Questo lavoro è stato sostenuto da una borsa di studio a Ehrman HY. H., Jane Coffin Childs Memorial Fund per la borsa di studio post-dottorato di ricerca medica RJJ, NIH Grant F32EY016309 a DV, Fellowship Tesi di New York University di Dean a DJ, NIH GrantR01 EY13010 su CD e una borsa di studio per DFG JR (RI 2208/1- 1). Ringraziamo Nina Vogt e Pamela Boodram per i commenti sul manoscritto.

1. Introduzione In questo video, si descrivono i metodi per dissezioni retiniche e immunoistochimica in tre periodi definiti di sviluppo: il terzo instar larvali, la midpupal e la fase adulta. Anche se il nostro protocollo funziona anche per le altre fasi di pupa (per i dettagli su fasi precedenti, vedi 16), abbiamo scelto lo stadio midpupal, come è ottimale per l&#39;imaging tutte le PR in un unico piano focale e la loro nuclei sono facilmente identificabili, che facilita la visualizzazione di fattore di trascrizione espressione modelli. La dissezione di ritardo pupa retina è molto simile alla dissezione dei retine adulti. In primo luogo, si dimostra come raccogliere larve e pupe nelle fasi desiderati. Poi, ci spiega come ottenere dissezioni ottimali di larvale 17-18 midpupal e retine adulti. In secondo luogo, si dimostra come visualizzare sviluppo PR in queste fasi usando la microscopia confocale e immunoistochimica. Il periodo di sviluppo degli insetti dipende dalla temperatura. Per facilitare la messa in scena riproducibile di larve e pupe, si raccomanda di mettere tutte le scorte di mosca a 25 ° C sotto un hr/12 12 ore ciclo luce / buio. Terzo instar larve: A proposito di 96 ore dopo la deposizione delle uova (a 25 ° C), fine del III alimentazione larve instar stop e vagare sulle pareti della fiala alimentare, dove alla fine pupate. Utilizzare una spazzola morbida o una pinza per rimuovere delicatamente un vagare terzo instar larve dalla parete del flaconcino cibo. 50% pupe (&#39;midpupae&#39;) sono stati osservati circa 48 ore dopo impupamento. Per i tempi esatti, si può girare intorno appena formata pupe bianco con un pennarello sul flaconcino cibo. In alternativa, spostare tutti pupe bianco a un nuovo flacone per ottenere una fiala di messa in scena uniforme (per la messa in scena di pupe in base a criteri morfologici, vedere 19). Dopo 48 ore, con attenzione remove il midpupae dal flacone utilizzando una pinza. Mosche adulte: anestetizzare mosche giovani adulti (2-5 giorni dopo eclosion) su un tappetino di CO 2. Rimuovere testine di stampa utilizzando una pinza e memorizzarli in un bicchiere e servire freddo contenente 1x PBS. 3. Dissezione Procedura (Circa 1-2 ore per Experiment) Dopo aver raccolto il campione, metterli in una goccia di PBS freddo su un piatto dissezione Sylgard. Dissect circa 10-15 retine per genotipo. Si prega di consultare le schede di sicurezza, nonché la salute ambientale del proprio istituto e Office di sicurezza per la corretta manipolazione dei reagenti e delle attrezzature (vedi tabelle alla fine di questo protocollo). 1. Dischi Eye larvali immaginale <ol><li> Individuare l&#39;estremità anteriore della larva, che contiene i ganci bocca. Utilizzare una pinza per premere la parte centrale della larva dolcemente contro la base del piatto Sylgard. Utilizzare another pinza per afferrare i ganci bocca e tirare delicatamente e lentamente dal corpo. Scartare la parte principale del corpo e utilizzare l&#39;apparato boccale per tenere la parte anteriore in modo da rimuovere il tessuto estraneo dai dischi immaginali che rimangono attaccate al cervello. </li><li> Trasferimento cervello con i dischi immaginali afferrando i ganci bocca con una pinza a 1x PBS in un tre ben piatto di vetro. Eseguire le operazioni di fissaggio (passo 4.1.). </li><li> Dopo la fissazione, rimuovere ghiandole salivari, tessuto adiposo e tirare il cervello lontano dagli occhi-antennale disco immaginale. Per migliorare l&#39;accessibilità delle &quot;difficili&quot; anticorpi, la membrana peripodial, che si trova sulla superficie dell&#39;epitelio, può essere rimosso premendo la parte antennale del disco e la membrana peeling via con un perno dissezione. Successivamente, eseguire immunoistochimica (punto 5.1.). </li></ol> 2. 50% ninfosi / Midpupal retine <ol><li> Tenere estremità posteriore del case pupa con una pinza.Rimuovere delicatamente la superficie anteriore del case pupa afferrando la cuticola con altre pinze e tirare lentamente dal pupario. Staccare cuticola residua per esporre la testa. </li><li> Utilizzare i suggerimenti pinze &quot;per perforare il fondo, sacco morbido nella regione dorsale anteriore. Questo fornisce l&#39;accesso alla testa. Delicatamente separare la testa dal torace lentamente succhiando la testa con una pipetta P20. Espellere testa dalla punta della pipetta nel fresco freddo 1x PBS e rimuovere il tessuto in eccesso; afferrare la cuticola testa e rimuovere proboscide. Procedere alla fase di fissaggio (passo 4.1.). </li><li> Pierce il lobo ottico con un perno di dissezione per stabilizzare il tessuto. Inserire l&#39;altro perno dissezione tra la lamina e retina per separare la retina dalla lamina / lobo ottico. Eseguire immunoistochimica (punto 5.1.). </li></ol> 3. Occhi adulti <ol><li> Afferra il centro della capsula posteriore testa con una pinza e rimuovere le parti della bocca (proboscide), Tiss grassiue e trachee. </li><li> Afferra la cuticola dorsale testa con una pinza e usare l&#39;altra per separare gli occhi di tessuto cerebrale, tirando delicatamente lateralmente. Nella maggior parte dei casi, la lamina, una guaina sottile e trasparente di neuropilo cervello, rimarrà attaccato alla retina. È più facile rimuovere la lamina dopo fissazione. </li><li> Rimuovere le cuticole al margine occhio, ma lascia un piccolo pezzo dietro per facilitare il trasferimento di retine a soluzioni diverse. Effettuare fissaggio (passo 4.1.). </li><li> Rimuovere la lamina afferrandolo da un lato con una pinza e delicatamente tirando lateralmente con le pinze altri senza compromettere la retina. Rimuovere cuticola residuo, in quanto potrebbe ripiegare sulla retina durante il montaggio. Questi due passaggi sono fondamentali per la visualizzazione fotorecettori, che altrimenti oscurato dalla lamina e rimane della cuticola durante il processo di imaging. Procedere alla immunoistochimica (punto 5.1.). </li></ol> 4. Fissazione e Washing (circa 1 ora) <ol><li> Conservare le retine sezionati in 1x PBS in tre ben piatto di vetro immessi sul ghiaccio mentre appena la preparazione del fissativo (diluire 40 microlitri di soluzione di formaldeide al 37% con 360 microlitri di PBS 1x, vortice e memorizzare sul ghiaccio). Si consiglia di procedere con il protocollo di fissazione e di evitare lunghi tempi di conservazione in ghiaccio (&gt; 15 min). </li><li> Sostituire 1x PBS con 200 ml di soluzione di formaldeide 3,7%. Assicurarsi che il tessuto viene immerso in fissativo. Questo è critico, in quanto può non essere altrimenti fissato correttamente. Se necessario, rimuovere le bolle d&#39;aria e trachee con le pinze. </li><li> Incubare su agitatore per 15 min a temperatura ambiente. </li><li> Sostituire il fissativo con 1x PBS e lavare due volte con PBS 1x. Continuare il lavaggio in PBS per 30 minuti (su agitatore, a temperatura ambiente). </li><li> Sostituire PBS con PBST. Risciacquare due volte con PBST. </li><li> Procedere con la rimozione della membrana peripodial dai dischi degli occhi larvali (3.1.3.) E le lamine di midpupal / adulto retine &lt;strong&gt; (3.2.3., 3.3.4.). </li></ol> 5. Immunoistochimica <ol><li> Bloccante: Sostituire PBS con 200 pl del 5% siero normale in PBST e incubare per 20 min retine su agitatore a temperatura ambiente. </li><li> Sostituire la soluzione di saturazione con la soluzione di anticorpo primario. Sigillare il tre ben piatto con parafilm e incubare per una notte su agitatore a temperatura ambiente. </li><li> Sostituire la soluzione con PBST e lavare 3 volte. Continuare lavaggio con PBST per almeno 1 ora su agitatore a temperatura ambiente. </li><li> Trasferimento in soluzione di anticorpo secondario ed incubare in un parafilm-piatto di vetro sigillato bene su agitatore notte a temperatura ambiente. </li><li> Sostituire la soluzione di anticorpi con PBST e lavare tre volte. Continuare lavaggio con PBST per un minimo di un hr su agitatore a temperatura ambiente. I campioni possono essere conservati in PBST per diversi giorni, ma è importante aggiungere PBST fresco almeno una volta al giorno per evitare che si secchi. </li></ol> 6. Montaggio <ol><li> Per il montaggio retine larvali e midpupal, utilizzare il P20 per trasferire retine al centro di un vetrino pulito. Rimuovere PBST eccesso. Utilizzare una pinza per allineare retine per facilitare l&#39;imaging. </li><li> Aggiungere 10 ml di mezzo di montaggio sopra le retine. Coprire delicatamente con un foglio 24x40-1 coperchio e sigillare con smalto trasparente. </li><li> Per montaggio retine adulti, preparare &#39;ponti&#39; 20: Aggiungere due gocce 5 ml di glicerolo al 50% su entrambe le estremità di un lato di vetro e coprire con 22x22-1 vetrini (0,13 a 0,17 mm di spessore). </li><li> Rimuovere il PBST con una pipetta P20, fare in modo che la retina non si seccano. Aggiungere 20 ml di mezzo di montaggio al pozzo. Utilizzare mezzo di montaggio per trasferire retine con il P20 al vetrino &#39;ponte&#39;. </li><li> Utilizzare una pinza per orientare (lenti devono affrontare il vetrino) e allineare retine per facilitare l&#39;imaging. Se necessario, rimuovere le bolle d&#39;aria con il P20. </li><li> Lentamenteposto antiscivolo 24x40-1 Coperchio (0,13 a 0,17 mm di spessore) da un lato in alto e sigillare i bordi con smalto trasparente o nastro adesivo. Conservare i campioni a 4 ° C in una casella di vetrino da microscopio saver (tenere al buio). </li></ol> 7. Risultati rappresentativi Ad esempio per l&#39;applicazione del presente protocollo, si mostrano i risultati rappresentativi per la visualizzazione di differenziazione dei fotorecettori i tre stadi di sviluppo nel video. A larvale eye-antennale disco e una retina midpupal state colorate con anticorpi che etichettano tipi fotorecettrici diversi durante il loro sviluppo (Figura 1A, B) e una retina adulta è stato colorato con due anticorpi Rhodopsin di visualizzare due sottotipi fotorecettrici (Figura 1C).

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Nessun conflitto di interessi dichiarati.

1. Risoluzione dei problemi Nella nostra esperienza, dissezioni richiedono una pratica (fino a diverse settimane) e sono facilitate da raggiungere una posizione comoda a mano 21 con appoggio i gomiti e gli avambracci sul tavolo e con le dita in contatto con il piatto dissezione. Dita In questo modo, solo il pollice, indice e medio compiere movimenti sottili. Rimozione della lamina senza danneggiare i fotorecettori è probabilmente il passo più difficile....

<table border="1"><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> Tampone fosfato salino (PBS1x, pH 7.4) </td><td> Sigma </td><td></td><td> Preparare soluzione di 10x 20. Diluire con acqua distillata per ottenere 1x PBS e conservare a temperatura ambiente. Raffreddare in ghiaccio prima dissezioni. </td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> 9002-93-1 </td><td> Attenzione: Irritante Indossare guanti. Preparare 50 ml 1xPBS con 0,3% Triton X-100 (PBST). Conservare a temperatura ambiente. </td></tr><tr><td> Soluzione di formaldeide al 37% </td><td> Fisher Scientific </td><td> F75P1GAL </td><td> Attenzione: Tossico, probabile cancerogeno umano Indossare guanti. Prima della fase di fissazione, di fresco preparare 3,7% soluzione in una cappa di diluizione con PBS, negozio su ghiaccio. </td></tr><tr><td> 5% di siero di cavallo normale </td><td> Jackson Immuno Research </td><td> 008-000-001 </td><td> Preparare 5% v / v di diluizione in PBST. Conservare a quattro gradi. </td></tr><tr><td> Gli anticorpi primari e secondari (ad esempio, Donkey anti-pecore Alexa Fluor 488, Donkey-coniglio anti Alexa Fluor 555, Donkey anti-topo Alexa Fluor 647) </td><td> Probes Invitrogen Molecular </td><td> A11015 A31572 A31571 </td><td> Diluire 1:800 anticorpi secondari in PBST e conservare a quattro gradi. </td></tr><tr><td> Alexa Fluor 488 falloidina </td><td></td><td> A12379 </td><td> Diluire 1:100 in soluzione anticorpo secondario. </td></tr><tr><td> Slowfade Oro Antifade reagente </td><td> Probes Invitrogen Molecular </td><td> S36936 </td><td> Mezzo di montaggio. Conservare a -20 gradi. </td></tr><tr><td> Glycerol </td><td> Fisher Scientific </td><td> G31-1 </td><td> Per il montaggio. Preparare 10 ml di 50% di diluizione con acqua distillata, conservare a temperatura ambiente. </td></tr><tr><td> CO 2 </td><td></td><td></td><td> Per anestetizzare mosche adulte. </td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> Due pinze taglienti (Dumont # 55) </td><td> Strumenti Scienza Belle </td><td> 11255-20 </td><td></td></tr><tr><td> Sylgard 184 Kit di silicone elastomero </td><td> Dow Corning </td><td></td><td> Preparare piatto dissezione Sylgard compilando un piatto di plastica con la miscela Sylgard Petri. </td></tr><tr><td> Tre piatti e vetro dissezione </td><td> Fisher Scientific </td><td> 21-379 </td><td></td></tr><tr><td> Two perni minutien dissezione (0,1 mm di diametro) e due pinholders (12 cm) </td><td> Strumenti per le scienze Belle </td><td> 26002-10 </td><td> Inserire un pin minutien in ciascuno dei due pinholders e piegare uno dei perni per formare un gancio. </td></tr><tr><td> Microscopio vetrini coprioggetto (22x22-24x40-1 e 1) </td><td> Fisher Scientific </td><td> 12-542A </td><td></td></tr><tr><td> Vetrini portaoggetto (25x75x1.0 mm; predepurata) </td><td> Fisher Scientific </td><td> 12-550-143 </td><td></td></tr><tr><td> 1,5 ml microcentrifuga tubi </td><td></td><td></td><td></td></tr><tr><td> Cancellare smalto o nastro adesivo </td><td></td><td></td><td></td></tr><tr><td> Agitatore orbitale </td><td> Bellco </td><td></td><td></td></tr><tr><td> Far scorrere il porta finestra </td&gt; <td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Parafilm </td><td> Bemis </td><td> PM996 </td><td></td></tr><tr><td> Pennello sottile </td><td></td><td></td><td></td></tr><tr><td> P20 e P200 micropipette e suggerimenti </td><td></td><td></td><td></td></tr><tr><td> Dissezione microscopio </td><td></td><td></td><td></td></tr><tr><td> Piccolo secchio con ghiaccio </td><td></td><td></td><td> Per raffreddare le lastre di vetro e, retine sezionati e soluzioni. </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

L&#39;occhio composto di Drosophila melanogaster è composto da circa 750 ommatidi (occhi unità). Ogni ommatidium è composto da circa 20 cellule, inclusa la lente-secernenti coni, cellule del pigmento, una cella a setole e otto fotorecettori (PR) R1-R8 2. Il PRS hanno le strutture di microvillar, le rhabdomeres, che contengono pigmenti sensibili alla luce, i rodopsine (sd). Le rhabdomeres di sei PR (R1-R6) formano un trapezio e contengono Th1 3 4. Le rhabdomeres di R7 e R8 sono posizionati in tandem nel centro del trapezio e condividono lo stesso percorso della luce. R7 e R8 PR stocasticamente esprimono diverse combinazioni di Rhs in due sottotipi principali 5: Nel sottotipo la &#39;p&#39;, in p RH3 R7s è accoppiato con RH5 in p R8s, mentre nel sottotipo del &#39;y&#39;, Rh4 in y R7s è associato RH6 in y R8S 6 7 8. Prime caratteristiche di PR e sviluppo di ommatidi inizia nella larvale eye-antennali disco immaginale, un monostrato di cellule epiteliali. Un&#39;ondata di differenziazione spazza tutto il disco 9 e avvia l&#39;assemblaggio di cellule indifferenziate in ommatidi 10-11. La R8 &#39;cella fondatore&#39; è specificato prima e recluta R1-6 e poi R7 12-14. Successivamente, durante lo sviluppo pupale, PR differenziazione porta a vasti cambiamenti morfologici 15, tra cui la formazione di rhabdomere, sinaptogenesi e di espressione alla fine rh. In questo protocollo, si descrivono i metodi per dissezioni retiniche e immunoistochimica in tre periodi definiti di sviluppo della retina, che possono essere applicati per affrontare una serie di questioni concernenti la formazione della retina e percorsi di sviluppo. Qui, usiamo questi metodi per visualizzare la differenziazione graduale PR a livello di singola cellula per intero monte larvale, midpupal e adulti retine ( <sTrong&gt; Figura 1).

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Il<em&gt; Drosophila</emRetina&gt; è un reticolo cristallino composto da un piccolo numero di tipi cellulari che sono generati in modo stereotipato<sup&gt; 1</sup&gt;. La sua riconducibilità a sofisticate analisi genetica permette lo studio di complessi programmi di sviluppo. Questo protocollo descrive le dissezioni e immunoistochimica di retina in tre distinte fasi di sviluppo, con particolare attenzione alla differenziazione dei fotorecettori.

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 ...

dissection-immunohistochemistry-larval-pupal-adult-drosophila

Research

JoVE Journal

Neuroscience

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

25.2K Views.  New York University. Il Drosophila è un reticolo cristallino composto da un piccolo numero di tipi cellulari che sono generati in modo stereotipato 1. La sua riconducibilità a sofisticate analisi genetica permette lo studio di complessi programmi di sviluppo. Questo protocollo descrive le dissezioni e immunoistochimica di retina in tre distinte fasi di sviluppo, con particolare attenzione alla differenziazione dei fotorecettori.

Video: Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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.

Visualizzazione dei nervi larvali segmentale in 3 Rd Instar Drosophila I preparativi larvali

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

Ricerca

Neuroscienze

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.

Mappatura e applicazione di Enhancer-trappola Espressione Flippase in larvali e adulti Drosophila SNC

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

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 ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

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 ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

Dissezione di utricolo topo adulto e adenovirus-mediata non protagonista infezione delle cellule

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila adulti olfattiva Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

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.

Analisi funzionale del circuito di alimentazione larvale 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.

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 ...

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

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

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

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

Acquisizione di alta qualità video digitale di<em&gt; Drosophila</em&gt; Comportamenti larvali e adulti da una prospettiva laterale

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

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Tutta Monte dissezione e immunofluorescenza del topo adulto Coclea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Dissezione e immunoistochimica di larvale, pupa e adulto

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Capitoli in questo video

Visualizzazione dei nervi larvali segmentale in 3^Rd Instar Drosophila I preparativi larvali

Mappatura e applicazione di Enhancer-trappola Espressione Flippase in larvali e adulti Drosophila SNC

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

Singolo Drosophila Ommatidium Dissection e Imaging

Dissezione di utricolo topo adulto e adenovirus-mediata non protagonista infezione delle cellule

Drosophila adulti olfattiva Shock Learning

Analisi funzionale del circuito di alimentazione larvale in Drosophila

Immagini dal vivo di

Acquisizione di alta qualità video digitale di

Tutta Monte dissezione e immunofluorescenza del topo adulto Coclea