Die Autoren danken RIKEN für die Finanzierung. Wir danken auch Cagri Yalgin, Caroline Delandre und Jay Parrish für Diskussionen über genetische und Immunhistochemie-Protokollen.

1.Vorbereitung von Reagenzien <ol><li> Bereiten Ca + +-freien HL3.1 Kochsalzlösung 28. <ol><li> In mM: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 Saccharose und 5 Trehalose; pH 7,2. Filter sterilisieren und lagern bei 4 ° C. Hinweis: Ca + +-freier Lösung verhindert Muskelkontraktion beim Präparieren. </li></ol></li><li> Machen Poly-L-Lysin (PLL) Deckgläser. <ol><li> Lösen 100mg PLL in 4.2ml Wasser und machen 300μl Aliquots in Eppendorf-Röhrchen und bei -20 ° C. </li><li> Vor der Beschichtung Deckgläser zunächst Tauwetter ein Aliquot, bringen Sie es bis zu 10ml in ddH 2 O, und fügen Sie 20 &amp;mgr; l von Kodak Photo-Flo; die endgültige PLL-Konzentration liegt bei 0,7 mg / ml. </li><li> Tauchen Deckgläser für 30 Minuten in PLL-Lösung, zu entfernen und trocken, dann kurz mit Wasser abspülen und trocknen. Zweimal wiederholen. Behandelte Deckgläser dauern ca. 1 Monat. </li></ol></li></ol> 2. Genetische Kreuzungen <ol><li> To generieren MARCM Klone. Hier ist ein Beispiel Kreuz mit einem pan-DA Fahrer 11,27: FRT2A x hsFLP; Gal4 109 (2) 80, UAS-mCD8:: GFP; Wanne-GAL80 FRT2A/SM5-TM6B </li><li> Zur Erzeugung von Flp-out-Klone. Hier ist ein Beispiel Kreuz mit einem Klasse IV-spezifische Treiber 8: ppk-Gal4 x yw, hsFLP; UAS-FRT-CD2, y +-stop-FRT-mCD8:: GFP </li></ol> 3. Sammlung von Embryonen <ol><li> Keep Drosophila Kreuze in einer Sammlung Flasche bei 25 ° C und sammeln Embryonen auf einem Apfelsaft-Agar Platte 29 verteilt mit einer dünnen Schicht von Hefepaste 30,31. </li></ol> 4. Hitze-Schock-Behandlung von Embryonen <ol><li> Stellen Sie eine leere Platte im Gegensatz zum Apfelsaft-Agar-Platte, auf der die Embryonen gelegt worden. Seal um diese beiden Platten mit Parafilm (Abb. 2). </li><li> Während Hitzeschockprotein, tauchen the Platte in das Wasserbad und halten Sie ihn mit dem Metall Gewicht. </li><li> Für MARCM Embryonen <ol><li> Sammeln Embryonen für 2h und inkubieren in einer 10cm Petrischale umgeben von feuchten Gewebe bei 25 ° C für eine zusätzliche 2h. Hinweis: Einstellung des Hitzeschock-Protokoll verändert die Frequenz, bei der Klone generiert werden. </li><li> Für eine kleinere Anzahl von Klonen, mit dem Ziel für einzelne isolierte Nervenzellen, tauchen und Hitzeschock im Wasserbad für 1 Stunde bei 38 ° C. </li><li> Für eine größere Anzahl von Klonen Hitzeschock für 45 Minuten bei 38 ° C, bei RT 30 Minuten, dann Hitzeschock wieder erholen für einen zusätzlichen 30 Minuten. </li></ol></li><li> Sammeln Flp-out Embryonen für 24 Stunden und Hitzeschock für 1h bei 38 ° C. </li></ol> 5. Screening nach Klonen <ol><li> Nach Hitzeschock, entfernen Sie den Deckel des Wasser-dichte Anordnung und Ort der Apfelsaft-Agar-Platte in eine 10 cm Petrischale mit angefeuchteten Gewebe umgeben. Kultur der Embryonen und Larven anschließende bei 25 ° C bis zu wandernIng. 3 rd instar. Hinweis: Kulturbedingungen, insbesondere Ernährung, haben gezeigt, dass DA dendritischen Dorn Morphologie 32,33 zu ändern. Stellen Sie sicher, dass wachsenden Larven Zugang zu Hefepaste zu allen Zeiten; überwachen und füllt die Hefepaste wie erforderlich. </li><li> Von diesem Zeitpunkt an das Protokoll ab, manipulieren Larven vorsichtig mit einer Pinzette Insekten. </li><li> Kurz und sanft spülen die Larven in Leitungswasser, und platzieren Sie dann auf eine Agarplatte. Hinweis: Dieser Schritt reduziert Hintergrundgeräusche Autofluoreszenz aus der Nahrung oder Schmutz auf der Oberfläche Larven. </li><li> Untersuchen Sie die Larven unter einem starken Fluoreszenz Präpariermikroskop. Identifizieren Larven mit GFP-positive Neurone / Zellen im Körper Wand. </li></ol> 6. In-vivo-Bildgebung von Dendriten <ol><li> Legen Sie die Larve in eine Depression Objektträger mit einem kleinen Tropfen von 80% Glycerin. Legen Sie ein Deckglas auf dem Objektträger um die Larve zu immobilisieren. Stellen Sie sicher, dass keine Luft zwischen der Larve und die coversli bleibtSeite </li><li> Schieben Sie das Deckglas auf die Larve Rolle der Visualisierung des Neurons von Interesse erlauben. Bild der mCD8:: GFP-markierten dendritischen Dorn über die konfokale Mikroskopie. </li></ol> 7. Larven Dissektion Beachten Sie vor Beginn: DA Neuron Dendriten verschlechtern sich rasch nach Beginn der Dissektion. Dissect jede einzelne Larve in weniger als 5min zum guten Dendriten Morphologie gewährleisten. <ol><li> Dissect wandernde 3 rd Larvenstadium auf einem Sylgard Platte wie zuvor 34 mit den folgenden Modifikationen: <ol><li> Für die Darstellung von DA Neuron Axon-Termini, sicherzustellen, dass das ZNS intakt ist und der segmentalen Nerven sind nicht kaputt. Nach dem Öffnen der Larven, zunächst vorsichtig schneiden die Tracheal-Verbindungen, um den Körper Wand und dann vorsichtig die gesamte Darm. </li><li> Wenn die Abbildung der Dendriten allein, entfernen Sie das ZNS zu erlauben eine flachere Montage. </li></ol></li></ol> 8. Fixation und Sperrung von Larven Filets <ol><li> Mit der Larve noch auf die Platte Sylgard merken, fix it in 4% PFA in PBS für 20 Minuten bei RT auf einem sanft rotierenden Schüttler. </li><li> Entfernen Spuren von Larven Gewebe (fat Körper, Luftröhre, etc.) nach der Fixierung. </li><li> Wash in PBST (PBS mit 0,1% Triton X-100) 3x 10 Minuten auf dem Sylgard Platte und Shaker. Wenn die Prüfung Axon-Termini, halten Sie das Filet auf dem Teller Sylgard. (Wenn Färbung Dendriten ist es möglich, die Larven Filets auf einem 0,5 ml Röhrchen füllen bei diesem Schritt 34) </li><li> Blockieren Sie die Larven für 20 Minuten bei RT in 5% normalem Esel-Serum (NDS) in PBST auf einem Schüttler. </li></ol> 9. Die Färbung der Larven Filets <ol><li> Blocking-Lösung und Inkubation in Primärantikörper (in 5% NDS / PBST) über Nacht bei 4 ° C in einer kleinen Tupperdose, umgeben von feuchten Gewebe. </li><li> Entfernen Sie die Rohre von 4 ° C inkubieren eine weitere Stunde bei RT. </li><li> Wash 6x 10 Minuten in PBST. Fügen Sie den sekundären antibOdy (in 5% NDS / PBST) und Abdeckung, um zu verhindern Fluorophor Photobleaching 34. </li><li> Inkubieren entweder bei RT für 2 Stunden oder über Nacht bei 4 ° C eine Stunde bei RT folgten. </li><li> Waschen Sie die Larven 6x 10 Minuten in PBST und fahren Sie mit Montage. </li></ol> 10. Montage der Larven Filets für die Prüfung der Dendriten Laube <ol><li> Montieren Sie je Larven Filet so flach wie möglich mit Sezieren einer Schere oder einem Skalpell den Kopf abzuschlagen (einschließlich der Mund Haken) und der hintere (einschließlich der Luftlöcher) 34. </li><li> Setzen Sie den Larven Filet auf der Folie Nagelhaut-Seite nach unten, in 80% Glycerin montieren und Dichtung den Seiten des Deckglases mit Nagellack für einen &quot;schnellen&quot; Halterung 34. </li><li> Für eine dauerhafte Befestigung und ein klareres Bild <ol><li> Setzen Sie den sezierten Larven Filet Muskel-Seite nach unten auf einen Tropfen PBS auf eine PLL Deckglas, wird es schnell zu dem Deckglas haften. </li><li> Entfernen Sie so viel Flüssigkeit wie möglich nach der Montage, however nicht lassen Sie es vollständig trocknen. </li><li> Nehmen Sie das Deckglas (mit angeschlossenem Larven Filet) durch eine Ethanol-Serie: 35% Ethanol, 50%, 70%, 95%, und schließlich 2x 100% Ethanol für jeweils 10 Minuten (2. 100% EtOH Lösung häufig geändert werden soll, gefolgt ), und schließlich, waschen 2-3x 10 Minuten in Xylol. </li><li> Gib einen Tropfen des DPX Deckfluessigkeit (Distyrene Weichmacher Xylol) auf einen sauberen Objektträger und legen Sie es vorsichtig Deckglas Filet-side-down an der Spitze. Halten Sie in der Dunkelheit, und warten Sie einen Tag für die DPX vor Bildgebung gesetzt. </li></ol></li></ol> 11. Montage der Larven Filets für die Prüfung der Axon-Termini <ol><li> Halten Sie die segmentalen Nerven, die zwischen den PNS und ZNS intakt während der Präparation, Fixierung und Färbung, damit die Verfolgung der Axone von peripheren sensorischen Neuronen Zellkörper zu den VNC. </li><li> Montieren Sie je Larven Filet Nagelhaut-Seite nach unten in 80% Glycerin in eine Depression gleiten. Verfolgen Sie die Axone aus der DA Zellkörper zu denVNC unter dem konfokalen Mikroskop. Hinweis: PNS Neuronen in jeder Körperzelle Wandsegment Projekt auf die verwandten Segment des VNC. </li></ol> 12. Repräsentative Ergebnisse: Repräsentative Ergebnisse sind in den Abbildungen 3-5 dargestellt. Abb.. 3 zeigt die ganze Laube einer Klasse IV da Neuron, in vivo unter dem konfokalen Mikroskop aufgenommen. Abb.. 4 zeigt eine Nahaufnahme eines Teils des Dendriten Laube eines immunhistochemisch markierten Klasse III Neuron, das richtig ist fest mit der Morphologie erhalten. Die zugehörigen Einschub zeigt die Erniedrigung, die nach einem erfolglosen Dissektion und Fixierung (beide gefärbt mit anti-GFP-Antikörper) auftreten können. Abb.. 5 zeigt eine einzelne Klasse IV Axon-Terminus in das ZNS (anti-GFP, grün), alle Klasse IV Termini sind mit anti-CD2 (magenta) co-gefärbt. <img alt="Abbildung 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> Abbildung 1 Protocol Überblick. a) Sammeln und dann Hitzeschock-Embryonen. b)<html> Wählen Sie eine Larve mit einem GFP-positive DA Neuron zu klonen, und dann Bild der GFP-markierten Dendriten Laube in der Live-Larve. c) Präparieren Sie die Larve, und dann immunhistochemisch färben das Filet. d) Montieren Sie die Kirchenfenster Filet, und dann Bild der dendritischen Dorn und axonalen Projektionen der DA Neuron Klone. <img alt="Abbildung 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> Abbildung 2: Herstellung von Apfelsaft-Agar-Platte für Hitzeschock. Nehmen Sie die Platte (weißer Pfeil, ab), fügen Sie eine zweite Petrischale auf (roter Pfeil b) und Dichtung um mit Parafilm (blauer Pfeil, b). <img alt="Abbildung 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> Abbildung 3 in vivo Bild des Dendriten Laube eines mCD8:: GFP-markierten MARCM clone (Genotyp in 2,1), die eine Klasse IV (v&#39;ada) Neuron einer 3 rd instar Larve. Scale-Bar ist 50 um. d/3111/3111fig4.jpg &quot;/&gt; Abbildung 4 mCD8:: GFP-markierten MARCM Klon eines Klasse-III-Neuron (ddaa) mit anti-GFP Antikörper gefärbt und zeigt gute Morphologie nach einer erfolgreichen Präparation und Befestigung (grün). Der Einschub zeigt Dendriten Abbau (weiße Pfeile), die nach erfolglosen Dissektion und Fixierung (magenta). Scale-Bar ist 25 um. <img alt="Abbildung 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> Abbildung 5 Die 3. Larvenstadium Larven VNC. Eine Flp-out-Klon aus einer einzigen Klasse VI (VDAB) Axon-Terminus (Genotyp in 2,2) wird unter Verwendung anti-GFP-Antikörper (grün). Anti-CD2 Etiketten aller Klasse IV Termini (magenta). Scale-Bar ist 25 um.

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

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Die Drosophila Larve DA Neuronenmodell liefert eine hervorragende genetische System von Mechanismen zu untersuchen, dass die Kontrolle Neuron Morphologie und Schaltung Bildung. MARCM ist in der Regel für die Kennzeichnung und zur Erzeugung von Mutanten DA Neuron Klone verwendet. Für MARCM wir verwenden entweder eine pan-neuronalen (zB Gal4 C155) oder DA Neuronen-spezifische Treiber. Mit einem pan-neuronaler Fahrer ist es möglich, direkt zu verwenden mehrere Bestände weit aus öff...

<table border="1"><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> SZX16 Fluoreszenz Dissektionsmikroskop (mit GFPHQ Filter) </td><td> Olymp </td><td> SZX16 </td><td></td></tr><tr><td> Live-Insect Zange </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26mm x 76mm Depression Objektträger </td><td> Toshinriko Co. </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (oder Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poly-L-Lysin </td><td> Sigma </td><td> P-1524 </td><td> Dieses Produkt hat sich als am effektivsten </td></tr><tr><td> DPX Eindeckmedium </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> Rabbit anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Verdünnung 1:500 </td></tr><tr><td> Rat anti-CD8 </td><td> Caltag </td><td> 5h10 </td><td> Verdünnung 1:200 </td></tr><tr><td> Maus anti-CD2 </td><td> AbD serotec </td><td> MCA443R </td><td> Verdünnung 1:700 </td></tr><tr><td> Maus anti-Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td> Verdünnung 1:10 </td></tr></table>

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

Entwicklung des Nervensystems erfordert die korrekte Spezifikation der Neuron Position und Identität, durch genaue Neuron-Klasse-spezifische dendritische Entwicklung und axonalen Verdrahtung gefolgt. Vor kurzem hat die dendritische Verzweigung (DA) sensorischen Neuronen der Drosophila Larve peripheren Nervensystem (PNS) haben sich leistungsfähige genetische Modelle, in denen sowohl allgemeine und Klassen-spezifische Mechanismen der neuronalen Differenzierung aufzuklären. Es gibt vier Haupt-DA Neuron Klassen (I-IV) 1. Sie sind in der Reihenfolge zunehmender Dendriten Laube Komplexität, benannt und haben klassenspezifische Unterschiede in der genetischen Kontrolle ihrer Differenzierung 2-10. Die DA sensorischen Systems ist ein praktisches Modell, um die molekularen Mechanismen hinter der Kontrolle von dendritischen Morphologie 11-13 zu untersuchen, weil: 1) es Vorteile der leistungsstarken genetischen Werkzeuge in der Fruchtfliege, 2) nehmen die DA Neuron Dendriten Laube breitet sich aus in nur 2 Dimensionen unter einem optisch clear Larvencuticula macht es einfach, mit hoher Auflösung in vivo, 3 Visualisierung) der Klasse-spezifische Vielfalt in dendritische Morphologie erleichtert eine vergleichende Analyse zu den wichtigsten Elementen der Kontrolle der Bildung von einfachen vs stark verzweigte dendritische Bäume zu finden, und 4) dendritischen Dorn stereotypen Formen der verschiedenen DA Neuronen zu erleichtern morphometrische statistischen Analysen. DA Neuronen-Aktivität modifiziert die Ausgabe eines larvalen Fortbewegung central pattern generator 14-16. Die verschiedenen DA Neuron Klassen haben verschiedene sensorische Modalitäten und deren Aktivierung auslöst verschiedenen Verhaltensreaktionen 14,16-20. Außerdem verschiedene Klassen schicken axonalen Projektionen stereotyp in die Drosophila Larve des zentralen Nervensystems in der Bauchmark (VNC) 21. Diese Projektionen enden mit topographischen Darstellungen beider DA Neuron Sinnesmodalität und die Position in der Körperwand der dendritischen Bereich 7,22, 23. Daher Untersuchung von DA axonalen Projektionen können verwendet werden, um Mechanismen, die topographische Kartierung 7,22,23, sowie die Verkabelung einer einfachen Schaltung modulierenden Larven Fortbewegung 14-17 aufzuklären. Wir stellen Ihnen hier eine praktische Anleitung zur Generierung und Analyse genetischer Mosaike 24 Kennzeichnung DA Neuronen über MARCM (Mosaic Analyse mit einem reprimierbaren Zell Marker) 1,10,25 und Flp-out 22,26,27 Techniken (zusammengefasst in Abb. 1)..

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

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

Die dendritischen Verzweigung sensorischen Neuronen des<em&gt; Drosophila</em&gt; Larven des peripheren Nervensystems sind nützliche Modelle, um sowohl allgemeine als auch Neuron-Klasse-spezifische Mechanismen der neuronalen Differenzierung aufzuklären. Wir präsentieren eine praktische Anleitung zur Generierung und Analyse dendritischer Verzweigung Neuron genetischen Mosaiken.

Morphologische Analyse Drosophila Larven peripheren sensorischen Neuron Dendriten und Axone mit Genetic Mosaike

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

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

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

15.2K Aufrufe.  RIKEN Brain Science Institute. Die dendritischen Verzweigung sensorischen Neuronen des Drosophila Larven des peripheren Nervensystems sind nützliche Modelle, um sowohl allgemeine als auch Neuron-Klasse-spezifische Mechanismen der neuronalen Differenzierung aufzuklären. Wir präsentieren eine praktische Anleitung zur Generierung und Analyse dendritischer Verzweigung Neuron genetischen Mosaiken.

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

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

In-vivo-Visualisierung von synaptischen Vesikeln Innerhalb Drosophila Larven Segmentale Axone

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

Forschung

Neurowissenschaften

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

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

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

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

Immunhistologische Markierung von Mikrotubuli in Sensory Neuron Dendriten, Tracheen und Muskeln in der Drosophila Larva Körperwand

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

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

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

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

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

Visualisierung und genetische Manipulation von Dendriten und Dornen in der Maus Hirnrinde und im Hippocampus mit<em&gt; In utero</em&gt; Die Elektroporation

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

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

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

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

Voltage-sensitive Dye Aufnahme von Axonen, Dendriten und dendritischen Spines einzelner Neuronen in Hirnschnitten

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

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

Funktionelle Analyse der Larvenfütterung Circuit in Drosophila</i

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Ganze Berge Immunmarkierung der olfaktorischen Rezeptorneuronen in der<em&gt; Drosophila</em&gt; Antennen

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

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

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

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

Produktion und Isolierung der Axone von Sinneszellen für Biochemische Analyse mit Porous Filter

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

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

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

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

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

Entfernung von<em&gt; Drosophila</em&gt; Muskelgewebe aus Larval Filets für Immunfluoreszenzanalyse von sensorischen Neuronen und epidermalen Zellen

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

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Linear Agarose Kanäle zu studieren<em&gt; Drosophila</em&gt; Larval Krabbeln Verhalten

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

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

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

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

Stoffwechselanalyse von Drosophila Melanogaster Schildzecken und erwachsenen Gehirn

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