Questo lavoro è stato sostenuto da un finanziamento della DFG a GMT

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+grasshopper+to+Drosophila:+a+common+plan+for+neuronal+development.">From grasshopper to Drosophila: a common plan for neuronal development.</a> Nature. 310, 203-207 (1984).</li><li>Whitington, P., Harris, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+axonogenesis+in+the+embryo+of+a+primitive+insect,+the+silverfish+Ctenolepisma+longicaudata.">Early axonogenesis in the embryo of a primitive insect, the silverfish Ctenolepisma longicaudata.</a> Development Genes and Evolution. 205 (5-6), 272-281 (1996).</li><li>Whitington, P., Meier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation,+neurogenesis+and+formation+of+early+axonal+pathways+in+the+centipede,+Ethmostigmus+rubripes+(Brandt).">Segmentation, neurogenesis and formation of early axonal pathways in the centipede, Ethmostigmus rubripes (Brandt).</a> Development Genes and Evolution. 199, 349-363 (1991).</li><li>Whitington, P. M., Seifert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+growth+from+limb+motorneurons+in+the+locust+embryo:+the+effect+of+target+limb+removal+on+the+pattern+of+axon+branching+in+the+periphery.">Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery.</a> Developmental Biology. 106, 438-449 (1984).</li><li>Whitington, P. M., Leach, D., Sandeman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+change+in+neural+development+within+the+arthropods:+axonogenesis+in+the+embryos+of+two+crustaceans.">Evolutionary change in neural development within the arthropods: axonogenesis in the embryos of two crustaceans.</a> Development. 118, 449-461 (1993).</li><li>Williams, D. 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Gli autori dichiarano di non avere conflitto di interessi finanziari.

Uno dei principali vantaggi di Drosophila come sistema modello è che permette l&#39;analisi di sviluppo e funzione del livello di singole cellule. Questo è particolarmente utile per quanto riguarda il sistema nervoso, in cui la diversità di tipi di cellule è eccezionalmente elevata e la funzione e la morfologia delle cellule vicine può essere totalmente differente. Il metodo qui presentiamo permette l&#39;etichettatura dei singoli neuroni con un colorante che può essere trasformato in una macchia permanente o esaminato direttamente mediante microscopia a fluorescenza. Rivela la morfologia dei processi neurali in grande dettaglio (Figura 4). Uno dei principali vantaggi del metodo è che consente la morfologia di qualsiasi tipo di neuroni nel SNC da esaminare (per il cavo embrionale nervoso ventrale, vedere Rickert et al, 2011;. Per il cervello embrionale, vedere Kunz et al., 2012). Inoltre, non richiede complesse combinazioni dielementi genetici di essere presenti nell&#39;embrione, dato che alcune delle tecniche di marcatura genetica turistiche. Iniezioni tintura può essere effettuata in cellule di animali che portano costrutti reporter, consentendo morfologie cellulari individuali da visualizzare nel contesto di particolari modelli di espressione genica (Figure 4D e 5) in entrambi wild type o mutanti sfondi. Abbiamo anche utilizzato la tecnica di monitorare le dinamiche di conseguenza assoni nel vivere neuroni embrionali 11, 12. Il metodo può essere facilmente adattato per etichettare i singoli neuroni in embrioni di altri organismi, purché l&#39;embrione è sufficientemente trasparente da esaminare sotto illuminazione DIC. Lo abbiamo usato per visualizzare la morfologia neurale in una vasta gamma di embrioni artropodi, tra cui cavallette 18, millepiedi 17, crostacei 19 e pesciolini d&#39;argento 16. Lo svantaggio principale del tecnicoque sta nella sua difficoltà tecnica. Confidiamo che il conto corrente dettagliata del metodo assistere i ricercatori interessati a padroneggiarlo.

La conoscenza della morfologia neuronale a livello delle singole celle è un presupposto fondamentale per la comprensione connettività neuronale e la funzione del SNC. Così, fin dai primi giorni di neuroscienze, i ricercatori hanno cercato di sviluppare singole tecniche di marcatura delle cellule (vedi 7 per una trattazione storica di questo numero). Metodi classici come colorazione di Golgi forniscono eccellente risoluzione di morfologia neuronale ma non sono adatti se si cerca di etichettare un particolare tipo di neurone in modo diretto, come colorazione avviene in modo casuale. Lo sviluppo di metodi per la colorazione di cellule singole mediante iniezione intracellulare o juxtacellular di coloranti da un microelettrodo affrontato l&#39;obbligo di etichettatura specifica. L&#39;applicazione di una singola iniezione di Drosophila neurone colorante rappresentato una sfida importante, a causa delle piccole dimensioni sia dell&#39;organismo e suoi neuroni. Tuttavia, la colorazione singolo neurone di embrionale Drosophila </em&gt; Neuroni è stato raggiunto a metà degli anni 1980 nel laboratorio di Corey Goodman 15. Mentre il metodo è stato eminentemente utile e ha fornito alcuni spunti importanti sui meccanismi per lo sviluppo neuronale in Drosophila nel corso degli ultimi 20-30 anni (ad esempio, 2, 10), molti lavoratori hanno evitato che, in gran parte a causa delle sue esigenze tecniche. La disponibilità in anni più recenti di tecniche genetiche per l&#39;etichettatura neuronale in Drosophila ha anche contribuito l&#39;impopolarità di singola iniezione colorante neurone. Espressione GAL4-diretto di membrana mirati costrutti GFP in grado di fornire un&#39;eccellente risoluzione della morfologia neurale 1, 20. Tuttavia, questo metodo ha alcune limitazioni: la spesso inevitabile espressione di GFP in cellule multiple possono oscurare la struttura dei singoli neuroni e una linea pilota GAL4 possono non essere disponibili per guidare l&#39;espressione in un neurone specifico di interesse. Il marcm (Analisi Mosaico con un addettoMarker ressible Cell) Metodo 8 può fornire etichettatura di ogni neurone essenzialmente a livello di cella singola, ma non può essere usato con successo nell&#39;embrione e larva presto a causa della lenta rotazione della proteina GAL80. Date queste limitazioni di etichettatura genetica, noi crediamo che una singola iniezione colorante neurone nell&#39;embrione Drosophila rimane una tecnica preziosa e merita una più ampia applicazione. Per promuovere questo obiettivo, forniamo qui una descrizione dettagliata del metodo. Un&#39;illustrazione della sua potenza è fornito dal nostro conto recente della morfologia del set completo di interneuroni in neuromeres addominali dell&#39;embrione ritardo Drosophila 14. Questo studio, che ha rivelato sia la variabilità morfologica dei singoli tipi di cellule neuronali e dei principi di organizzazione neuromere nel SNC dell&#39;embrione, non sarebbe stato possibile con qualsiasi altro metodo di etichettatura attualmente disponibile.

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td></td><td></td><td></td><td> REAGENTI </td></tr><tr><td> DAB </td><td> Sigma-Aldrich </td><td> <a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank">D-5905</a> </td><td> 2-3 mg / ml in 100 mM Tris-HCl, pH 7,4 </td></tr><tr><td> DiI </td><td> Probes Invitrogen / Molecular </td><td> <a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank">D-282</a> </td><td> 1 mg / ml in etanolo </td></tr><tr><td> <a href="http://www.merckmillipore.com/germany/chemicals/formaldehydloesung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWpOEfVhTl" target="_blank">Formaldeide</a> </td><td> Merck Millipore </td><td></td><td> 7,4% in PBS </td> &lt;/ Tr&gt; <tr><td> Glicerina </td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">Roth</a> </td><td> 3783 </td><td> 70% in PBS </td></tr><tr><td> Eptano Colla </td><td> Beiersdorf AG </td><td> Cello 31-39-30 * </td><td> diluire ca. 1 a 1 con n-eptano </td></tr><tr><td> PBS </td><td></td><td></td><td> 1x </td></tr><tr><td> TRIS </td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">Roth</a> </td><td> 4855 </td><td></td></tr><tr><td> Vectashield </td><td> Vector Laboratories </td><td> <a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank">H1000</a> </td><td></td></tr><tr><td></td><td></td><td></td><td> ATTREZZATURE </td></tr><tr><td> Microscopio confocale </td><td> Leica </td><td> TCS SP2 </td><td> per visualizzare e documentare labelings in fluorescenza </td></tr><tr><td> DC - unmplifier </td><td> Dragan Corporation </td><td> Cornerstone ION-100 </td><td> effettuare le labelings </td></tr><tr><td> Coprioggetto </td><td> <a href="http://www.menzel.de/" target="_blank">Menzel</a> </td><td> BB018018A1 </td><td> 18 x 18 mm </td></tr><tr><td> Coprioggetto </td><td> <a href="http://www.menzel.de/" target="_blank">Menzel</a> </td><td> BB024060A1 </td><td> 24 x 60 mm </td></tr><tr><td> Dissezione Microscopio </td><td> Leica </td><td> MZ8 </td><td> per preparare e disect embrioni </td></tr><tr><td> Appartamento Capillari </td><td> <a href="http://www.hilgenberg-gmbh.de/" target="_blank">Hilgenberg</a> </td><td></td><td> diametro esterno 1 mm, spessore vetro 0,1 millimetri </td></tr><tr><td> Iniezione Capillari </td><td> Scienza Prodotti </td><td> GB 100 TF 8P </td><td></td></tr><tr><td> Micromanipolatore R </td><td> Leica </td><td></td><td> effettuare le labelings </td></tr><tr><td> Modello P-97 </td><td> Sutter Instruments </td><td></td><td> per tirare capillari </td></tr><tr><td> Oggetto Slides </td><td> <a href="http://www.marienfeld-superior.com/" target="_blank">Marienfeld Superior</a> </td><td> 1000000 </td><td></td></tr><tr><td> Microscopio scientifico </td><td> Zeiss </td><td> Axioskop 2 mot </td><td> per visualizzare labelings dopo fotoconversione </td></tr><tr><td> Microscopio scientifico </td><td> Olimpo </td><td> BX50 </td><td> effettuare le labelings </td></tr><tr><td> Sony MC3255 Videocamera </td><td> Sony / AVT Horn </td><td> Sony MC3255 </td><td> registrare labelings dopo fotoconversione </td></tr></tbody></table> Tabella 1.

labeling of single cells in the central nervous system of drosophila melanogaster

In questo articolo viene descritto come etichettare singolarmente i neuroni del sistema nervoso centrale dell&#39;embrione di Drosophila melanogaster mediante iniezione juxtacellular del DiI lipofilo fluorescente marker di membrana. Questo metodo permette la visualizzazione della morfologia delle cellule neuronali nei minimi dettagli. È possibile etichettare qualsiasi cella nel CNS: corpi cellulari dei neuroni bersaglio sono visualizzati sotto DIC ottiche o mediante espressione di un marcatore fluorescente genetico come GFP. Dopo etichettatura, la DiI può essere trasformato in un permanente macchia marrone da fotoconversione per permettere la visualizzazione della morfologia cellulare con luce trasmessa e ottica DIC. In alternativa, le cellule DiI-etichetta possono essere osservati direttamente con microscopia confocale, permettendo geneticamente introdotte proteine ​​reporter fluorescenti da colocalised. La tecnica può essere utilizzata in qualsiasi animale, indipendentemente dal genotipo, rendendo possibile analizzare fenotipi mutanti a risoluzione singola cella.

La figura 4 illustra i risultati tipici della tecnica, si descrive qui. Figura 4A illustra un esempio di una piena interneurone DiI singolo che è stato pulito photoconverted. Si dimostra in modo esauriente la quantità di dettagli questi offrono preparazioni. Se visto sotto ottiche DIC contesto spaziale della cella etichettata all&#39;interno del tessuto non marcato circostante diventa visibile, ad esempio, la posizione del corpo cellulare all&#39;interno della corteccia e della proiezione fibra nella neuropile. Figura 4B è un caso dove la goccia colorante era un po &#39;troppo grande causando diverse celle vicine diventando etichettati simultaneamente. Ciò rende spesso difficile mettere in relazione proiezioni singoli corpi cellulari distinti. Essa mostra anche che quando visualizzate direttamente sotto il microscopio a fluorescenza (senza fotoconversione) risoluzione di fondo è molto più basso. Il campione in Figura 4C </strong&gt; illustra un caso in cui più celle essendo stati etichettati singolarmente in segmenti vicini. Su fotoconversione la preparazione è stata successivamente trattata con un anticorpo contro Fas2 5following protocolli standard da 13 a fornire punti di riferimento (fascicoli) nel neuropile. Sebbene in questa preparazione organismi più cellulari sono ben separati, e consentendo la mappatura affidabile dei loro proiezioni, dimostra anche ciò può verificarsi quando il periodo fotoconversione è prolungato (per convertire anche le cellule debolmente marcate): le cellule più intensamente marcate tendono a ottenere overstained e iniziare a gonfiarsi (confrontare con A). La figura 4D mostra che l&#39;osservazione al microscopio confocale rivela la morfologia delle cellule marcate in dettaglio. DiI etichettatura è stata eseguita in un ceppo che portava un costrutto reporter GFP. Questo può fornire importanti informazioni sul contesto spaziale (e l&#39;identità) della cella colorante occupati entro specifiche popolazioni di neuroni o glial tipi cellulari (come definito dalla espressione genica). Come paradigma per valutare la variabilità morfologica di un neurone identificato abbiamo scelto uno dei neuroni atteri positivi 14. Questo neurone si trova in una posizione vicina dorsale e mediale alla neuropile (dAP, Figura 5A, Lundgren et al., 1995) separato dagli altri neuroni positivi atteri ed è quindi facilmente identificabile in apGal4-UASGFP animali. Figura 5B mostra una cella dAP che era pieno di DII e photoconverted. Il corpo cellulare si trova a livello della commessura anteriore e mostra un assone che cresce prima verso la linea mediana e poi si trasforma in una regione anteriore mediale connettivo. Ha cono di crescita, come rigonfiamenti sulla punta e il punto di svolta. 19 etichette di questa cellula, disegnata come proiezioni massime di stack di immagini, sono riassunti nella figura 5C. Morfologia cellulare diventa visibile al grande dettaglio. Solo poche cellule cono di crescita, come mostrano<html> strutture; sei delle 19 celle anche inviare un posteriore ramo che è sempre più corto del ramo anteriore e potrebbe essere una caratteristica transitoria. In Figura 5D tutte le celle 19 DAP etichettati sono accatastati con ogni cella avente solo una opacità del 12%. Ciò si traduce in zone che sono più scure le cellule più condividerli. Mentre il corpo della cella varia più del diametro di cella intorno alla posizione centrale, la posizione mediolaterale dell&#39;assone nel neuropile varia molto meno - quando la larghezza neuropile è diviso in nove parti che risiede sempre in una delle due posizioni mediali. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50150/50150fig1highres.jpg" src="/files/ftp_upload/50150/50150fig1.jpg" /> Figura 1. Schema delle fasi del metodo etichettatura neurone e le sue due varianti. &quot;Fo: content-width =&quot; 4in &quot;fo: src =&quot; / files/ftp_upload/50150/50150fig2highres.jpg &quot;src =&quot; / files/ftp_upload/50150/50150fig2.jpg &quot;/&gt; Figura 2. Diagramma che illustra le varie fasi di preparazione di embrioni di Drosophila per iniezione neurone colorante, a seconda della variante B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50150/50150fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 3" fo:content-width="5.5in" fo:src="/files/ftp_upload/50150/50150fig3highres.jpg" src="/files/ftp_upload/50150/50150fig3.jpg" /> Figura 3. . Rappresentazione schematica della configurazione di iniezione colorante L&#39;inserto superiore destro è una vista ingrandita della disposizione del campione, la micropipetta iniezione e l&#39;elettrodo vasca A:. Micromanipolatore, B: microscopio scena fissa. Il micromanipolatore è fissato a un angolo basso - circa il 10 &amp; de. g C: Porta Micropipetta con micropipetta, D: campione sul vetrino, E: elettrodo Bagno fissato con plastilina, F: Collegamento al amplificatore DC. <img alt="Figura 4" fo:content-width="5in" fo:src="/files/ftp_upload/50150/50150fig4highres.jpg" src="/files/ftp_upload/50150/50150fig4.jpg" /> Figura 4. Esempi di interneuroni DiI marcati nel cordone nervoso ventrale della fase 17 embrioni. Dorsal vista, anteriore è a sinistra. Linea mediana: linee tratteggiate. <ol class="ualpha"><li> Un esempio di una cella perfettamente photoconverted singola, mostrando un prominente proiezione controlaterale assonale e fibre dendritiche omolaterali (freccia). Ottica DIC. </li><li> mostra una preparazione in cui è stato etichettato più di una cella - questo può oscurare la chiara assegnazione di un corpo singola cellula alla sua proiezione. </li><li> 8 celle sono stati etichettati in 3 segmenti consecutivi. Inoltre, in questa preparazione il fotoconversione è stato seguito da una colorazione standard di protocollo di anticorpi contro Fas2 come punto di riferimento. </li><li> mostra un singolo neurone DiI pieno documentato con microscopia confocale in un ceppo mosca porta un reporter GFP costrutto. </li></ol><img alt="Figura 5" fo:content-width="5in" fo:src="/files/ftp_upload/50150/50150fig5highres.jpg" src="/files/ftp_upload/50150/50150fig5.jpg" /> Figura 5. Mirata DiI etichettatura (contrassegnato da espressione GFP) rivela il range di variabilità morfologica di un neurone identificato 14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50150/50150fig5large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <ol class="ualpha"><li> La cella dAP (colorati artificialmente blu) può essere riconosciuta in modo inequivocabile nel atteri Gal4-UASGFP modello nell&#39;addome (DAP, Figura 5A, Lundgren et unl. 1995). Cellule che esprimono GFP sono stati colorati con un anticorpo anti-GFP. La linea mediana è segnato con linee tratteggiate. ac = commissura anteriore, pc = commissura posteriore. </li><li> mostra un esempio di un photoconverted DiI-riempita cella dAP. La cella è stato identificato prima tingere compilando un atteri Gal4-UASGFP embrione con la sua espressione di GFP. </li><li> Una galleria di 19 riempie di questa cellula, disegnata come proiezioni massime di stack di immagini. Visualizzazioni dorsali. Anteriore è in alto. Il neuropile è di colore grigio chiaro, la zona corteccia è grigio più scuro. </li><li> Le celle 19 DAP sono accatastati con ogni cella avente una opacità del 12%. La regione occupata dalla maggior parte delle cellule ha la tonalità più scura del grigio. Il neuropile di tutti i campioni (blu) è stata scalata per una larghezza simile e le commissure anteriori accanto alla cella sono state allineate in modo che si sovrapponevano. Mentre il corpo della cella varia più del diametro di cella intorno alla posizione centrale, la posizione mediolaterale dell&#39;assone all&#39;interno neuropile mostrameno variazioni. </li></ol>

Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

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

Presentiamo una tecnica per l&#39;etichettatura singoli neuroni nel sistema nervoso centrale (SNC)<em&gt; Drosophila</em&gt; Embrioni, che consente l&#39;analisi della morfologia neuronale da una luce trasmessa o microscopia confocale.

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 ...

labeling-single-cells-central-nervous-system-drosophila

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

12.7K Views.  University of Mainz. Presentiamo una tecnica per l'etichettatura singoli neuroni nel sistema nervoso centrale (SNC) Drosophila Embrioni, che consente l'analisi della morfologia neuronale da una luce trasmessa o microscopia confocale.

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

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 ...

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Neuroscienze

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualizzazione del sistema nervoso embrionale in Whole-mount Drosophila Embrioni

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Resezione del nervo ottico: un modello di apoptosi Neuron adulti nel Sistema Nervoso Centrale

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

Imaging multimodale impianto di cellule staminali nel sistema nervoso centrale di topi

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Produzione di vettori lentivirali per Celle di trasduzione del Sistema Nervoso Centrale

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Un paradigma Infortunio per Indagare riparazione del sistema nervoso centrale in<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

In vivo Interrogatorio del Sistema Nervoso Centrale Translatome da Polyribosome Frazionamento

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Clearing ottica del sistema nervoso centrale del mouse Utilizzando CHIAREZZA passivo

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...