Les auteurs tiennent à remercier Alexandra Opie pour l&#39;assistance technique et le soutien, James Barton pour fournir la vidéo narration, et Ramona Flatz et Joellen Sweeney pour apparaître dans la vidéo qui l&#39;accompagne. Ce travail a été financé par le Fonds fiduciaire de bienfaisance MJ Murdock (Grant No. 2012205 JED).

1 Le système de Microscope stéréo Remarque: Bien que ce protocole est facilement adaptable à n&#39;importe quel système de microscope stéréo couplé à un appareil photo numérique avec la possibilité d&#39;acquérir la vidéo, les détails sont fournis sur le système utilisé dans notre laboratoire (Table des Matières / Équipement). <ol><li> Acquisition vidéo numérique en utilisant un microscope stéréoscopique trinoculaire couplé à un appareil photo numérique commercial. </li><li> Afin de coupler l&#39;appareil photo numérique commerciale au port trinoculaire du microscope stéréo, retirez le ½x monture C du port de Phototube du microscope stéréo et le remplacer par un 1X C-mount. </li><li> Monter un coupleur d&#39;appareil photo numérique (43 mm de fil) à la 1X C-mount. </li><li> Montez deux anneaux abaisseurs, 58 mm à 48 mm, et 48 mm à 43 mm, à l&#39;attelage de la caméra pour combler la connexion du coupleur d&#39;appareil photo numérique à un kit adaptateur d&#39;objectif pour l&#39;appareil photo numérique. </li><li> Monter l&#39;appareil photo numérique le kit d&#39;adaptateur de lentille. </li><li> Acquérir vidéo avec le grossissement du microscope et un zoom optique de l&#39;ensemble de l&#39;appareil photo numérique pour un grossissement combinée d&#39;environ 12X (30 images par seconde, 640 x 480 pixels). Remarque: Le grossissement du microscope stéréo doit être rémunéré conformément à la 1X C-mount nouvellement reconfiguré du port trinoculaire. </li></ol> 2. imagerie Drosophila troisième stade larvaire <ol><li> Ruban d&#39;un marqueur permanent pour la plaque de platine noir d&#39;un microscope stéréo couplé à un appareil photo numérique de sorte que le côté du capuchon de marqueur occupe environ ¼ à ⅓ du champ de vision vertical observé sur l&#39;écran LCD de l&#39;appareil. Utilisation des dessus de marqueurs comme l&#39;étape d&#39;effectuer une imagerie des larves car elles sont dans un assortiment de couleurs qui peut être utilisée pour le code de couleur et de différencier les génotypes des larves est imagé. </li><li> Délimiter le champ de vision observé sur l&#39;écran LCD de l&#39;appareil numérique sur la surface de la partie supérieure de marqueur à pointe finemarqueur. </li><li> Sélectionnez un troisième stade larvaire à l&#39;image. Les critères de sélection des larves au troisième stade larvaire est la longueur du corps, l&#39;émergence de la source d&#39;alimentation au cours de la phase larvaire du cycle de vie, la présence de antérieures et postérieures stigmates, et la structure des crochets mandibulaires de l&#39;appareil de la bouche 11. S&#39;assurer que la larve est propre en le lavant soigneusement à l&#39;eau. </li><li> Illuminer l&#39;étage de haut marqueur permanent d&#39;en haut avec de la lumière à partir d&#39;un système d&#39;éclairage à fibres optiques. Ajustez l&#39;angle de la lumière incidente pour fournir un éclairage optimal. </li><li> Concentrer le microscope sur le bord de la partie supérieure de marqueur permanent. Commencez acquisition vidéo numérique. </li><li> Placer la chenille sur le côté du capuchon de marqueur d&#39;environ 75 ° par rapport à l&#39;axe vertical, à l&#39;extérieur du champ de vision, avec la partie antérieure de la larve, tournée vers le champ de vue (figure 1). Note: Placement de la larve sur le côté de la casquette de marqueur permet à la caméra d&#39;enregistrer le mouvement de ee larve d&#39;un point de vue latérale. Il aide à garder la larve humide avec de l&#39;eau afin qu&#39;ils ne tombent pas sur le côté de la casquette de marqueur. Il faut être prudent, cependant, de ne pas utiliser trop d&#39;eau que des quantités excessives adhèrent à la larve comme il rampe sur le terrain. </li><li> Pousser doucement et pousser la larve d&#39;un petit pinceau pour contraindre à ramper à travers le champ de vision. Soyez patient car les larves coopérer rarement et doivent souvent être revenu au point de départ de nombreuses fois avant qu&#39;ils rampent directement dans le champ. </li><li> Fiche d&#39;environ 10-15 minutes de séquences vidéo numériques sans interruption et culture et enlever toutes les séquences inutiles post-acquisition avec le logiciel de montage vidéo numérique. </li></ol> 3. Imaging adultes Drosophila <ol><li> Placez une seule drosophile adulte dans un jetable de 1,5 ml spectroscopique polystyrène cuvette. Remarque: CO 2 anesthésie de la drosophile adulte immédiatement avant un comporioral protocole d&#39;analyse peut compromettre les résultats 12. Il est recommandé que la drosophile adulte d&#39;un délai de 24 heures pour se remettre de CO 2 anesthésie avant d&#39;effectuer un test de comportement 13. </li><li> Branchez l&#39;autre extrémité de la cuvette avec une petite boule de coton. S&#39;assurer que la boule de coton est emballé assez serré pour occuper le grand espace de la PAC et confine à la volée dans le compartiment de réduction du volume de la cuve. </li><li> Placer la cuvette sur la plaque de platine blanc d&#39;un microscope stéréo et aligner correctement le compartiment à volume réduit de la cuvette avec le champ de vision observé sur l&#39;écran LCD de l&#39;appareil numérique. </li><li> Éclairer la cuvette au-dessus de la lumière d&#39;un système d&#39;éclairage à fibres optiques. Ajustez l&#39;angle de la lumière incidente pour fournir un éclairage optimal. </li><li> La mise au microscope et commencer à acquérir la vidéo numérique. </li><li> Fiche d&#39;environ 30-45 min de séquences vidéo numériques sans interruption et cultures et retirez tous les inutilesimages post-acquisition avec le logiciel de montage vidéo numérique. </li></ol>

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	<li>Zhang, B., Freeman, M. R., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila neurobiology: a laboratory manual. , (2010).</li><li>Frank, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+for+studying+synaptic+development,+function,+and+plasticity+using+Drosophila+as+a+model+system.">New approaches for studying synaptic development, function, and plasticity using Drosophila as a model system.</a> J Neurosci. 33, 17560-17568 (2013).</li><li>Mudher, A., Newman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila : a toolbox for the study of neurodegenerative disease. , (2008).</li><li>Bilen, J., Bonini, N. 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(24), (2009).</li><li>Colomb, J., Reiter, L., Blaszkiewicz, J., Wessnitzer, J., Brembs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+tracking+and+analysis+of+adult+Drosophila+locomotion+in+Buridan's+paradigm+with+and+without+visual+targets.">Open source tracking and analysis of adult Drosophila locomotion in Buridan's paradigm with and without visual targets.</a> PLoS ONE. 7, e42247 (2012).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1965).</li><li>Barron, A. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microtubule+Regulatory+Protein+Stathmin+Is+Required+to+Maintain+the+Integrity+of+Axonal+Microtubules+in+Drosophila.">The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.</a> 8, e683244 (2013).</li><li>Belmont, L. D., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+interacts+with+tubulin+dimers+and+increases+the+catastrophe+rate+of+microtubules.">Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.</a> Cell. 84, 623-631 (1996).</li><li>Cassimeris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncoprotein+18/stathmin+family+of+microtubule+destabilizers.">The oncoprotein 18/stathmin family of microtubule destabilizers.</a> Curr Opin Cell Biol. 14, 18-24 (2002).</li></ol>

Les auteurs ont déclaré que aucun conflit d&#39;intérêts existe.

La force de Drosophila melanogaster comme un système modèle pour l&#39;étude de la fonction du système nerveux provient en grande partie de la convergence des outils génétiques puissants disponibles et la large gamme de tests comportementaux robustes développé. Nous présentons ici une technique simple et largement accessible microscopie pour l&#39;acquisition vidéo numérique de haute qualité de la drosophile adulte et phénotypes de locomotives larvaires dans une perspective latérale. Nous...

Le fruit mouche Drosophila melanogaster commun est un système de modèle expérimental puissant pour étudier la fonction du système nerveux 1-3. Conservation évolutive de la structure et la fonction du système nerveux chez les humains, ainsi que la facilité de la manipulation génétique et une vaste gamme d&#39;outils génétiques chez la drosophile fait l&#39;organisme de première pour modéliser les maladies neurodégénératives humaines 4. Les mutations génétiques qui provoquent un dysfonctionnement du système nerveux entraînent souvent des larves mutant viable et adultes de Drosophila avec une altération de la locomotion. Phénotypes observés dans le système nerveux mutants défectueux, notamment le taux de locomotion, coordination anormale, et les mouvements spasmodiques chez les adultes, ainsi que les déficits de la contraction péristaltique de la paroi musculature du corps, et une paralysie partielle des larves réduits. Ces phénotypes ont été exploités dans le développement à haut débit de cribles génétiques et des dosages de la locomotion des larves mutant 5, 6 et 7 à 10 adultes de Drosophila visant à quantifier l&#39;altération de la locomotion et l&#39;identification de gènes nécessaire pour la fonction du système nerveux. Bien que ces approches sont extrêmement utiles pour quantifier les comportements locomotive larves et les adultes, ils ne parviennent pas à transmettre des informations qualitatives sur chaque comportement aberrant spécifique. Par exemple, alors que la troisième stade larvaire mutant peut présenter des paramètres de locomotion modifiées dans un test de comportement, il peut être difficile de savoir si cela est le résultat des altérations de contractions péristaltiques rythmiques pendant le cycle de ramper, manque général de coordination, ou une paralysie partielle du corps postérieur musculature de la paroi. Nous décrivons ici une technique simple et largement accessible microscopie pour l&#39;acquisition vidéo numérique de haute qualité de la drosophile adulte et phénotypes de locomotives larvaires dans une perspective latérale. Vidéo numérique acquise à partir d&#39;un point de vue latérale permet l&#39;observation directe et l&#39;analyse des distinctions subtiles dans locomotivcomportements e d&#39;une plus informative vue latérale orientation.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trinocular Stereozoom Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ6145TR</td>
			<td>&#189; C-mount was removed and replaced with 1x C-mount</td>
		</tr>
		<tr>
			<td>1X C-mount</td>
			<td>Leeds Precision Instruments</td>
			<td>LSZ-1XCMT2</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera Coupler (43mm thread)</td>
			<td>Qioptiq Imaging Solutions</td>
			<td>25-70-10-02</td>
			<td></td>
		</tr>
		<tr>
			<td>58mm to 48mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR5848</td>
			<td></td>
		</tr>
		<tr>
			<td>48mm to 43mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR4843</td>
			<td></td>
		</tr>
		<tr>
			<td>Lensmate Adapter Kit for Canon G10</td>
			<td>LensMateOnline.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Canon PowerShot G10 Digital Camera</td>
			<td>Canon U.S.A., Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Spectroscopic PolystereneCuvette</td>
			<td>Denville Scientific</td>
			<td>U8650-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

Drosophila melanogaster est un système de modèle expérimental puissant pour étudier la fonction du système nerveux. Les mutations génétiques qui provoquent un dysfonctionnement du système nerveux produisent souvent des larves viables et les adultes qui ont locomotion phénotypes défectueux qui sont difficiles à décrire de manière adéquate avec le texte ou représenter complètement avec une seule image photographique. Les formes actuelles de l&#39;édition scientifique, cependant, soutiennent la présentation de supports vidéo numérique comme documentation supplémentaire pour accompagner un manuscrit. Nous décrivons ici une technique simple et largement accessible microscopie pour l&#39;acquisition vidéo numérique de haute qualité des deux phénotypes larves et les adultes de Drosophila du point de vue latérale. Vidéo de la locomotion des larves et des adultes dans une vue de côté est avantageuse car elle permet l&#39;observation et l&#39;analyse des distinctions et de subtiles variations dans les comportements aberrants des locomotives. Nous avons utilisé avec succès la technique de visualiser et de quantifier aberrant rampant comportements en troisième stade larvaire, en plus de phénotypes mutants et les comportements des adultes, y compris le toilettage.

Nous avons utilisé avec succès cette technique pour acquérir et quantifier le phénotype comportemental larvaire associée à la perte de fonction du gène de la stathmine (figure 2) 14. Le gène code pour une protéine stathmine réglementaire microtubules qui partitionne la tubuline des pools de tubuline soluble, et se lie microtubules et favorise leur démontage 15,16. Stathmin fonction est nécessaire pour maintenir l&#39;intégrité des m...

Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Nous décrivons ici une technique simple et largement accessible microscopie à acquérir la vidéo numérique de haute qualité de la drosophile adulte et phénotypes mutants larvaires dans une perspective latérale.

Acquisition de haute qualité vidéo numérique de<em&gt; Drosophila</em&gt; Comportements larvaire et adulte à partir d&#39;un point de vue latéral

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

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Neuroscience

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

8.7K vues.  Willamette University.  Nous décrivons ici une technique simple et largement accessible microscopie à acquérir la vidéo numérique de haute qualité de la drosophile adulte et phénotypes mutants larvaires dans une perspective latérale. 

Vidéo: Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Visualisation des larves nerfs segmentaires en 3 Rd Instar Drosophile Les préparatifs des larves

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

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Neurosciences

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

Application de cartographie et de Enhancer-trap expression flippase dans larvaire et adulte Drosophile SNC

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissection et immunohistochimique de larve, de nymphe et adulte<em&gt; Drosophila</em&gt; Rétines

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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 ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imagerie en direct de<em&gt; Drosophila</em&gt; larves Neuroblastes

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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 ...

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 ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Focal Macropatch enregistrements de courants synaptiques de la jonction neuromusculaire larves de drosophile

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

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

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

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

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

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

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

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 ...