Wir bedanken uns bei RIKEN für die Finanzierung. P10-Gal4 war eine Art Geschenk Alain Vincent (Université Paul Sabatier, Toulouse, Frankreich).

1. Vorbereitung der Reagenzien Hinweise vor Beginn: Dissection und immunhistochemische Färbung sind in einem magnetischen Kammer durchgeführt und die Larve sich mit speziell geformten Insektennadeln merken. Detaillierte Anweisungen für den Bau eines magnetischen Kammer und Vorbereitung dieser Stifte können in Zusammenhang mit Verweisen 14,15 gefunden werden. Kurz gesagt, ist ein 1x1cm quadratisches Loch in ein magnetisches Blatt und einem Deckglas angebracht auf der Rückseite des Blattes in eine kleine Kammer zu schneiden. Die Seiten der Kammer sind mit Epoxy-Kleber versiegelt; nach dieser Leim abgebunden hat die Kammer mehrmals mit 70% Ethanol vor Gebrauch gewaschen werden. Dissection Insekten Pins sind durch Biegen in die gewünschte Form hergestellt und anschließend verklebt auf eine Metall-Reiter 14,15. Alternativ zu einem Metall-Reiter haben wir ein umgekehrtes Flat-Head Stahl Reißzwecke mit einem Handgriff von einem cut-off gelben Spitze gemacht werden. Die Nutzung dieser magnetische Kammer Anordnung ermöglicht genaue Kontrolle over pin Positionierung und Gewebe Stretching beim Präparieren. Zum Antrieb der Reportergen-Expression in den verschiedenen Untergruppen der DA Neuronen Ermittler können mit mehreren verschiedenen Gal4-Linien (zusammengefasst von Shimono und Kollegen 16). Viele dieser Linien sind aus dem öffentlichen Lager-Zentren zur Verfügung. In dieser repräsentativen Protokoll führen wir Immunfärbung einer Zeile, in der zwei gegensätzliche Klassen von DA Neuronen sind Co-markierten: die einfachste Klasse I und komplexeste Klasse IV (P10-Gal4 17,18, UAS-mCD8: Kusabira-Orange (KO)). <ol><li> Bereiten Ca + +-freien HL3.1 Kochsalzlösung <ol><li> In mM: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 Saccharose und 5 Trehalose; pH 7.2 19. Filter-sterilisiert und bei 4 ° C. Hinweis: Ca + +-freier Lösung verhindert Muskelkontraktion beim Präparieren. </li></ol></li><li> Bereiten Sie 2x PHEM Puffer <ol><li> In mM: 130 PIPES, 60 HEPES, 20 EGTA, 4 MgSO4; pH 7,0. Filter-sterilisiert und bei 4 ° C. Hinweis: Diese Materialien werden nicht auflösen, bis der pH-Wert nähert 7,0. </li></ol></li><li> Bereiten Sie das Fixativ frisch unmittelbar vor der Fixierung. <ol><li> Um eine 25ml Lösung, erste Mix 2g Paraformaldehyd, 100 &amp;mgr; l 1M NaOH und 10 ml Wasser in einem 50 ml-Falcon-Röhrchen vorbereiten. </li><li> Schütteln Sie in einem 55 ° C Wasserbad mit Schüttler Fixativ, bis die Lösung klar ist. (Gesamtvolumen wird bei etwa 11,5 ml sein.) </li><li> Coole Fixativ auf Eis. </li><li> Add 12.5ml 2x PHEM Puffer. </li><li> Der pH-Wert auf 7,0 mit 1 M HCl. </li><li> Füllen Sie die Lösung mit Wasser, bis das Volumen ist 25ml. </li><li> Filtern Sie die Lösung mit Whatman-Papier. </li></ol></li></ol> 2. Larven Dissektion Beachten Sie vor Beginn: Mikrotubuli-Netzwerke und insbesondere in sensorischen Dendriten, werden Zusammenbruch rasch nach Beginn der Dissektion. Achieving schnelle Zerlegung in weniger als fünf Minuten durch eine sofortige Fixierung gefolgt sind Schlüsselfaktoren für den Erfolg dieses Protokolls. <ol><li> Waschen Sie die Larve im Wasser schnell und verschieben Sie sie in dieser Rückgang mit einer Schleife des Haares. </li><li> Orient die Larve dorsal und ventral auf dem Glas. Hinweis: Diese Orientierung ist die ventrale Cluster Neuronen zu untersuchen. Um zu untersuchen, dorsalen Cluster Neuronen, invertieren die Orientierung. </li><li> Verwenden Sie das Zentrum Insekten-polig auf dem vorderen Ende in der Nähe der Mündung Haken pin. Legen Sie den Stift kurz vor dem Ende für die besten Ergebnisse. </li><li> Geben Sie einen Tropfen Kochsalzlösung HL3.1 in der Sektion Kammer. </li><li> Schneiden Sie das sehr hinteren Spitze der Larve off mit Mikroschere. Hinweis: Dieser Schritt eröffnet eine Öffnung am hinteren Ende der Larve, die den Zugang für die Mikroschere (Schritt 2,7) ermöglichen wird. </li><li> Schnappen Sie mit der Zange der Region gut, dass jetzt Stossen ist aus der Öffnung am hinteren Ende der Larve. Ziehen Sie die gesamte Darm. </li><li> Legen Sie dieSpitze von einem Blatt der Mikroschere durch die Öffnung und entlang der ventralen Mittellinie in Richtung der vorderen. </li><li> Mit der Ecke Stifte, Bolzen der nun freien Ecken, posterior, dann anterior. Gleichzeitig sanft dehnen öffnen Sie die Larven Filet. </li></ol> 3. Fixation, Sperrung, Färbung und Montage von Larven Filets Hinweise vor Beginn: Alle Fixierung und Färbung Schritte werden in der Sektion Kammer durchgeführt. Während dieses Prozesses darauf achten, nicht das Insekt Stifte halten die Larve, da dies zu Gewebeschäden führen kann klopfen. Um zu verhindern, das Experiment vor dem Austrocknen, das tut alles Färbeschritte in eine kleine Tupperware Behälter befeuchtet Gewebe umgeben. <ol><li> Saugen Sie das Ca + +-freien HL3.1 Puffer unter Verwendung eines gelben Spitze. Unmittelbar fügen Sie die Fixiermittel mit einem anderen pipetteman. </li><li> Gently Pipette auf und ab, um das Fixiermittel mit den restlichen Spuren von Ca + +-freien HL3.1 Puffer-Mixin der Kammer. Unmittelbar absaugen und dann fügen Sie frisches Fixativ in die Kammer. </li><li> Inkubieren bei Raumtemperatur für 20 Minuten. </li><li> Wash 6x 10 Minuten in PBST (0,1% Triton X-100 in PBS). </li><li> Block mit 5% Ziegenserum in PBST für 20 min bei RT. </li><li> Ersetzen Sie die Blocking-Lösung mit primärem Antikörper in 5% Ziegenserum in PBST verdünnt. Die primären Antikörper verwendet werden Maus-anti-α-Tubulin (DM1A) und Ratte anti-CD8 (5h10), beide verdünnt 1 / 1000. Hinweis: Der Prüfer kann wollen Maus-anti-α-Tubulin (DM1A) mit Maus-Anti-futsch (22C10) 20,21 verdünnt 1 / 1000 unter bestimmten Umständen ersetzt (siehe Diskussion). </li><li> Inkubieren über Nacht (mindestens 16h) bei 4 ° C. </li><li> Wash 6x 10 Minuten in PBST. </li><li> Add sekundären Antikörper-Lösung in 5% Ziegenserum in PBST verdünnt. Die sekundären Antikörper verwendet werden, Alexa Fluor 488 Ziege anti-Maus IgG und Cy3 donkey anti-Ratte-IgG. Halten Sie die Probe abgedeckt, um zu verhindern FluorophorFoto-Bleiche 22. </li><li> Inkubieren entweder bei RT für 2 Stunden oder über Nacht bei 4 ° C eine Stunde bei Raumtemperatur. </li><li> Wash 6x 10 Minuten in PBST. </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; zu montieren. Hinweis: Für eine verbesserte Gewebe Lichtung, und eine dauerhafte stabile Probe, mount in DPX wie bisher 23 beschrieben. </li></ol> 4. Repräsentative Ergebnisse: Fluoreszenzfärbung wurde unter einem konfokalen Mikroskop untersucht. In den Abbildungen 1-2, verschiedene Zweige innerhalb eines Dendriten Laube haben verschiedene Zytoskelett-Organisation. Abbildung 1 zeigt einen Bereich der Laube einer Klasse IV DA Neuron bei der 1. Larvenstadium Larvenstadium. Die ganze Laube ist mit mCD8 markiert:: KO erkannt und mit einem Anti-CD8-Antikörper und fluoreszierende sekundäre (Cy3). Tubulin wird unter Verwendung anti α-Tubulin-Antikörper und Fluorescent sekundären (Alexa Fluor 488). Die wichtigsten Branchen sind für Tubulin positiv, einige dünne Seitenäste Tubulin-negativ. Movie 1 ist eine Reihe von seriellen Schnitten durch eine ähnliche Färbung einer Klasse-I-DA Neurons. Abbildung 2 zeigt einen Bereich der Laube einer Klasse IV DA Neuronen mit Antikörpern gegen futsch und CD8 an der 1. Larvenstadium Larvenstadium gefärbt. Die wichtigsten Branchen sind futsch-positiv, einige dünne Seitenäste futsch-negativ. Abbildung 3. Tracheen in der larvalen Körper Wand zeigen eine komplexe Mikrotubuli Organisation. Filme 2 und 3 zeigen Schnittserien der Färbung durch Körperwand Muskeln und Luftröhre. <img alt="Abbildung 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> Abbildung 1. Abb.. 1 zeigt eine Region von der Laube einer Klasse IV DA Neuron bei der 1. Larvenstadium Larvenstadium. : Die ganze Laube ist mit mCD8 gekennzeichnet: KO erkannt und mit anti-CD8-Antikörper und fluoreszierende sekundäre (Cy3). Tubulin wird unter Verwendung anti-&amp;-Tubulin Antikörper und fluoreszierende sekundäre (Alexa 488). Panels AC sequentielle konfokalen z-Abschnitte (0,5 um), C&#39;-C &quot;, single Antikörper-Färbung von Feld C Beispiel von Ästen mit einem (gelbe Pfeilspitze) oder ohne (lila Pfeil) Mikrotubuli sind hervorgehoben. Roten Pfeile markieren Mikrotubuli in der zugrunde liegenden Epithelzellen. <img alt="Abbildung 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> Abbildung 2. Abb.. 2 zeigt eine ähnliche Region von der Laube einer Klasse IV da Neuron mit Antikörpern gegen futsch und CD8 an der 1. Larvenstadium Larvenstadium gefärbt. : Die ganze Laube ist mit mCD8 gekennzeichnet: KO erkannt und mit anti-CD8-Antikörper und fluoreszierende sekundäre (Cy3). Futsch erkannt wird mit anti-futsch-Antikörper und fluoreszierende sekundäre (Alexa 488). Die wichtigsten Branchen sind futsch-positiv, einige dünne Seitenäste futsch-negativ. Beispiel für Branchen mit (gelbe Pfeilspitze) oder ohne (lila Pfeil) futsch sind hervorgehoben. Abbildung 3. Trachea befleckt mit dem anti-Tubulin-Protokoll beschrieben. Diese Larve ist der dritte Larvenstadium und seziert wie zuvor 23,24 beschrieben. Movie 3 zeigt vergrößert Schnittserien aus dem Bereich von einem Quadrat markiert. Movie 1. Serienschnitte Tracing Tubulin-Färbung in der dendritischen Dorn einer Klasse I Neuron. Die ganze Laube ist mit mCD8 markiert:: KO (Magenta) erkannt und mit anti-CD8-Antikörper und fluoreszierende sekundäre (Cy3). Tubulin (grün) wird unter Verwendung eines Anti-α-Tubulin-Antikörper und fluoreszierende sekundäre (Alexa Fluor 488). Maßstab: die eine Seite des Videobildes entspricht 46.88μm im Abschnitt. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">Klicken Sie hier, um sich den Film anzusehen.</a> Movie 2. Serienschnitte Tracing Tubulin-Färbung in body Wand Muskeln eines dritten Larvenstadium Larve. Tubulin wird unter Verwendung anti-α-Tubulin-Antikörper und fluoreszierende sekundäre (Alexa Fluor 488). Maßstab: die eine Seite des Videobildes entspricht 46.88μm im Abschnitt. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">Klicken Sie hier, um sich den Film anzusehen.</a> Movie 3. Serienschnitte Tracing Tubulin-Färbung in einer Körperwand Luftröhre eines dritten Larvenstadium Larve, mit einem quadratischen in Abbildung 3 markiert. Tubulin wird unter Verwendung anti-α-Tubulin-Antikörper und fluoreszierende sekundäre (Alexa Fluor 488). Maßstab: die eine Seite des Videobildes entspricht 46.88μm im Abschnitt. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">Klicken Sie hier, um sich den Film anzusehen.</a>

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Um zu verstehen, wie komplex Zellformen sind es erreicht ist wichtig in der Lage sein, genau Assay Mikrotubuli Organisation. Hier beschreiben wir eine robuste immunhistologische Markierung Methode Assay Mikrotubuli Organisation von dendritischen Verzweigung Neuron sensorischen Dendriten. Neben Färbung sensorischen Neuronen erreicht diese Methode robust immunhistologische Färbung der Luftröhre, Muskeln und andere Körperteile Wand Gewebe. Wir verwenden dieses Protokoll, um Mikrotubuli Orga...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog Anzahl </th><th> Kommentare (Optional) </th></tr><tr><td> Zange </td><td> Dumont </td><td> 11251-20 </td><td></td></tr><tr><td> Mikroschere </td><td> FST </td><td> 15000-08 </td><td></td></tr><tr><td> Maus anti-α-Tubulin (Clone: ​​DM1A) </td><td> Sigma </td><td> T9026 </td><td> Verdünnung 1 / 1000 </td></tr><tr><td> Maus anti-futsch (Clone: ​​22C10), Überstand </td><td> Entwicklungsbiologie Studium Hybridoma der Bank </td><td> 22C10 </td><td> Verdünnung 1 / 1000 </td></tr><tr><td> Rat anti-CD8 (Klon: 5h10) </td><td> Caltag </td><td> MCD0800 </td><td> Verdünnung 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 Anti-Maus-IgG </td><td> Invitrogen </td><td> A-11001 </td><td> Verdünnung 1 / 500 </td></tr><tr><td> Cy3 anti-Rat IgG </td><td> Jackson Immunoresearch </td><td> 712-166-150 </td><td> Verdünnung 1 / 200 </td></tr></tbody></table>

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

Um zu verstehen, wie sich Unterschiede in komplexen Zelle Formen erreicht werden, ist es wichtig, genau zu verfolgen Mikrotubuli Organisation. Die Drosophila Larve Körper Wand enthält verschiedene Zelltypen, die Modelle, um Zellen und Gewebe Morphogenese Studie sind. Zum Beispiel Tracheen werden verwendet, um Rohr Morphogenese 1, und die dendritische Verzweigung (DA) sensorischen Neuronen der Drosophila Larve zu einem primären System für die Aufklärung der allgemeinen und Neuron-Klasse-spezifische Mechanismen der dendritischen Differenzierung 2-5 und Degeneration 6 prüfen . Die Form der Dendriten Niederlassungen erheblich variieren kann zwischen Neuron Klassen und sogar zwischen verschiedenen Niederlassungen eines einzelnen Neurons 7,8. Genetische Studien in DA Neuronen lassen vermuten, dass Differential Zytoskelett-Organisation kann morphologischen Unterschiede in dendritische Verzweigung Form 4,9-11 zugrunde liegen. Wir bieten eine robuste immunologische Kennzeichnung Methode, um einessay in vivo Mikrotubuli Organisation in DA sensorischen Neurons Dendriten Laube (Abbildungen 1, 2, Movie 1). Dieses Protokoll zeigt die Zerlegung und Immunfärbung des ersten Stadiums Larve, eine Bühne, wenn sie aktiv sensorischen Neurons Dendriten Auswuchs und Verzweigung Organisation auftritt 12,13. Neben Färbung sensorischen Neuronen erreicht diese Methode robust Kennzeichnung von Mikrotubuli Organisation in den Muskeln (Filme 2, 3), Luftröhre (Abbildung 3, Movie 3), und andere Körperteile Wand Gewebe. Es ist für die Ermittler wollen Mikrotubuli Organisation in situ Analyse in die Wand des Körpers bei der Untersuchung von Mechanismen, die wertvolle Kontrolle von Geweben und Zellen zu gestalten.

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

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

Um zu verstehen, wie komplex Zelle Formen, wie neuronalen Dendriten, während der Entwicklung erreicht werden, ist es wichtig, in der Lage sein, genau Assay Mikrotubuli Organisation. Hier beschreiben wir eine robuste immunhistologische Markierung Methode, um Mikrotubuli Organisation von dendritischen Verzweigung Neuron sensorischen Dendriten, Luftröhre, Muskeln zu untersuchen, und andere<em&gt; Drosophila</em&gt; Larve Körperwand Gewebe.

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 ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

Research

JoVE Journal

Neuroscience

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

12.3K Aufrufe.  RIKEN Brain Science Institute. Um zu verstehen, wie komplex Zelle Formen, wie neuronalen Dendriten, während der Entwicklung erreicht werden, ist es wichtig, in der Lage sein, genau Assay Mikrotubuli Organisation. Hier beschreiben wir eine robuste immunhistologische Markierung Methode, um Mikrotubuli Organisation von dendritischen Verzweigung Neuron sensorischen Dendriten, Luftröhre, Muskeln zu untersuchen, und andere Drosophila Larve Körperwand Gewebe.

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

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

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

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

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

Live-Cell-Imaging von Sinnesorgan Vorläuferzellen im Intact Drosophila Puppen

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

Forschung

Neurowissenschaften

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

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

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

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

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 ...

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.

Visualisierung von Propriozeptoren in<em&gt; Drosophila</em&gt; Larven und Puppen

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

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

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

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

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

Retrograde Fluoreszenzmarkierung Ermöglicht Gezielte Extracellular Einzel-Einheit Aufnahme von identifizierten Neuronen<em&gt; In vivo</em

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

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

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

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

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

Beschriften einzelner Zellen im zentralen Nervensystem<em&gt; Drosophila melanogaster</em

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

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 ...

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

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

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

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

Olfaktorischen Behaviors Assayed von Computer-Tracking<em&gt; Drosophila</em&gt; In einem Vier-Quadranten-Olfaktometer

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

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

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

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

Ballistische Kennzeichnung von Pyramidal neurons in Gehirnscheiben und in der primären Zellkultur

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

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

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

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

Semi-quantitative Bestimmung der dopaminergen Neuronendichte in der Substantia Nigra von Nagetiermodellen mittels automatisierter Bildanalyse

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

Betrachtung von Mikrotubuli-Netzwerken in neuromuskulären Verbindungen und Muskelzellen von Drosophila

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

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

Autor im Rampenlicht: Verständnis des Mikrotubuli-Netzwerks in neuromuskulären Verbindungen von Drosophila 

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