Die Autoren möchten sich Alexandra Opie für technische Hilfe und Unterstützung, James Barton für die Bereitstellung von Video-Erzählung anerkennen und Ramona Flatz und Joellen Sweeney für im zugehörigen Video erscheinen. Diese Arbeit wurde von der MJ Murdock Charitable Trust (Grant No 2012205 zu JED) unterstützt.

1. Die Stereomikroskopsystem Anmerkung: Obwohl dieses Protokoll einfach an praktisch jedem Stereomikroskopsystem mit der Fähigkeit zum Erfassen Video in einer Digitalkamera gekoppelt ist, werden die Details von der in unserem Labor verwendet (Tabelle Materialien / Equipment) System. <ol><li> Erwerben Sie digitale Videos mit einer trinokularen Stereomikroskop zu einem kommerziellen Digitalkamera gekoppelt ist. </li><li> Zur Kopplung des kommerziellen Digitalkamera an den trinokularen Anschluss des Stereo-Mikroskop, entfernen Sie die ½x C-Mount-Anschluss der Photozelle des Stereomikroskops und ersetzen Sie es mit einem 1X C-Mount. </li><li> Montieren Sie eine Digitalkamera Koppler (43 mm Gewinde) an die 1X C-Mount. </li><li> Halterung zwei Abwärts-Ringe, 58 mm bis 48 mm und 48 mm bis 43 mm, zu der Kamera-Koppler, um die Verbindung von der Digitalkamera Koppler an einem Linsenadaptersatz für die digitale Kamera brücken. </li><li> Montieren Sie die Digitalkamera an den Objektiv-Adapter-Kit. </li><li> Erwerben Video mit dem Mikroskop Vergrößerung und optischen Zoom der Digitalkamera-Set für eine kombinierte Vergrößerung von ca. 12x (30 Bilder pro Sekunde, 640 x 480 Pixel). Hinweis: Die Vergrößerung des Stereomikroskops muss in Übereinstimmung mit der neu rekonfiguriert 1X C-Mount-Anschluss des trinokularen kompensiert werden. </li></ol> 2. Imaging Drosophila Dritte Larvenstadium <ol><li> Band ein Permanent-Marker auf die schwarze Tischplatte von einem Stereomikroskop mit einer digitalen Kamera gekoppelt, so dass die Seite der Marker Kappe nimmt etwa ⅓ auf ¼ der vertikalen Bereich der in der Kamera LCD-Monitor beobachtet Blick. Verwenden Sie Marker Spitzen wie die Bühne, um Larven Bildgebung durchzuführen, weil sie in einer Auswahl von Farben, die Farbcode verwendet werden kann und differenzieren die Genotypen von Larven, die abgebildet sind. </li><li> Grenzen den Bereich der in der Digitalkamera LCD-Monitor auf der Oberfläche des Markers oben beobachtet Ansicht mit einer feinen SpitzeMarker. </li><li> Wählen Sie einen dritten Larvenstadium zu Bild. Die Kriterien für die Auswahl der dritten Larvenstadium war Körperlänge Austritt aus der Nahrungsmittelquelle während des Larven Phase des Lebenszyklus, die Anwesenheit von vorderen und hinteren Luftlöcher, und die Struktur der Unterkiefer Haken des Mundvorrichtung 11. Stellen Sie sicher, die Larve ist sauber durch Waschen Sie es gründlich in Wasser. </li><li> Beleuchten den Permanentmarker obersten Stufe von oben mit Licht aus einem Glasfaser-Beleuchtungssystem. Stellen Sie den Winkel des einfallenden Lichts, um eine optimale Ausleuchtung sorgen. </li><li> Fokussieren Sie das Mikroskop auf dem Rand des Permanentmarker Spitze. Beginnen Erfassen digitaler Video. </li><li> Legen Sie die Larve auf der Seite der Marker Kappe ca. 75 ° von der vertikalen Achse, etwas außerhalb des Blickfeldes, mit dem vorderen der Larve zugewandten Sichtfeld (Abbildung 1). Hinweis: Die Platzierung der Larve auf der Seite des Markers Kappe ermöglicht die Kamera auf Bewegung thE-Larve aus einer seitlichen Perspektive. Es hilft, die Larve mit Wasser feucht zu halten, damit sie nicht herunterfallen die Seite der Marker Kappe. Vorsicht ist jedoch, nicht zu viel Wasser, wie übermäßige Mengen werden der Larve halten, wie es über das Feld kriecht. </li><li> Sanft zu stoßen und prod die Larve mit einem kleinen Pinsel, um sie zu zwingen, in das Blickfeld zu kriechen. Geduld, da die Larven nur selten zusammenarbeiten und müssen oft an den Ausgangspunkt zurückgebracht werden viele Male, bevor sie gerade über das Feld zu kriechen. </li><li> Rekord ca. 10-15 min ununterbrochenen digitalen Videomaterial und Pflanzen und entfernen Sie alle unnötigen Material nach der Übernahme mit digitalen Video-Editing-Software. </li></ol> 3. Imaging Erwachsene Drosophila <ol><li> Legen Sie eine einzelne erwachsenen Drosophila in einer Wegwerf 1,5 ml Polystyrol spektroskopischen Küvette. Hinweis: CO 2 Betäubung von erwachsenen Drosophila unmittelbar vor einer BehavtensAnalyseProtokoll kann Ergebnisse 12 beeinträchtigen. Es wird empfohlen, dass erwachsene Drosophila eine 24-Stunden-Zeitraum gegeben, um von CO 2 Betäubung erholen, bevor Sie in einem Verhaltenstest 13 werden. </li><li> Stecken Sie das Ende der Küvette mit einem kleinen Wattebausch. Sicherstellen, dass der Wattebausch ist fest genug, um die große Kappenraum besetzen verpackt und beschränkt die Fliege auf das reduzierte Volumen Fach der Küvette. </li><li> Setzen Sie die Küvette auf der weißen Tischplatte von einem Stereomikroskop und richten das reduzierte Volumen Fach der Küvette mit dem Gebiet der in der Digitalkamera LCD-Monitor beobachtet Blick. </li><li> Beleuchten die Küvette von oben mit Licht aus einem Lichtwellenleiter-Beleuchtungssystem. Stellen Sie den Winkel des einfallenden Lichts, um eine optimale Ausleuchtung sorgen. </li><li> Fokus des Mikroskops und beginnen Erfassung digitaler Video. </li><li> Rekord ca. 30-45 min ununterbrochenen digitalen Videomaterial und Pflanzen und entfernen Sie alle unnötigenVideo nach dem Erwerb mit digitalen Video-Editing-Software. </li></ol>

<ol>
	<li>Zhang, B., Freeman, M. R., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila neurobiology: a laboratory manual. , (2010).</li><li>Frank, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+for+studying+synaptic+development,+function,+and+plasticity+using+Drosophila+as+a+model+system.">New approaches for studying synaptic development, function, and plasticity using Drosophila as a model system.</a> J Neurosci. 33, 17560-17568 (2013).</li><li>Mudher, A., Newman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila : a toolbox for the study of neurodegenerative disease. , (2008).</li><li>Bilen, J., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+human+neurodegenerative+disease.">Drosophila as a model for human neurodegenerative disease.</a> Annu Rev Genet. 39, 153-171 (2005).</li><li>Jakubowski, B. R., Longoria, R. A., Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+and+sensitive+method+correlates+neuronal+disorder+genotypes+to+Drosophila+larvae+crawling+phenotypes.">A high throughput and sensitive method correlates neuronal disorder genotypes to Drosophila larvae crawling phenotypes.</a> Fly (Austin). 6, 303-308 (2012).</li><li>Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+larval+locomotion+in+Drosophila+chordotonal+organ+mutants).">Dynamic analysis of larval locomotion in Drosophila chordotonal organ mutants).</a> Proc Natl Acad Sci U S A. 100, 16053-16058 (2003).</li><li>Jahn, T. R., 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=Detection+of+early+locomotor+abnormalities+in+a+Drosophila+model+of+Alzheimer's+disease.">Detection of early locomotor abnormalities in a Drosophila model of Alzheimer's disease.</a> J Neurosci Methods. 197, 186-189 (2011).</li><li>Donelson, N. C., 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=High-resolution+positional+tracking+for+long-term+analysis+of+Drosophila+sleep+and+locomotion+using+the+&amp;#34;tracker&amp;#34;+program.">High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the &amp;#34;tracker&amp;#34; program.</a> PLoS ONE. 7, e37250 (2012).</li><li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+video+tracking+of+locomotion+in+adult+Drosophila+melanogaster.">High-resolution video tracking of locomotion in adult Drosophila melanogaster.</a> J Vis Exp. (24), (2009).</li><li>Colomb, J., Reiter, L., Blaszkiewicz, J., Wessnitzer, J., Brembs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+tracking+and+analysis+of+adult+Drosophila+locomotion+in+Buridan's+paradigm+with+and+without+visual+targets.">Open source tracking and analysis of adult Drosophila locomotion in Buridan's paradigm with and without visual targets.</a> PLoS ONE. 7, e42247 (2012).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1965).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J Insect Physiol. 46, 439-442 (2000).</li><li>Greenspan, 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=.">.</a> Fly pushing : the theory and practice of Drosophila genetics.. , (2004).</li><li>Duncan, J. E., Lytle, N. K., Zuniga, A., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microtubule+Regulatory+Protein+Stathmin+Is+Required+to+Maintain+the+Integrity+of+Axonal+Microtubules+in+Drosophila.">The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.</a> 8, e683244 (2013).</li><li>Belmont, L. D., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+interacts+with+tubulin+dimers+and+increases+the+catastrophe+rate+of+microtubules.">Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.</a> Cell. 84, 623-631 (1996).</li><li>Cassimeris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncoprotein+18/stathmin+family+of+microtubule+destabilizers.">The oncoprotein 18/stathmin family of microtubule destabilizers.</a> Curr Opin Cell Biol. 14, 18-24 (2002).</li></ol>

Die Autoren haben erklärt, dass keine Interessenkonflikte bestehen.

Drosophila melanogaster &#39;s Stärke als ein Modellsystem für die Untersuchung Funktion des Nervensystems stammt weitgehend aus der Konvergenz der mächtigen genetische Werkzeuge zur Verfügung, und die breite Palette von robusten Verhaltenstests entwickelt. Hier präsentieren wir Ihnen eine einfache und allgemein zugänglich Mikroskopietechnik für den Erwerb von qualitativ hochwertigen digitalen Video von Drosophila Erwachsenen-und Larven Lokomotive Phänotypen aus einer seitlichen Perspektive. Wi...

Die gemeinsame Fruchtfliege Drosophila melanogaster ist eine leistungsfähige experimentelle Modellsystem zur Untersuchung der Funktion des Nervensystems 1-3. Evolutionären Erhaltung der Struktur und Funktion des Nervensystems beim Menschen, als auch einfache genetische Manipulation und eine Vielzahl von genetischen Werkzeugen macht Drosophila die Premiere Organismus der menschlichen neurodegenerativen Erkrankungen 4 modellieren. Gen-Mutationen, die Funktionsstörungen des Nervensystems verursachen führen oft in lebensfähigen Mutanten Larven und adulte Drosophila mit eingeschränkter Fortbewegung. In Nervensystem Mutanten beobachteten Phänotypen sind reduzierte Geschwindigkeit der Fortbewegung, anomale Koordination und spastischen Bewegungen bei Erwachsenen, als auch Defizite in peristaltische Kontraktion der Muskulatur der Körperwand und teilweise Lähmung der Larven. Diese Phänotypen in der Entwicklung von Hochdurchsatz-genetischen Screens und Fortbewegung Assays Mutante Larven 5 ausgenutzt, 6 und Erwachsenen 7-10 Drosophila bei der Quantifizierung der Fortbewegung Wertminderung und die Identifizierung für die Funktion des Nervensystems notwendigen Gene abzielen. Während diese Ansätze sind äußerst nützlich für die Quantifizierung Larven und Erwachsenen Lokomotive Verhaltensweisen, versagen sie auf qualitative Informationen über die einzelnen spezifischen abweichendes Verhalten zu vermitteln. Zum Beispiel, während Mutante dritten Larvenstadium kann die Fortbewegung veränderten Parameter in einem Verhaltenstest zeigen, kann es unklar sein, wenn dies das Ergebnis von Veränderungen in rhythmischen Kontraktionen während der Schlauch Crawling Zyklus, allgemeine Mangel an Koordination oder teilweise Lähmung der hinteren Körper Wandmuskulatur. Hier beschreiben wir eine einfache und allgemein zugänglich Mikroskopietechnik für den Erwerb von qualitativ hochwertigen digitalen Video von Drosophila Erwachsenen-und Larven Lokomotive Phänotypen aus einer seitlichen Perspektive. Digitale Videosignale von einer seitlichen Perspektive erworben erlaubt die direkte Beobachtung und Analyse der feinen Unterschiede in Locomotive Verhaltensweisen von einem aussageSeitenAnsicht Orientierung.

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

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

Drosophila melanogaster ist eine leistungsfähige experimentelle Modellsystem zur Untersuchung der Funktion des Nervensystems. Gen-Mutationen, die Funktionsstörungen des Nervensystems verursachen produzieren häufig lebensfähigen Larven und Erwachsene, die Fortbewegung defekt Phänotypen, die schwierig ist, ausreichend mit Text beschreiben oder vollständig mit einem einzigen fotografischen Bild darstellen, sind zu haben. Aktuelle Modi des wissenschaftlichen Publizierens, unterstützen jedoch die Vorlage von digitalen Video-Medien als ergänzendes Material, ein Manuskript zu begleiten. Hier beschreiben wir eine einfache und allgemein zugänglich Mikroskopietechnik für den Erwerb von qualitativ hochwertigen digitalen Video beider Drosophila Larven und erwachsene Phänotypen aus einer seitlichen Perspektive. Video der Larven und erwachsene Fortbewegung aus einer Seitenansicht ist von Vorteil, weil sie die Beobachtung und Analyse der feinen Unterschiede und Variationen in abweichenden Lokomotive Verhaltensweisen. Wir haben erfolgreich die Technik zur Visualisierung und Quantifizierung aberrant kriechen Verhaltensweisen in Drittlarvenstadium, zusätzlich zu Erwachsenen Phänotypen und Verhaltensweisen einschließlich Pflege.

Wir haben erfolgreich diese Technik zur Erfassung und Quantifizierung der Larven Verhaltensphänotyp mit Verlust der Funktion des Gens Stathmin (Figur 2) 14 verbunden. Das Gen kodiert für ein Stathmin Mikrotubuli regulatorisches Protein, das Tubulindimeren Partitionen von Pools von löslichen Tubulin und Mikrotubuli bindet und fördert ihre Demontage 15,16. Stathmin Funktion ist erforderlich, um die Integrität des Mikrotubuli in den Axonen von peripheren Nerven ...

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

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

Hier beschreiben wir eine einfache und allgemein zugänglich Mikroskopietechnik, um hochwertige digitale Video von Drosophila Erwachsenen-und Larven Phänotypen aus einer seitlichen Perspektive zu erwerben.

Erwerb von qualitativ hochwertigen digitalen Video<em&gt; Drosophila</em&gt; Larven und Verhalten der Erwachsenen aus einer seitlichen Perspektive

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

acquisition-high-quality-digital-video-drosophila-larval-adult

Research

JoVE Journal

Neuroscience

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

8.7K Aufrufe.  Willamette University.  Hier beschreiben wir eine einfache und allgemein zugänglich Mikroskopietechnik, um hochwertige digitale Video von Drosophila Erwachsenen-und Larven Phänotypen aus einer seitlichen Perspektive zu erwerben. 

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

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

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

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

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

Visualisierung von Larven segmentalen Nerven in 3 Rd Instar Drosophila Larven Vorbereitungen

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

Forschung

Neurowissenschaften

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

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

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

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

Mapping und Anwendung von Enhancer-trap Flippase Expression in Larven und Erwachsene Drosophila CNS

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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

Dissektion und Immunhistochemie von Larven, Puppen und Erwachsenenbildung<em&gt; Drosophila</em&gt; Netzhäute

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

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

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 ...

Live Imaging of Drosophila Larval Neuroblasts

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

Live Imaging of Drosophila Larval Neuroblasts

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

Live-Imaging von<em&gt; Drosophila</em&gt; Larven Neuroblasten

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

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

Quantifizieren Fisch Schwimmverhalten in Reaktion auf akute Exposition von wässrigen Kupfer Form von computergestützten Video und digitale Bildanalyse

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

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 ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Geschmackspräferenz Assay für Erwachsene<em&gt; Drosophila</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

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 ...

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Funktionelle Analyse der Larvenfütterung Circuit in Drosophila

Live-Imaging von

Quantifizieren Fisch Schwimmverhalten in Reaktion auf akute Exposition von wässrigen Kupfer Form von computergestützten Video und digitale Bildanalyse

Olfaktorischen Behaviors Assayed von Computer-Tracking

Geschmackspräferenz Assay für Erwachsene

Entfernung von

Linear Agarose Kanäle zu studieren