This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1. Fixation de la tête sur les colliers et incorporation dans la paraffine Note: Toutes les étapes du processus de fixation doit être fait dans une hotte. Méthylbenzoate, tout en ne posant un risque pour la santé, a une odeur très distincte, ce qui peut être écrasante en cas de manipulation dans une hotte. <ol><li> Avant anesthésier les mouches, forment 50 ml de solution Carnoy en ajoutant 15 ml de chloroforme et 5 ml d&#39;acide acétique glacial à 30 ml d&#39;éthanol à 99% (ne pas mélanger le chloroforme et l&#39;acide acétique). Verser dans un récipient en verre à fond plat, comme un plat cristallisant, veiller à ce que les colliers peuvent poser à plat et sont complètement couvertes par la solution. </li><li> Anesthetize les mouches avec du CO 2 ou de l&#39; éther. </li><li> les mouches de la discussion (jusqu&#39;à 20 avec la plupart des colliers) par leur cou dans les colliers à l&#39;aide de pinces. Rappelez - vous d&#39;aligner toutes les têtes dans la même orientation, comme on le voit sur la figure 1A, et être doux pour assurer qu&#39;aucun dommage se produit à la tête ou les yeux. </li><li> Inclure les mouches sinus de oculis (flèches, Figure 1A) à des positions aléatoires de sorte que l&#39;ordre des mouches peut être facilement identifié dans les sections. En outre, si les mouches expérimentales ont une couleur des yeux clair ou blanc, enfiler des mouches aux yeux rouges, comme le type sauvage, entre eux pour veiller à ce que le pigment est présent suffisamment pour colorer la diapositive. Notez l&#39;ordre des mouches sur une feuille de protocole ainsi que le nombre de collier si vous utilisez plus d&#39;un collier. </li><li> Une fois un collier a été terminé, placez-le dans la solution préparée Carnoy pendant 3,5 - 4 h. </li><li> Déverser la solution Carnoy dans la disposition cartouche appropriée et commencer les lavages à l&#39;éthanol. Assurez-vous de verser lentement afin de ne pas perturber le placement des colliers dans le conteneur. </li><li> Laver les colliers pendant 30 minutes dans 99% d&#39;éthanol deux fois. </li><li> Laver les colliers dans 100% d&#39;éthanol pendant 1 h. Assurez-vous de changer les lavages à temps pour éviter overdehydration. </li><li> Mettez les colliers en méthylbenzoateO / N à la température ambiante. Fermer le récipient avec du parafilm pour empêcher l&#39;évaporation du benzoate de méthyle. </li><li> Verser le methylbenozate dans le récipient jetable approprié dans la hotte. Ajouter un mélange préalablement préparé de 1: 1 à faible point de fusion (56-57 ° C) de la paraffine de cire et méthylbenzoate. A partir de ce moment-là, les colliers doivent être conservés dans un incubateur à 65 ° C pour faire en sorte que la paraffine ne durcit pas. </li><li> Verser le methylbenozate et le mélange de paraffine dans le récipient jetable approprié et verser la cire de paraffine fondue pure, maintenue à 65 ° C sur les collerettes. </li><li> Modifier la paraffine après 30 minutes et répéter ce au moins 5 fois. Au moins 6 - 8 lavages doivent être effectués. </li><li> Une fois que les lavages sont complets, placez les colliers dans un bac à glaçons en caoutchouc avec des fentes d&#39;environ la taille des colliers. Versez la paraffine fondue sur eux jusqu&#39;à ce que complètement couvert et laisser durcir O / N (essayez d&#39;éviter les bulles d&#39;air). </li><li> Retirez les blocs de paraffine containing les colliers à partir du bac à glaçons. Séparer le bloc de paraffine du collier à l&#39;aide d&#39;une lame de rasoir, brisant doucement le col. Les têtes seront dans le bloc de paraffine tandis que les corps restent dans le col. Les blocs peuvent être maintenus à la température ambiante. </li><li> Pour nettoyer les colliers, les faire tremper dans un agent de deparafinization à 65 ° C pour éliminer la paraffine, propre avec lavage léger, et laver dans de l&#39;éthanol avant de le réutiliser. </li></ol> 2. Sectionnement et montage <ol><li> Chauffer une plaque chauffante à 50 ° C. Placez les porte-objets (ou des blocs métalliques de montage) et des lames de rasoir sur la plaque et laissez-les réchauffer. </li><li> En fonction de l&#39;orientation souhaitée pour sectionner, fixer le bloc de paraffine, soit avec les têtes vers le côté (pour les sections horizontales) ou vers le haut (pour les sections frontales) à un bloc de montage chauffé (fondre brièvement le bloc du côté de contact). Retirez le bloc de la plaque chauffante et laisser refroidir pendant au moins 10 min pour veiller à ce que la paraffine est durcie suffisamment pour une bonne étanchéité sur le bloc de montage. Gardez la rangée de têtes alignées parallèlement à la surface du bloc, autant que possible pour éviter les sections inégales. </li><li> Prenez une lame de rasoir et couper l&#39;excédent de paraffine à l&#39;écart des têtes de mouches de sorte que seule une petite rangée avec les têtes intégrés reste (la lame de rasoir peut être réchauffé pour faciliter la garniture). Assurez-vous de ne pas couper trop de sorte que la paraffine ne se casse pas pendant la coupe (plus parage peut être fait pendant la coupe). </li><li> Placez le bloc de montage dans le porte-objet de la microtome et veiller à ce que l&#39;alignement de la rangée de têtes est aussi parallèle que possible du bord de la lame. </li><li> Préparer des lames de microscope en les recouvrant d&#39;une fine couche de poly-L-lysine (PLL) solution et les laisser sécher pendant 5 min. Couvrez-les avec de l&#39;eau peu avant d&#39;utiliser. </li><li> Couper 7 um sections et transférer le ruban de sections à la diapositive flottant sur l&#39;eau. &lt;br /&gt; NOTE: Pour obtenir l&#39;ensemble du cerveau pour les sections horizontales, nous recueillons le ruban à partir de quand commencer à couper dans l&#39;oeil jusqu&#39;à ce que la tête a été complètement coupé (coupe de la trompe dans le cerveau). Plus d&#39;une diapositive peut être nécessaire pour l&#39;ensemble de la tête. </li><li> Placer la lame sur une plaque chauffante à 37 ° C et laisser le ruban d&#39;étendre pendant environ 1 min. </li><li> Retirer l&#39;excès d&#39;eau (en le versant hors ou à l&#39;aide d&#39;un tissu) et sécher les diapositives O / N. </li><li> Retirez la cire de paraffine de la diapositive en plaçant les lames dans un, vertical jar grand slide-coloration rempli d&#39;un agent de deparafinization (recouvrant complètement les sections). Effectuer 3 lavages de 30 min - 60 min chacun. </li><li> Retirer la lame du lavage final. Placez 2 gouttes de médias d&#39;enrobage sur la lame et le couvrir avec une grande lamelle. </li></ol> 3. Photographier et analyse les sections <ol><li> Laisser les lames préparées à sécher pendant 1 - 2 d. Ensuite, examinez-les sur une Microsc de fluorescenceope sous une lumière bleue. </li><li> Utilisez un faible grossissement pour déterminer l&#39;orientation des mouches et de trouver la région d&#39;intérêt se concentrant sur une région spécifique. NOTE: Pour les mouches de sws (voir la figure 2), on trouve la section qui contient la grande commissure et de prendre une image (généralement à un grossissement de 40X). Lors de l&#39; analyse de la totalité du cerveau (comme dans la figure 3), nous faisons défiler à travers toutes les sections de la tête et soit photographier la section avec le phénotype le plus sévère ou toutes les sections qui montrent vacuoles. </li><li> Pour une analyse en double aveugle, prendre et images numériques sans connaître le génotype et enregistrer le nombre de cols et de la position de la tête dans la ligne pour les identifier plus tard. </li><li> Une fois que les images ont été prises, de les analyser en utilisant un logiciel d&#39;imagerie. </li><li> Comptez le nombre de vacuoles par section ou par tête. Pour mesurer la taille de la vacuole, ouvrez les images dans un logiciel et sélectionnez les vacuoles avec une sélection tropl. Déterminer la quantité de pixels dans les vacuoles sélectionnés. </li><li> Pour la conversion en um 2, prendre une photo d&#39;un étalonnage micromètre d&#39;étape au grossissement utilisé pour acquérir des photos. Déterminer la quantité de pixels à 100 um 2 pour calculer un facteur de conversion. </li><li> Convertir le nombre total de pixels en um 2 en divisant le nombre de pixels par le facteur de conversion calculé à l&#39;étape précédente. </li></ol>

<ol>
	<li>Alexander, A. G., Marfil, V., Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Caenorhabditis+elegans+as+a+model+to+study+Alzheimer's+disease+and+other+neurodegenerative+diseases.">Use of Caenorhabditis elegans as a model to study Alzheimer's disease and other neurodegenerative diseases.</a> Front Genet. 5, 279 (2014).</li><li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neurodegenerative+disease+modeling+using+Drosophila.">Human neurodegenerative disease modeling using Drosophila.</a> Annu Rev Neurosci. 26, 627-656 (2003).</li><li>Calahorro, F., Ruiz-Rubio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+an+experimental+tool+for+the+study+of+complex+neurological+diseases:+Parkinson's+disease,+Alzheimer's+disease+and+autism+spectrum+disorder.">Caenorhabditis elegans as an experimental tool for the study of complex neurological diseases: Parkinson's disease, Alzheimer's disease and autism spectrum disorder.</a> Invert Neurosci. 11, 73-83 (2011).</li><li>Chen, X., Barclay, J. W., Burgoyne, R. D., Morgan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+C.+elegans+to+discover+therapeutic+compounds+for+ageing-associated+neurodegenerative+diseases.">Using C. elegans to discover therapeutic compounds for ageing-associated neurodegenerative diseases.</a> Chemistry Central Journal. 9, 65 (2015).</li><li>Gama Sosa, M. A., De Gasperi, R., Elder, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+neurodegenerative+diseases+in+transgenic+systems.">Modeling human neurodegenerative diseases in transgenic systems.</a> Hum Genet. 131, 535-563 (2012).</li><li>Jaiswal, M., Sandoval, H., Zhang, K., Bayat, V., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+mechanisms+that+underlie+human+neurodegenerative+diseases+in+Drosophila.">Probing mechanisms that underlie human neurodegenerative diseases in Drosophila.</a> Annu Rev Genet. 46, 371-396 (2012).</li><li>Konsolaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruitful+research:+drug+target+discovery+for+neurodegenerative+diseases+in+Drosophila.">Fruitful research: drug target discovery for neurodegenerative diseases in Drosophila.</a> Expert Opin Drug Discov. 8, 1503-1513 (2013).</li><li>Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+mutants+in+Drosophila:+a+means+to+identify+genes+and+mechanisms+involved+in+human+diseases.">Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases.</a> Invert Neurosci. 5, 97-109 (2005).</li><li>Kretzschmar, D., 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=Swiss+cheese+et+allii,+some+of+the+first+neurodegenerative+mutants+isolated+in+Drosophila.">Swiss cheese et allii, some of the first neurodegenerative mutants isolated in Drosophila.</a> J Neurogenet. 23, 34-41 (2009).</li><li>Li, J., Le, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neurodegenerative+diseases+in+Caenorhabditis+elegans.">Modeling neurodegenerative diseases in Caenorhabditis elegans.</a> Exp Neurol. 250, 94-103 (2013).</li><li>Prussing, K., Voigt, A., Schulz, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+organism+for+Alzheimer's+disease.">Drosophila melanogaster as a model organism for Alzheimer's disease.</a> Mol Neurodegener. 8, (2013).</li><li>Wentzell, J., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+and+tauopathy+studies+in+flies+and+worms.">Alzheimer's disease and tauopathy studies in flies and worms.</a> Neurobiol Dis. 40, 21-28 (2010).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J Vis Exp. , (2011).</li><li>Barone, M. C., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+neurodegenerative+phenotypes+in+Drosophila+dopaminergic+neurons+by+climbing+assays+and+whole+brain+immunostaining.">Assessing neurodegenerative phenotypes in Drosophila dopaminergic neurons by climbing assays and whole brain immunostaining.</a> J Vis Exp. , e50339 (2013).</li><li>Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+expression+of+Swiss-cheese+(SWS),+the+Drosophila+orthologue+of+Neuropathy+Target+Esterase,+is+required+for+neuronal+ensheathment+and+function.">Glial expression of Swiss-cheese (SWS), the Drosophila orthologue of Neuropathy Target Esterase, is required for neuronal ensheathment and function.</a> Dis Model Mech. , (2015).</li><li>Iijima, K., 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=Abeta42+mutants+with+different+aggregation+profiles+induce+distinct+pathologies+in+Drosophila.">Abeta42 mutants with different aggregation profiles induce distinct pathologies in Drosophila.</a> PLoS One. 3, e1703 (2008).</li><li>Krishnan, N., 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=Loss+of+circadian+clock+accelerates+aging+in+neurodegeneration-prone+mutants.">Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.</a> Neurobiol Dis. 45, 1129-1135 (2012).</li><li>Liu, H., 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=Automated+rapid+iterative+negative+geotaxis+assay+and+its+use+in+a+genetic+screen+for+modifiers+of+Abeta(42)-induced+locomotor+decline+in+Drosophila.">Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Abeta(42)-induced locomotor decline in Drosophila.</a> Neurosci Bull. 31, 541-549 (2015).</li><li>Papanikolopoulou, K., Skoulakis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+distinct+phosphorylations+differentiate+Tau-dependent+learning+deficits+and+premature+mortality+in+Drosophila.">Temporally distinct phosphorylations differentiate Tau-dependent learning deficits and premature mortality in Drosophila.</a> Hum Mol Genet. 24, 2065-2077 (2015).</li><li>Ping, Y., 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=Linking+abeta42-induced+hyperexcitability+to+neurodegeneration,+learning+and+motor+deficits,+and+a+shorter+lifespan+in+an+Alzheimer's+model.">Linking abeta42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer's model.</a> PLoS Genet. 11, e1005025 (2015).</li><li>Sujkowski, A., Rainier, S., Fink, J. K., Wessells, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Induction+of+Human+NTE+(PNPLA6)+Rescues+Neurodegeneration+and+Mobility+Defects+of+Drosophila+swiss+cheese+(sws)+Mutants.">Delayed Induction of Human NTE (PNPLA6) Rescues Neurodegeneration and Mobility Defects of Drosophila swiss cheese (sws) Mutants.</a> PLoS One. 10, e0145356 (2015).</li><li>Barone, M. C., Sykiotis, G. P., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+activation+of+Nrf2+signaling+is+sufficient+to+ameliorate+neurodegenerative+phenotypes+in+a+Drosophila+model+of+Parkinson's+disease.">Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease.</a> Dis Model Mech. 4, 701-707 (2011).</li><li>Coulom, H., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+rotenone+models+sporadic+Parkinson's+disease+in+Drosophila+melanogaster.">Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster.</a> J Neurosci. 24, 10993-10998 (2004).</li><li>Navarro, J. 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=Analysis+of+dopaminergic+neuronal+dysfunction+in+genetic+and+toxin-induced+models+of+Parkinson's+disease+in+Drosophila.">Analysis of dopaminergic neuronal dysfunction in genetic and toxin-induced models of Parkinson's disease in Drosophila.</a> J Neurochem. 131, 369-382 (2014).</li><li>Heisenberg, M., B&#246;hl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+anatomical+brain+mutants+of+Drosophila+by+histological+means.">Isolation of anatomical brain mutants of Drosophila by histological means.</a> Zeitschrift f&#252;r Naturforschung C. 34, 143 (1979).</li><li>Han, P. L., Meller, V., Davis, R. 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+Drosophila+brain+revisited+by+enhancer+detection.">The Drosophila brain revisited by enhancer detection.</a> J Neurobiol. 31, 88-102 (1996).</li><li>Lin, C. W., 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=Automated+in+situ+brain+imaging+for+mapping+the+Drosophila+connectome.">Automated in situ brain imaging for mapping the Drosophila connectome.</a> J Neurogenet. 29, 157-168 (2015).</li><li>Strausfeld, N. J., Sinakevitch, I., Vilinsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mushroom+bodies+of+Drosophila+melanogaster:+an+immunocytological+and+golgi+study+of+Kenyon+cell+organization+in+the+calyces+and+lobes.">The mushroom bodies of Drosophila melanogaster: an immunocytological and golgi study of Kenyon cell organization in the calyces and lobes.</a> Microsc Res Tech. 62, 151-169 (2003).</li><li>Bettencourt da Cruz, A., Wentzell, J., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swiss+Cheese,+a+protein+involved+in+progressive+neurodegeneration,+acts+as+a+noncanonical+regulatory+subunit+for+PKA-C3.">Swiss Cheese, a protein involved in progressive neurodegeneration, acts as a noncanonical regulatory subunit for PKA-C3.</a> J Neurosci. 28, 10885-10892 (2008).</li><li>Bolkan, B. J., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Tau+results+in+defects+in+photoreceptor+development+and+progressive+neuronal+degeneration+in+Drosophila.">Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila.</a> Dev Neurobiol. 74, 1210-1225 (2014).</li><li>Cook, M., Mani, P., Wentzell, J. S., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+RhoA+prenylation+in+the+loechrig+(loe)+mutant+leads+to+progressive+neurodegeneration.">Increased RhoA prenylation in the loechrig (loe) mutant leads to progressive neurodegeneration.</a> PLoS One. 7, e44440 (2012).</li><li>Wittmann, C. W., 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=Tauopathy+in+Drosophila:+neurodegeneration+without+neurofibrillary+tangles.">Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.</a> Science. 293, 711-714 (2001).</li><li>Rasmuson, B., Green, M. M., Ewertson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QUALITATIVE+AND+QUANTITATIVE+ANALYSES+OF+EYE+PIGMENTS+AND+PTERIDINES+IN+BACK-MUTATIONS+OF+THE+MUTANT+wa+IN+DROSOPHILA+MELANOGASTER.">QUALITATIVE AND QUANTITATIVE ANALYSES OF EYE PIGMENTS AND PTERIDINES IN BACK-MUTATIONS OF THE MUTANT wa IN DROSOPHILA MELANOGASTER.</a> Hereditas. 46, 635-650 (1960).</li><li>Bettencourt da Cruz, 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=Disruption+of+the+MAP1B-related+protein+FUTSCH+leads+to+changes+in+the+neuronal+cytoskeleton,+axonal+transport+defects,+and+progressive+neurodegeneration+in+Drosophila.">Disruption of the MAP1B-related protein FUTSCH leads to changes in the neuronal cytoskeleton, axonal transport defects, and progressive neurodegeneration in Drosophila.</a> Mol Biol Cell. 16, 2433-2442 (2005).</li><li>Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., Benzer, 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+swiss+cheese+mutant+causes+glial+hyperwrapping+and+brain+degeneration+in+Drosophila.">The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila.</a> J Neurosci. 17, 7425-7432 (1997).</li><li>Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+expression+of+Swiss+cheese+(SWS),+the+Drosophila+orthologue+of+neuropathy+target+esterase+(NTE),+is+required+for+neuronal+ensheathment+and+function.">Glial expression of Swiss cheese (SWS), the Drosophila orthologue of neuropathy target esterase (NTE), is required for neuronal ensheathment and function.</a> Dis Model Mech. 9, 283-294 (2016).</li><li>Davis, M. Y., 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=Glucocerebrosidase+Deficiency+in+Drosophila+Results+in+alpha-Synuclein-Independent+Protein+Aggregation+and+Neurodegeneration.">Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration.</a> PLoS Genet. 12, e1005944 (2016).</li><li>Dias-Santagata, D., Fulga, T. A., Duttaroy, A., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+mediates+tau-induced+neurodegeneration+in+Drosophila.">Oxidative stress mediates tau-induced neurodegeneration in Drosophila.</a> J Clin Invest. 117, 236-245 (2007).</li><li>Kretzschmar, D., 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=Glial+and+neuronal+expression+of+polyglutamine+proteins+induce+behavioral+changes+and+aggregate+formation+in+Drosophila.">Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.</a> Glia. 49, 59-72 (2005).</li><li>Li, Y., 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=A+Drosophila+model+for+TDP-43+proteinopathy.">A Drosophila model for TDP-43 proteinopathy.</a> Proc Natl Acad Sci U S A. 107, 3169-3174 (2010).</li></ol>

Les auteurs n&#39;ont rien à dévoiler.

Le procédé décrit fournit un moyen pour quantifier la neurodégénérescence dans le cerveau de la drosophile. Alors que d&#39;autres méthodes, telles que le comptage d&#39;un type de cellule spécifique, peuvent être utilisées pour identifier la neurodégénérescence, l&#39;avantage de cette méthode est qu&#39;elle peut être appliquée de façon plus générale. Le comptage des cellules exige que ces cellules puissent être identifiées de façon fiable en utilisant soit un anticorps spécifique ou l&...

Avec l&#39;augmentation de l&#39;espérance de vie, les maladies neurodégénératives comme la maladie d&#39;Alzheimer ou de Parkinson sont devenus une menace pour la santé de plus en plus pour la population générale. Selon les instituts nationaux de la santé, 115 millions de personnes dans le monde sont prévus d&#39;être affectées par la démence en 2050. Bien que des progrès significatifs ont été réalisés dans l&#39;identification des gènes et des facteurs de risque impliqués dans au moins certaines de ces maladies, pour beaucoup d&#39;entre eux, le sous-jacent mécanismes moléculaires sont encore inconnus ou mal compris. Organismes modèles invertébrés simples comme Caenorhabditis elegans et Drosophila melanogaster offrent une variété d&#39;avantages expérimentaux pour étudier les mécanismes des maladies neurodégénératives, y compris un cycle court de la vie, un grand nombre de descendants, et la disponibilité des méthodes génétiques et moléculaires bien établies et parfois uniques 1 -12. En outre, ces organismes se prêtent à impartialeécrans d&#39;interaction qui permettent d&#39;identifier les facteurs qui contribuent à ces maladies par leurs effets aggravants ou améliorer sur phénotypes neurodégénératives. L&#39;analyse de ces interactions génétiques et évaluer les effets du vieillissement nécessite des protocoles quantitatifs pour détecter la neurodégénérescence et de mesurer sa gravité. Cette évaluation peut se faire relativement facilement lorsque l&#39;on mesure les aspects comportementaux chez la drosophile, tels que l&#39; apprentissage olfactif, géotaxie négative, ou phototaxie rapide, qui fournissent une valeur de performance numérique 13-21. Il est également possible de déterminer les effets sur la survie neuronale en comptant les neurones. Cependant, ceci est seulement possible lorsqu&#39;on se concentre sur une population spécifique qui est clairement identifiable, comme les neurones dopaminergiques qui sont affectés dans la MP, et même alors, les résultats ont été controversés 22-24. Le protocole décrit ici utilise la méthode du collier pour effectuer des coupes en série de paraffine, un procédéqui a été à l&#39; origine développé par Heisenberg et Böhl, qui sert à isoler des mutants du cerveau anatomiques chez la drosophile 25. L&#39;utilisation de la méthode du collier a été ensuite adapté, y compris dans cryocoupes, vibratome sections, et les sections en plastique 26-28. Ici, cette méthode est utilisée pour obtenir des coupes en série de la tête de la mouche entière, qui peut ensuite être utilisé pour mesurer les vacuoles qui se développent dans les mouches avec des phénotypes neurodégénératives 16,21,29-32. Ces mesures peuvent être effectuées dans des zones spécifiques du cerveau ou peuvent couvrir l&#39;ensemble du cerveau; cette dernière approche permet d&#39;identifier même faibles phénotypes dégénératives, comme observé au cours du vieillissement. Enfin, lors de l&#39;utilisation des colliers, jusqu&#39;à 20 mouches peuvent être traités comme une préparation, ce qui est non seulement moins de temps, mais permet également à l&#39;analyse de contrôle et les mouches expérimentales dans la même préparation, en minimisant les artefacts dus à de légers changements dans la préparation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of the Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>Collar</td>
			<td>Genesee Scientific</td>
			<td>TS 48-100</td>
			<td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A discription to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&#160;</td>
		</tr>
		<tr>
			<td>Rubber ice cube tray for embedding</td>
			<td>Household store</td>
			<td></td>
			<td>The size can be made to fit by glueing in additional walls&#160;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Fisher Scientific company</td>
			<td>08-762-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Fisher Scientific Company</td>
			<td>E138-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories Inc.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Choloroform</td>
			<td>Fisher Scientific Company</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific Company</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylbenzoate</td>
			<td>Fisher Scientific Company</td>
			<td>M205-500</td>
			<td>Distinct Odor</td>
		</tr>
		<tr>
			<td>Use in fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Point Paraffin Wax</td>
			<td>Fisher Scientific Company</td>
			<td>T565</td>
			<td>Make sure to keep extra melted in a 65&#176;C waterbath</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>Reichert Jung 2040 Autocut</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Fisher Scientific Company</td>
			<td>12-550D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeClear</td>
			<td>Fisher Scientific Company</td>
			<td>314-629</td>
			<td>Three different containers for washes</td>
		</tr>
		<tr>
			<td>Vertical Staining Jar with Cover</td>
			<td>Ted Pella Inc.&#160;</td>
			<td>432-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific Company</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution</td>
			<td>Sigma Life Science</td>
			<td>P8290-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass histology to quantify neurodegeneration in drosophila

maladies progressives neurodégénératives comme la maladie d&#39;Alzheimer (AD) ou la maladie de Parkinson (PD) sont une menace croissante pour la santé humaine dans le monde entier. Bien que les modèles de mammifères ont fourni des informations importantes sur les mécanismes sous-jacents de la pathogénicité, la complexité des systèmes de mammifères ainsi que leurs coûts élevés limitent leur utilisation. Par conséquent, le modèle système Drosophila simple mais bien établie offre une alternative pour étudier les voies moléculaires qui sont affectés dans ces maladies. Outre les déficits comportementaux, les maladies neurodégénératives sont caractérisées par des phénotypes histologiques tels que la mort neuronale et axonopathy. Pour quantifier la dégénérescence neuronale et de déterminer comment elle est affectée par des facteurs génétiques et environnementaux, nous utilisons une approche histologique qui est basé sur la mesure des vacuoles dans le cerveau des mouches adultes. Pour minimiser les effets de l&#39;erreur systématique et de comparer directement les sections de contrôle et expmouches erimental dans une préparation, nous utilisons la méthode du «col» pour des coupes en paraffine. La neurodégénérescence est ensuite évaluée par la mesure de la taille et / ou le nombre de vacuoles qui se sont développées dans le cerveau de mouche. Cela peut être fait en se concentrant sur une région d&#39;intérêt spécifique ou en analysant l&#39;ensemble du cerveau en obtenant des coupes en série qui couvrent la tête complète. Par conséquent, cette méthode permet de mesurer non seulement la dégénérescence sévère, mais aussi des phénotypes relativement douces qui ne sont détectables que dans quelques sections, comme cela se produit au cours du vieillissement normal.

En utilisant les résultats de la méthode décrite dans les sections en série colorées par le pigment de l&#39; œil 33 qui englobent la tête de la mouche entière. Une partie de ceci est montré à la figure 1B, où les sections à partir d&#39; une tête individuelle sont présentés de haut en bas. Les sections de différentes mouches sont vu de gauche à droite dans cet exemple. Pour faciliter l&#39; orientation et l&#39; identification des mouches, un...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophile est largement utilisé en tant que système modèle pour étudier la neurodégénérescence. Ce protocole décrit un procédé par lequel une dégénérescence, comme déterminé par la formation de vacuoles dans le cerveau, peuvent être quantifiés. Il minimise également les effets dus à la procédure expérimentale par le traitement et le contrôle sectionner et les mouches expérimentales comme un échantillon.

Mass histologie à Quantifier neurodégénérescence<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

mass-histology-to-quantify-neurodegeneration-in-drosophila

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

10.1K vues.  Oregon Health & Sciences University.  Drosophile est largement utilisé en tant que système modèle pour étudier la neurodégénérescence. Ce protocole décrit un procédé par lequel une dégénérescence, comme déterminé par la formation de vacuoles dans le cerveau, peuvent être quantifiés. Il minimise également les effets dus à la procédure expérimentale par le traitement et le contrôle sectionner et les mouches expérimentales comme un échantillon. 

Vidéo: Mass Histology to Quantify Neurodegeneration in Drosophila

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Synapses Quantifier: un dosage à base d&#39;immunocytochimie Quantifier Nombre Synapse

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Recherche

Neurosciences

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Combinant colorant lipophile, In situ</emHybridation>, immunohistochimie et l&#39;histologie

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Déficits locomoteur Dosage, apprentissage et mémoire dans Drosophile Modèles de la neurodégénérescence

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo laser axotomie en C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Méthodes de dosage à Drosophila Comportement

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

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 ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Utilisant la technologie 3D Impression de fusion IRM avec histologie: Un protocole pour Brain Sectionnement

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Une nouvelle méthode pour modéliser l&#39;encéphalopathie traumatique chronique dans<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Modélisation de toxicité amyloïde-β42 et neurodégénérescence dans le cerveau de poisson-zèbre adulte

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Analyse quantitative de la complexité d’arborisation dendritique neuronale chez la drosophile

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...