Nous remercions RIKEN pour le financement. P10-Gal4 a été un cadeau genre d&#39;Alain Vincent (Université Paul Sabatier, Toulouse, France).

1. Préparation des réactifs Remarques avant de commencer: la dissection et la coloration immunohistochimique sont effectués dans une chambre magnétique et la larve est coincé utilisant des broches insectes de forme spéciale. Des instructions détaillées sur la construction d&#39;une chambre magnétique, et la préparation de ces broches peuvent être trouvés dans les références liées 14,15. En bref, un trou carré 1x1cm est coupé dans une feuille magnétique et d&#39;une lamelle apposée à l&#39;arrière de la feuille pour faire une petite chambre. Les côtés de la chambre sont scellés avec de la colle époxy, après cette colle a mis la chambre est lavé plusieurs fois avec de l&#39;éthanol à 70% avant l&#39;utilisation. Dissection broches insectes sont préparés par la flexion de la forme requise et ensuite collés sur un onglet 14,15 métal. Alternativement à une languette métallique, nous avons utilisé une tête plate inversée punaise d&#39;acier avec une poignée faite d&#39;une pointe de coupure jaune. L&#39;utilisation de cet arrangement chambre magnétique permet un contrôle étroit over goupille de positionnement et de tissus d&#39;étirement lors de la dissection. Pour diriger l&#39;expression du gène rapporteur en différents sous-ensembles de neurones DA enquêteurs peuvent utiliser différents Gal4 lignes (résumé par Shimono et ses collègues 16). Beaucoup de ces lignes sont disponibles auprès des centres d&#39;actions publiques. Dans ce protocole représentatifs, nous effectuons immunomarquage d&#39;une ligne dans laquelle deux classes contrastantes de DA neurone sont co-marqués: le plus simple de classe I et les plus complexes de classe IV (P10-Gal4 17,18, SAMU-mCD8:: Kusabira-Orange (KO)). <ol><li> Préparer Ca + + libre HL3.1 salines <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 19. Filtre-stériliser et conserver à 4 ° C. Note: Ca + solution + libre empêche la contraction des muscles lors de la dissection. </li></ol></li><li> Préparer 2x tampon PHEM <ol><li> En mm: 130 TUBES, 60 HEPES, 20 EGTA, 4 MgSO4; pH 7,0. Filtre-stériliser et conserver à 4 ° C. Note: Ces matériaux ne se dissoudra pas jusqu&#39;à ce que le pH des approches 7.0. </li></ol></li><li> Préparer le fixateur fraîchement immédiatement avant la fixation. <ol><li> En vue de préparer une solution de 25ml, d&#39;abord paraformaldéhyde 2g mélanger, 100 ul de NaOH 1 M et de l&#39;eau dans un tube 10ml 50ml Falcon. </li><li> Agiter fixatif dans une 55 ° C avec bain d&#39;eau jusqu&#39;à ce shaker la solution est claire. (Le volume total sera d&#39;environ 11,5 ml). </li><li> Fixatif refroidir sur glace. </li><li> Ajouter 12,5 ml de tampon 2x PHEM. </li><li> Ajuster le pH à 7,0 avec HCl 1M. </li><li> Remplissez la solution avec de l&#39;eau jusqu&#39;à ce que le volume est de 25ml. </li><li> Filtrer la solution en utilisant du papier Whatman. </li></ol></li></ol> 2. Dissection des larves Notez avant de commencer: les réseaux de microtubules, et en particulier ceux dans les dendrites sensoriels, se décompose rapidement après l&#39;initiation de la dissection. Achieving dissection rapide en moins de cinq minutes, suivie d&#39;une fixation immédiate sont des facteurs clés dans la réussite de ce protocole. <ol><li> Laver la larve dans l&#39;eau et rapidement les déplacer dans cette chute en utilisant une boucle de cheveux. </li><li> Orientez le côté jusqu&#39;à la larve dorsale et la face ventrale sur le verre. Note: Cette orientation est d&#39;examiner les neurones pôle ventral. Pour examiner les neurones pôle dorsale, inverser l&#39;orientation. </li><li> Utilisez le centre d&#39;insectes-broche à broche à l&#39;extrémité antérieure près de la bouche des crochets. Placez la tige près de la fin pour obtenir les meilleurs résultats. </li><li> Déposer une goutte de sérum physiologique HL3.1 dans la chambre de dissection. </li><li> Couper la pointe très postérieure de la larve hors d&#39;microciseaux. Remarque: Cette étape ouvre une ouverture à l&#39;extrémité postérieure de la larve qui va permettre l&#39;accès à l&#39;microciseaux (étape 2.7). </li><li> Grab avec la pince de la région de l&#39;intestin qui est maintenant piquer hors de l&#39;ouverture à l&#39;extrémité postérieure de la larve. Tirez doucement sur le tube digestif tout entier. </li><li> Placez lela pointe d&#39;une pale de l&#39;microciseaux à travers l&#39;ouverture et coupé le long de la ligne médiane ventrale vers la partie antérieure. </li><li> En utilisant les broches angle, broches les coins maintenant libre, postérieure d&#39;abord, puis antérieure. Simultanément étirer doucement le filet ouvert larvaire. </li></ol> 3. Fixation, le blocage, la coloration, et le montage de filets larvaires Remarques avant de commencer: Toutes les étapes de fixation et de coloration sont effectuées dans la chambre de dissection. Pendant ce processus, faites attention de ne pas heurter les broches insectes tenant la larve, car cela pourrait conduire à des lésions tissulaires. Afin d&#39;éviter l&#39;expérience de se dessécher, faire toutes les étapes de coloration dans un petit récipient Tupperware entourées de tissus humidifiés. <ol><li> Aspirer le Ca + + libre HL3.1 tampon en utilisant une pointe de jaune. Ajoutez immédiatement le fixateur en utilisant un autre pipetteman. </li><li> Délicatement la pipette de haut en bas pour mélanger le fixateur avec les traces restantes de Ca + + libre HL3.1 tampondans la chambre. Immédiatement l&#39;aspirer et ensuite ajouter fixateur frais dans la chambre. </li><li> Incuber à température ambiante pendant 20 minutes. </li><li> Lavez 10mins 6x dans le PBST (0,1% de Triton X-100 dans du PBS). </li><li> Bloc avec du sérum de chèvre de 5% en PBST pendant 20 min à température ambiante. </li><li> Remplacer la solution de blocage avec des anticorps primaire dilué dans du sérum de chèvre de 5% en PBST. Les anticorps primaires utilisés sont de souris anti-α-tubuline (DM1A) et de rat anti-CD8 (5h10) à la fois dilué au 1 / 1000. Remarque: L&#39;investigateur peut souhaiter substitut de souris anti-α-tubuline (DM1A) avec souris anti-Futsch (22C10) 20,21 dilué au 1 / 1000 dans certaines circonstances (voir la discussion). </li><li> Incuber une nuit (au moins 16h) à 4 ° C. </li><li> Lavez 10mins 6x dans le PBST. </li><li> Ajouter une solution d&#39;anticorps secondaire dilué dans du sérum de chèvre de 5% en PBST. Les anticorps secondaires utilisés sont Alexa Fluor 488 de chèvre anti-IgG de souris et Cy3 âne anti-IgG de rat. Conserver l&#39;échantillon couvert pour empêcher fluorophorephoto-décoloration 22. </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> Lavez 10mins 6x dans le PBST. </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. Note: Pour la compensation des tissus améliorés, et un échantillon permanent stable, monter dans DPX 23 comme décrit précédemment. </li></ol> 4. Les résultats représentatifs: Fluorescent coloration a été examiné sous un microscope confocal. Dans les figures 1-2, les différentes branches au sein d&#39;une tonnelle dendrites ont différentes organisation du cytosquelette. La figure 1 montre une région de la tonnelle d&#39;un neurone de classe IV DA à l&#39;étape 1 er stade larvaire. La tonnelle entière est marquée par mCD8:: KO et détectés à l&#39;aide d&#39;un anticorps anti-CD8 et secondaire fluorescent (Cy3). La tubuline est détectée à l&#39;aide anti-α-tubuline anticorps et Fluorescent secondaire (Alexa Fluor 488). Les branches principales sont positives pour la tubuline, certaines branches latérales minces sont tubuline-négatif. Film 1 est un ensemble de sections de série grâce à une coloration semblable d&#39;un neurone de classe I DA. La figure 2 montre une région de la tonnelle d&#39;un neurone de classe IV DA colorées avec des anticorps contre le Futsch et CD8 au stade 1 er stade larvaire. Les branches principales sont Futsch-positifs, certains branches latérales minces sont Futsch-négatif. Figure 3. Trachées dans la paroi du corps larvaire montrent une organisation des microtubules complexes. Films 2 et 3 montrent des coupes en série de la coloration grâce muscles de la paroi du corps et de la trachée. <img alt="Figure 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> Figure 1. Fig. 1 montre une région de la tonnelle d&#39;un neurone de classe IV DA à l&#39;étape 1 er stade larvaire. La tonnelle entière est marquée par mCD8:: KO et détectés à l&#39;aide d&#39;anticorps anti-CD8 et secondaire fluorescent (Cy3). La tubuline est détecté utilisant des anticorps anti-&amp;-tubulid&#39;anticorps fluorescents n et secondaire (Alexa 488). Panneaux AC séquentielle confocale z-sections (0.5μm), C&#39;-C &quot;, la coloration seul anticorps à partir du panneau C. Exemple de branches avec (flèche jaune) ou sans (violet pointe de flèche) microtubules sont mis en surbrillance. Red microtubules pointes de flèches en évidence dans le sous-jacentes les cellules épithéliales. <img alt="Figure 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> Figure 2. Fig. 2 montre une région similaire de la tonnelle d&#39;un neurone de classe IV da colorées avec des anticorps contre le Futsch et CD8 au stade 1 er stade larvaire. La tonnelle entière est marquée par mCD8:: KO et détectés à l&#39;aide d&#39;anticorps anti-CD8 et secondaire fluorescent (Cy3). Futsch est détecté à l&#39;aide d&#39;anticorps anti-Futsch et secondaire fluorescent (Alexa 488). Les branches principales sont Futsch-positifs, certains branches latérales minces sont Futsch-négatif. Exemple de branches avec (flèche jaune) ou sans (violet pointe de flèche) Futsch sont mis en évidence. Figure 3. Trachée teinté en utilisant le protocole anti-tubuline décrit ci-dessus. Cette larve est le troisième stade larvaire et disséqués, comme décrit précédemment 23,24. Movie 3 montre élargie des sections en série à partir du champ marqué par un carré. Sections Film 1. Serial traçage coloration tubuline travers la tonnelle dendritiques d&#39;un neurone de classe I. La tonnelle entière est marquée par mCD8:: KO (Magenta) et détectés à l&#39;aide d&#39;anticorps anti-CD8 et secondaire fluorescent (Cy3). Tubuline (vert) est détecté en utilisant un anticorps anti-α-tubuline et secondaire fluorescent (Alexa Fluor 488). Échelle: d&#39;un côté de l&#39;image vidéo correspond à 46.88μm dans la section. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">Cliquez ici pour voir le film.</a> Film 2 sections. Serial traçage coloration tubuline en DBOY muscles de la paroi d&#39;une larve de troisième stade larvaire. La tubuline est détectée à l&#39;aide anti-α-tubuline anticorps et secondaire fluorescent (Alexa Fluor 488). Échelle: d&#39;un côté de l&#39;image vidéo correspond à 46.88μm dans la section. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">Cliquez ici pour voir le film.</a> Movie 3. Sections de série traçage coloration tubuline dans un corps mur trachée d&#39;une larve de troisième stade larvaire, marquée d&#39;un carré dans Figure.3. La tubuline est détectée à l&#39;aide anti-α-tubuline anticorps et secondaire fluorescent (Alexa Fluor 488). Échelle: d&#39;un côté de l&#39;image vidéo correspond à 46.88μm dans la section. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">Cliquez ici pour voir le film.</a>

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Nous n&#39;avons rien à révéler.

Pour comprendre comment des formes complexes cellulaires sont qu&#39;elle atteint est important d&#39;être en mesure de précision de dosage organisation des microtubules. Nous décrivons ici une méthode de marquage robuste immunohistologiques à l&#39;organisation des microtubules dendritiques dosage des dendrites des neurones sensoriels arborisation. En plus de coloration des neurones sensoriels, cette méthode permet d&#39;obtenir robustes coloration immunohistochimique de la trachée, muscles et autres tissus de l...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Catalogue le numéro de </th><th> Commentaires (Optionnel) </th></tr><tr><td> Forceps </td><td> Dumont </td><td> 11251-20 </td><td></td></tr><tr><td> Microciseaux </td><td> TSF </td><td> 15000-08 </td><td></td></tr><tr><td> De souris anti-α-tubuline (Clone: ​​DM1A) </td><td> Sigma </td><td> T9026 </td><td> Dilution 1 / 1000 </td></tr><tr><td> Souris anti-Futsch (Clone: ​​22C10), surnageant </td><td> Développement Etudes Hybridomes Banque </td><td> 22C10 </td><td> Dilution 1 / 1000 </td></tr><tr><td> Rat anti-CD8 (clone: ​​5h10) </td><td> Caltag </td><td> MCD0800 </td><td> Dilution 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 IgG anti-souris </td><td> Invitrogen </td><td> A-11001 </td><td> Dilution 1 / 500 </td></tr><tr><td> Cy3 anti-IgG de rat </td><td> Jackson Immunoresearch </td><td> 712-166-150 </td><td> Dilution 1 / 200 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

Pour comprendre comment les différences dans les formes cellulaires complexes sont atteints, il est important de suivre avec précision l&#39;organisation des microtubules. Le mur de la drosophile corps des larves contient plusieurs types de cellules qui sont des modèles pour étudier des cellules et la morphogenèse des tissus. Par exemple trachées sont utilisées pour examiner une morphogenèse du tube, et l&#39;arborisation dendritique (DA) des neurones sensoriels de la larve de drosophile sont devenus un système de primaires pour l&#39;élucidation des généraux et des neurones spécifiques à la classe des mécanismes de différenciation dendritiques de 2-5 et une dégénérescence 6 . La forme des branches des dendrites peut varier considérablement entre les classes de neurones, et même entre les différentes branches d&#39;un seul neurone 7,8. Les études génétiques suggèrent que les neurones DA différentiel de l&#39;organisation du cytosquelette peuvent sous-tendre les différences morphologiques en forme la branche dendritiques 4,9-11. Nous fournissons une méthode immunologique robuste à un étiquetagessay dans l&#39;organisation des microtubules in vivo chez DA sensorielle tonnelle neurone dendrite (figures 1, 2, Film 1). Ce protocole illustre la dissection et immunomarquage du stade larvaire d&#39;abord, un stade où les actifs excroissance sensorielle dendrites des neurones et l&#39;organisation de branchement se produit 12,13. En plus de coloration des neurones sensoriels, cette méthode permet d&#39;obtenir l&#39;étiquetage robuste de l&#39;organisation des microtubules dans les muscles (Cinéma 2, 3), la trachée (figure 3, Film 3), et d&#39;autres tissus de la paroi du corps. Il est précieux pour les chercheurs souhaitant analyser l&#39;organisation des microtubules in situ dans la paroi du corps où l&#39;étude des mécanismes de contrôle et que le tissu forme des cellules.

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

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

Pour comprendre comment les formes cellulaires complexes, comme les dendrites neuronales, sont atteints au cours du développement, il est important d&#39;être en mesure de précision de dosage organisation des microtubules. Nous décrivons ici une méthode de marquage immunohistochimique robustes afin d&#39;examiner l&#39;organisation des microtubules des cellules dendritiques dendrites des neurones sensoriels arborisation, la trachée, les muscles et les autres<em&gt; Drosophile</em&gt; Tissus du corps larvaire mur.

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 ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

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JoVE Journal

Neuroscience

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

12.3K vues.  RIKEN Brain Science Institute. Pour comprendre comment les formes cellulaires complexes, comme les dendrites neuronales, sont atteints au cours du développement, il est important d'être en mesure de précision de dosage organisation des microtubules. Nous décrivons ici une méthode de marquage immunohistochimique robustes afin d'examiner l'organisation des microtubules des cellules dendritiques dendrites des neurones sensoriels arborisation, la trachée, les muscles et les autres ...

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Imagerie des cellules vivantes des cellules sensorielles de précurseurs d&#39;organes en Intact Drosophile Les chrysalides

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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Neurosciences

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

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

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

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisation des propriocepteurs dans<em&gt; Drosophila</emLes larves&gt; et pupes

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Marquage fluorescent rétrograde Permet ciblée extracellulaire seule unité d&#39;enregistrement de neurones identifiés<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Étiquetage des cellules isolées dans le système nerveux central des<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of 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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportements olfactifs dosées par Tracking Computer Of<em&gt; Drosophila</em&gt; Dans un Olfactométre quatre quadrants

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Étiquetage balistique des neurones pyramidaux dans les tranches de cerveau et dans la culture cellulaire primaire

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Détermination semi-quantitative de la densité des neurones dopaminergiques dans la Substantia Nigra des modèles de rongeurs à l’aide de l’analyse automatisée d’images

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Observation des réseaux de microtubules dans les jonctions neuromusculaires et les cellules musculaires de la drosophile

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Pleins feux sur l’auteur : Comprendre le réseau de microtubules dans les jonctions neuromusculaires de la drosophile 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.