Wir danken Bernard Charroux für Diskussionen. Diese Arbeit wurde von der KCL Biomedical Schule finanziert.

1. Dissektion der Drosophila Netzhaut <ol><li> Füllen Sie eine Vertiefung einer 3-Well Objektträger mit Phosphat-gepufferter Kochsalzlösung (PBS 1X) und dann zu betäuben fliegt von CO 2. Sowohl Männchen als auch Weibchen können für dieses Experiment verwendet werden. </li><li> Sobald die Fliegen betäubt sind, nehmen Sie eine Fliege mit einer Pinzette und legen Sie es in das Schälchen PBS. </li><li> Unter einem Binokular, kneifen den Rüssel mit einer Pinzette, und dann nehmen Sie den Kopf vom Körper durch Abtrennen der Hals mit einem zweiten Satz von Zangen. </li><li> Halten Sie die Fliege Kopf aus dem Rüssel, geschnitten längs der linken / rechten Mittellinie the fly Kopf mit Mikroschere an das Gehirn offenbaren. </li><li> Verwenden Sie einen Schnitt Pipettenspitze abholen der Netzhaut und die Übertragung auf eine zweite und der 3-Well Objektträger aus Glas mit Schneider Medium gefüllt ist. </li><li> Zunächst abziehen meisten der Kopf Nagelhaut und entfernen Sie dann das Gehirn mit einer Pinzette. Halten Sie die Netzhaut zwischen der Lamina und der Hornhaut eingeklemmt. </li><li> Anschließend Transfer der Netzhaut auf einen Tropfen (50 ul) von frischen Schnieder Medium direkt von der Glasplatte gelegt. </li></ol> 2. Dissection der Ommatidien <ol><li> Abhängig vom endgültigen Ziel der Dissektion (ob sofort ein Bildbearbeitungs-oder Kultur) und auf die Optik des Fluoreszenz-Mikroskops (direkt oder umgekehrt), kann dieser Schritt erfolgt entweder auf einen Objektträger oder in eine 24-Well-Platte. Für die endgültige Zweck dieser Sektion wird der Ommatidien direkt auf das Glas gleiten seziert werden. </li><li> Fassen Sie entweder die dorsalen oder ventralen Ende der Netzhaut mit einer Pinzette. Mit Mikroschere, schneiden die Netzhaut in zwei Hälften entlang der dorsalen / ventralen Mittellinie der Ommatidien in der Mitte des Auges aussetzen. </li><li> Weiter auf die Netzhaut, so dass die Hornhaut nach unten und in der Lamina nach oben zu erfassen. Mit einem feinen Wolfram-Nadel, nehmen Sie den Facettenaugen von der Lamina und der Hornhaut. </li><li> Einzel Facettenaugen und Gruppen von Ommatidien wird auf dem Grund des Brunnens oder schieben zu sammeln. Entfernen Sie alle Fremdkörper, die größer, bevor Sie fortfahren gesammelt haben. </li></ol> 3. Die Behandlung, Färbung und Imaging <ol><li> Zur Analyse Autophagie, verdünnte 1:1000 LysoTracker in der Schneider &amp; Drop (erste Verdünnung 1:10 in Schneider und fügen Sie dann 0,5 ul der Tropfen auf dem Objektträger), mischen und 10 Sekunden warten. </li><li> Entfernen Sie die überschüssige Flüssigkeit aus der Folie, wobei darauf geachtet, die Facettenaugen hinter sich zu lassen. Dies geschieht am besten mit einer feinen Spitze für Gel durchgeführt. </li><li> Fügen Sie einen Tropfen Vectashield die Facettenaugen decken, Deckglas auflegen und Dichtung mit Nagellack. </li></ol> 4. Repräsentative Ergebnisse: Die gute Ausführung des Protokolls sollten verlassen eine Reihe von einzelnen Ommatidien und kleine Gruppen von Einzelaugen zusammen gehalten durch Fragmente von Lamina aber schön auf der Hornhaut Seite getrennt. Diese können wie in diesem Beispiel dargestellt werden, um ATG8 visualisieren: GFP und LysoTracker, zeigt Autophagosomen, Lysosomen und autophagolysosomes (Abb. 3). Die Methode für die Live-Bildgebung verwendet werden kann, aber in diesem Fall ist zu beachten, dass Autofluoreszenz der Schneider-Medium wird es schwierig machen, mit niedriger Intensität fluoreszierender Signale zu unterscheiden. Ein weiteres Problem ist, dass die Pigmentkörnchen, die die Photorezeptoren verbunden bleiben intensiv fluoreszierende, vor allem in den roten Kanal sind. Ein Hellfeld Bild erforderlich, um sie von echten Organellen unterscheiden. <img src="/files/ftp_upload/2882/2882fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Flussdiagramm der Dissektion Verfahren auf einzelne Ommatidien oder kleine Gruppen von Facettenaugen von der Fliege Netzhaut zu erhalten. <img src="/files/ftp_upload/2882/2882fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Mögliche Variationen des einfachen Protokolls hier zu beschreiben, dass einbeziehen fliegen Genetik und Altern vor der Dissektion und Färbung oder Kultivierung für bis zu 24 Stunden and Drug Administration nach dem Sezieren. <img src="/files/ftp_upload/2882/2882fig3.jpg" alt="Abbildung 3"/> Abbildung 3 konfokale Scan Autophagosomen und Lysosomen in einem Ommatidium von aw seziert; GMR-Gal4, UAS-Atro75QN; UAS-GFP:.: ATG8 / + fliegen, Ausdruck einer Polyglutamin Atrophin Mutante und im Alter von 12 Tagen bei 29 ° C. Die Hellfeld-Panel (oben rechts) zeigt die nahezu intakte Struktur des Ommatidium und der Rahmen zeigt das gescannte Gebiet. Rote Markierungen Lysosomen, grün die Autophagosomen, Auto-Lysosomen, was durch die Fusion der beiden Organellen, gelb erscheinen. Die Pfeilspitzen zeigen zwei laufende Fusion Ereignisse zwischen Autophagosomen und Lysosomen.

<ol>
	<li>Marsh, J. L., Thompson, L. 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+in+the+study+of+neurodegenerative+disease.">Drosophila in the study of neurodegenerative disease.</a> Neuron. 52, 169-178 (2006).</li><li>Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>Nisoli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+by+polyglutamine+Atrophin+is+not+rescued+by+induction+of+autophagy.">Neurodegeneration by polyglutamine Atrophin is not rescued by induction of autophagy.</a> Cell Death Differ. 17, 1577-1587 (2010).</li><li>Charroux, B., Fanto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+line+between+waste+disposal+and+recycling:+DRPLA+fly+models+illustrate+the+importance+of+completing+the+autophagy+cycle+for+rescuing+neurodegeneration.">The fine line between waste disposal and recycling: DRPLA fly models illustrate the importance of completing the autophagy cycle for rescuing neurodegeneration.</a> Autophagy. 6, 667-669 (2010).</li><li>Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+potassium+channels+in+Drosophila+photoreceptors.">Voltage-sensitive potassium channels in Drosophila photoreceptors.</a> J Neurosci. 11, 3079-3095 (1991).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends in neurosciences. 24, 251-254 (2001).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1237 (1993).</li><li>Gambis, A., Dourlen, P., Steller, H., Mollereau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+in+vivo+imaging+of+photoreceptor+apoptosis+and+development+in+Drosophila.">Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila.</a> Dev Biol. 351, 128-1234 (2011).</li><li>Napoletano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyglutamine+Atrophin+provokes+neurodegeneration+in+Drosophila+by+repressing+fat.">Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat.</a> The EMBO journal. 30, 945-958 (2011).</li><li>Ravikumar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+mTOR+induces+autophagy+and+reduces+toxicity+of+polyglutamine+expansions+in+fly+and+mouse+models+of+Huntington+disease.">Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease.</a> Nature. 36, 585-595 (2004).</li><li>Zou, S., Meadows, S., Sharp, L., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+study+of+aging+and+oxidative+stress+response+in+Drosophila+melanogaster.">Genome-wide study of aging and oxidative stress response in Drosophila melanogaster.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 13726-13731 (2000).</li></ol>

Keine Interessenskonflikte erklärt.

Die einzelnen Ommatidium Dissektion hier vorgestellten ermöglicht das Speichern von tiefer zellbiologische Informationen über Prozesse wie Neurodegeneration, wenn in der Drosophila Auge nachempfunden. Der Photorezeptor Neuronen sind leichter zugänglich als andere Neuronen und sie Zytoplasma ist besonders groß, und damit geeignet, um Vesikel Dynamik Studie, in der selben Zellen kompetent in vielen Modellen verwendet werden, um Neurodegeneration in vivo zu quantifizieren. Der kritische Aspekt der Dis...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Tragbare CO2 Dispenser </td><td> Flystuff </td><td> 59-150 </td><td> Refills auch </td></tr><tr><td> DUMONT Pinzette Nr. 5 </td><td> AGAR Scientific </td><td> T5034 </td><td></td></tr><tr><td> 3-Well Glas Slide </td><td> EMS </td><td> 71561-01 </td><td></td></tr><tr><td> Micro Schere </td><td> VWR </td><td> HAMMHSB516-09 </td><td></td></tr><tr><td> Schneider-Medium </td><td> VWR </td><td> 733-1663 </td><td></td></tr><tr><td> LysoTracker Red DND-99 </td><td> Invitrogen </td><td> L7528 </td><td></td></tr><tr><td> Superfrost Slides </td><td> VWR </td><td> 631-0108 </td><td></td></tr><tr><td> Vectashield mit Dapi </td><td> Vector Labs </td><td> H-1200 </td><td></td></tr><tr><td> Deckgläser </td><td> VWR </td><td> 631-1336 </td><td></td></tr></tbody></table>

single drosophila ommatidium dissection and imaging

Die Fruchtfliege Drosophila melanogaster hat unschätzbare Beiträge zur neurowissenschaftlichen Forschung gemacht und wurde weithin als Modell für neurodegenerative Erkrankungen wegen seiner leistungsfähigen Genetik 1 verwendet. Die Fliege Auge in allem hat das Organ der Wahl für Neurodegeneration Forschung, wobei der am besten zugänglichen und Leben entbehrlich Teil der Drosophila Nervensystem. Doch die große Einschränkung der intakten Augen ist die Schwierigkeit, wegen der intensiven Autofluoreszenz des Pigments, in der Bildgebung intrazelluläre Ereignisse wie Autophagie Dynamik 2, die ausschlaggebend für das Verständnis der Neurodegeneration sind. Wir haben vor kurzem die Präparation und Kultur der einzelnen Ommatidien 3, die wichtig ist für unser Verständnis von autophagischen Dysfunktionen in einer Fliege Modell Dentatorubro-Pallidoluysian Atrophie (DRPLA) 3, 4 verwendet. Wir berichten nun über eine umfassende Beschreibung dieser Technik (Abb. 1), von elektrophysiologischen Studien 5, die voraussichtlich erheblich erweitern die Möglichkeit des Fly-Modelle für Neurodegeneration angepasst ist. Diese Methode lässt sich an Bild zu leben subzelluläre Ereignisse und wirksames Medikament Verwaltung auf Sehzellen Monitor (Abb. 2). Wenn in Kombination mit Mosaik-Techniken 8.6 verwendet, können die Reaktionen von genetisch unterschiedlichen Zellen parallel getestet werden (Abb. 2).

Watch this Scientific Journal Video about Single Drosophila Ommatidium Dissection and Imaging at JoVE.com

Single Drosophila Ommatidium Dissection and Imaging

Der limitierende Faktor in der Verwendung der Erwachsenen<em&gt; Drosophila</em&gt; Blick auf Neurodegeneration und Zellbiologie Studie ist die schwierige Darstellung von intrazellulären Prozessen. Wir beschreiben die Zerlegung der einzelnen Ommatidien zu einer bona-fide primären neuronalen Zellkulturen, die Gegenstand der medikamentösen Behandlung und fortschrittliche Bildgebung erzeugen können.

Single Drosophila Ommatidium Dissection und Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

single-drosophila-ommatidium-dissection-and-imaging

Research

JoVE Journal

Neuroscience

Single Drosophila Ommatidium Dissection and Imaging

14.0K Aufrufe.  King's College London. Der limitierende Faktor in der Verwendung der Erwachsenen Drosophila Blick auf Neurodegeneration und Zellbiologie Studie ist die schwierige Darstellung von intrazellulären Prozessen. Wir beschreiben die Zerlegung der einzelnen Ommatidien zu einer bona-fide primären neuronalen Zellkulturen, die Gegenstand der medikamentösen Behandlung und fortschrittliche Bildgebung erzeugen können.

Serie: Single Drosophila Ommatidium Dissection and Imaging

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Einzel Sensillum Recordings in der Insekten Drosophila melanogaster Und Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Forschung

Neurowissenschaften

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissection und Imaging von aktiven Zonen in der Drosophila Neuromuskulären Synapse

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

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 ...

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 ...

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

Ein Single-fly-Assay für Sammelverhalten in<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Eine einfache Ein-Schritt-Dissection-Protokoll für Whole-mount Vorbereitung von Adult<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

In Vivo Einzelmolekül-Tracking bei Drosophila präsynaptischen motorischen Nerv Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

Einzellige Auflösung Fluoreszenz Imaging von Drosophila zirkadiane Uhren in Larven Gehirn Kultur Leben

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...