neurones sensoriels et les cellules épidermiques

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

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 ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Analyse métabolique des larves de Drosophila melanogaster et un cerveau adulte

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...