Ringraziamo Bernard Charroux per le discussioni. Questo lavoro è stato finanziato dalla Scuola LKC biomedica.

1. Dissezione della retina Drosophila <ol><li> Riempire un pozzo di un 3-Bene vetrino con tampone fosfato (PBS 1X), e poi anestetizzare vola da CO 2. Sia i maschi che le femmine possono essere utilizzati per questo esperimento. </li><li> Una volta che le mosche sono anestetizzati, prendere una mosca con pinze e inseritelo nel piccolo piatto di PBS. </li><li> In un ambito di dissezione, pizzicare la proboscide con una pinza, e poi staccare la testa dal corpo eliminando il collo con una seconda serie di pinze. </li><li> Tenendo la testa vola dalla proboscide, tagliata a metà lungo la linea mediana a sinistra / destra della testa volare con microscissors per rivelare il cervello. </li><li> Utilizzare un puntale tagliato a raccogliere la retina, e trasferirli in un secondo pozzo della 3-Bene vetrino di vetro pieni di medio Schneider. </li><li> In primo luogo, buccia via la maggior parte della cuticola testa e poi rimuovere il cervello con una pinza. Mantenere la retina a sandwich tra la lamina e la cornea. </li><li> Poi, il trasferimento della retina su una goccia (50 ml) di media Schnieder fresca posizionato direttamente del vetrino. </li></ol> 2. Dissezione del ommatidi <ol><li> A seconda dello scopo finale della dissezione (sia immagini immediato o la cultura) e l&#39;ottica del microscopio a fluorescenza (diretta o invertita), questo passaggio può avvenire sia su un vetrino o in una 24-pozzetti. Per lo scopo finale di questa dissezione, il ommatidi verrà sezionato direttamente sul vetrino. </li><li> Afferrare una delle estremità più dorsale o ventrale della retina con una pinza. Utilizzando microscissors, tagliare la retina a metà lungo la linea mediana dorsale / ventrale per esporre la ommatidi al centro dell&#39;occhio. </li><li> Continua a cogliere la retina in modo che la cornea è rivolto verso il basso e la lamina è rivolto verso l&#39;alto. Utilizzando un ago di tungsteno fine, staccare il ommatidi dalla lamina e la cornea. </li><li> Singoli ommatidi e gruppi di ommatidi raccoglierà sul fondo del pozzo o diapositiva. Rimuovere eventuali residui che potrebbero essere più grandi raccolte prima di procedere. </li></ol> 3. Trattamento, colorazione e di imaging <ol><li> Per analizzare l&#39;autofagia, diluire 1:1000 Lysotracker nel menu Schneider (prima diluita 1:10 in Schneider e poi aggiungere 0,5 microlitri alla goccia sul vetrino), mescolare e attendere 10 secondi. </li><li> Togliere il liquido in eccesso dalla diapositiva, facendo attenzione a lasciarsi alle spalle ommatidi. Questo viene fatto meglio con una punta fine caricamento del gel. </li><li> Aggiungere una goccia di Vectashield per coprire il ommatidi, applicare un vetrino coprioggetti e sigillare con nailpolish. </li></ol> 4. Rappresentante dei risultati: La buona esecuzione del protocollo dovrebbe lasciare un numero di ommatidi singoli e di piccoli gruppi di ommatidi tenuti insieme da frammenti di lamina ma ben separati dal lato della cornea. Questi possono essere ripreso come in questo esempio per visualizzare Atg8: GFP e Lysotracker, mostrando autophagosomes, lisosomi e autophagolysosomes (Fig. 3). Il metodo può essere utilizzato per l&#39;imaging dal vivo, però in questo caso è da notare che autofluorescenza di media di Schneider renderà difficile distinguere i segnali a bassa intensità fluorescente. Un ulteriore problema è che i granuli di pigmento che rimangono attaccati ai fotorecettori sono intensamente fluorescenti, soprattutto nel canale rosso. Una foto brightfield può essere richiesto per distinguerli dagli organelli genuina. <img src="/files/ftp_upload/2882/2882fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso della procedura di dissezione di ottenere ommatidi singoli o piccoli gruppi di ommatidi dalla retina volare. <img src="/files/ftp_upload/2882/2882fig2.jpg" alt="Figura 2"/> Figura 2. Eventuali variazioni del semplice protocollo qui descritto che coinvolgono la genetica e l&#39;invecchiamento volare a monte della dissezione e colorazione o coltura per un massimo di 24 ore ed a valle della droga amministrazione della dissezione. <img src="/files/ftp_upload/2882/2882fig3.jpg" alt="Figura 3"/> Figura 3 scansione confocale di autophagosomes e lisosomi in un singolo ommatidium sezionato da aw; GMR-GAL4, UAS-Atro75QN; UAS-GFP:.: Atg8 / + volo, esprimendo un poliglutamine Atrophin mutante e dai 12 giorni a 29 ° C. Il pannello di campo chiaro (in alto a destra) mostra la struttura quasi intatta della ommatidium e la cornice indica l&#39;area acquisita. Segni rossi lisosomi, il verde autophagosomes, auto-lisosomi, risultante dalla fusione dei due organelli, di colore giallo. Le frecce indicano due eventi di fusione in corso tra autophagosomes e lisosomi.

<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>

Nessun conflitto di interessi dichiarati.

La dissezione ommatidium singolo presentato qui permette la raccolta di informazioni delle cellule più profonde sui processi biologici come la neurodegenerazione, quando modellato negli occhi Drosophila. I neuroni dei fotorecettori sono più facilmente accessibili di altri neuroni e citoplasma è particolarmente grande, e quindi adatto per studiare la dinamica delle vescicole, nelle cellule stesse utilizzate proficuamente in molti modelli di quantificare neurodegenerazione in vivo. L&#39;aspetto criti...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Dispenser portatile di CO2 </td><td> Flystuff </td><td> 59-150 </td><td> Ricariche disponibili </td></tr><tr><td> DUMONT Tweezer n. 5 </td><td> AGAR scientifico </td><td> T5034 </td><td></td></tr><tr><td> 3-Bene vetrino </td><td> SME </td><td> 71561-01 </td><td></td></tr><tr><td> Micro forbici </td><td> VWR </td><td> HAMMHSB516-09 </td><td></td></tr><tr><td> Media Schneider </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 diapositive </td><td> VWR </td><td> 631-0108 </td><td></td></tr><tr><td> Vectashield con DAPI </td><td> Vector Labs </td><td> H-1200 </td><td></td></tr><tr><td> Coprioggetto </td><td> VWR </td><td> 631-1336 </td><td></td></tr></tbody></table>

single drosophila ommatidium dissection and imaging

La mosca della frutta Drosophila melanogaster ha dato un contributo inestimabile alla ricerca neuroscientifica ed è stato ampiamente utilizzato come modello per le malattie neurodegenerative a causa della sua potente genetica 1. L&#39;occhio vola in particolare è stato l&#39;organo di scelta per la ricerca neurodegenerazione, essendo la parte più accessibile e di vita indispensabili del sistema nervoso Drosophila. Ma l&#39;avvertimento principale degli occhi intatto è la difficoltà, a causa della intensa autofluorescenza del pigmento, in eventi di imaging intracellulari, come autofagia dinamica 2, che sono di primaria importanza per la comprensione della neurodegenerazione. Recentemente abbiamo usato la dissezione e la cultura dei singoli ommatidi 3 che è stato essenziale per la comprensione delle disfunzioni autofagici in un modello di volo Dentatorubro-Pallidoluysian Atrofia (DRPLA) 3, 4. Ora segnalare una descrizione completa di questa tecnica (Fig. 1), adattato da studi elettrofisiologici 5, che rischia di ampliare notevolmente la possibilità di modelli di volo per neurodegenerazione. Questo metodo può essere adattato a vivere immagine eventi subcellulare e per monitorare la somministrazione efficace farmaco su cellule fotorecettrici (Fig. 2). Se usato in combinazione con tecniche di mosaico 6-8, le risposte di cellule geneticamente diverse possono essere analizzati in parallelo (Fig. 2).

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

Single Drosophila Ommatidium Dissection and Imaging

Il fattore limitante l&#39;uso degli adulti<em&gt; Drosophila</emOcchio&gt; per studiare neurodegenerazione e biologia cellulare è l&#39;imaging difficile dei processi intracellulari. Descriviamo la dissezione dei singoli ommatidi per generare una buona fede colture primarie di cellule neuronali, che possono essere soggetti a trattamento farmacologico e di imaging avanzate.

Singolo Drosophila Ommatidium Dissection e 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 Views.  King's College London. Il fattore limitante l'uso degli adulti Drosophila per studiare neurodegenerazione e biologia cellulare è l'imaging difficile dei processi intracellulari. Descriviamo la dissezione dei singoli ommatidi per generare una buona fede colture primarie di cellule neuronali, che possono essere soggetti a trattamento farmacologico e di imaging avanzate.

Video: 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).

Registrazioni Sensillum singolo in Insetti Drosophila melanogaster E 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 ...

Ricerca

Neuroscienze

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.

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

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

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.

Dissezione e immunoistochimica di larvale, pupa e adulto<em&gt; Drosophila</em&gt; Retine

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.

Etichettatura di singole cellule nel sistema nervoso centrale di<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.

A Single-fly saggio per comportamento alimentare 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.

Immagini dal vivo di<em&gt; Drosophila</em&gt; larvali neuroblasti

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.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<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 Singola molecola Tracking al terminale presinaptico nervo motore Drosophila

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.

Cella singola risoluzione fluorescenza Live Imaging di Drosophila orologi circadiani nel cervello larvale cultura

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

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Dissezione di fibre muscolari scheletriche singole per analisi immunofluorescenti e morfometriche di giunzioni neuromuscolari a montaggio intero

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...