Questo lavoro è stato finanziato da una sovvenzione NIH assegnato a R. Brewster (1R01GM085290-01A1).

1.Microinjection <ol><li> Diluire plasmide codifica membrana mirati Green Fluorescent Protein (mGFP, per gentile concessione di Richard Harland) ad una concentrazione di 40 ng / ml. Il DNA è preparato da linearizzazione plasmide (mGFP/PCS2 +, per gentile concessione di Richard Harland) purificato utilizzando il kit Qiagen Maxi Prep. L&#39;aggiunta di rosso fenolo (diluito volume totale 1 / 10) alla soluzione è preferibile per visualizzare la soluzione. Mantenere soluzione di DNA su ghiaccio. </li><li> Sotto uno stereomicroscopio, calibrare l&#39;ago di iniezione (90mm di vetro capillari da Narishinge scientifico; Cat. No. NA-GD-1), allineando l&#39;ago su un vetrino graduato e picchiettando delicatamente la parte dell&#39;ago in cui il diametro è 1μm usando un rasoio pulito lama (per ottenere una punta di dimensioni adeguate). </li><li> Al fine di calibrare l&#39;ago per iniettare un volume noto (2NL), collegare l&#39;ago per l&#39;apparato microiniezione (PCI 100 Microinjector; Harvard Apparatus) e davanti riempire l&#39;ago con 0.5μl di una soluzione contenente rosso fenolo e acqua posto su un pezzo di paraffina. La soluzione può essere dispensato con un pedale. Impostare la pressione a 7 KPa e variare il tempo (10msec 30msec) in modo che ci vogliono 250 pedali per erogare 0.5μl della soluzione. </li><li> Accuratamente posto l&#39;ago calibrato su una striscia di cera in un piatto 100 millimetri umidificato Petri. Per umidificare il piatto, posto Kimwipes bagnato intorno ai bordi (questo impedisce l&#39;evaporazione della soluzione l&#39;ago). </li><li> Utilizzare una micropipetta per caricare 0,5-1ml di DNA in fine dell&#39;ago (che alla fine non è stata calibrata). </li><li> Una volta che la soluzione ha raggiunto la punta dell&#39;ago, collegare l&#39;estremità dell&#39;ago che è stato utilizzato per il caricamento di un apparato di microiniezione. Regolare le impostazioni sul microinjector per fornire circa 2 nl / iniezione utilizzando un comando a pedale. </li><li> Successivamente, zebrafish embrioni posizione nel bel mezzo di una piastra di Petri piena di medio degli embrioni (Stock 1,0 ml di Hank # 1, 0,1 ml di Hank Stock # 2, Stock 1,0 ml di Hank # 4, 95,9 ml DDH 2 O, Stock 1,0 ml di Hank # 5 , Stock 1,0 ml fresca di Hank # 6, Usa circa 10 gocce di NaOH 1 M a pH 7,2). </li><li> Una volta che gli embrioni si sono sviluppati allo stadio desiderato, gentilmente presa il corion con una pinza sottile e situare l&#39;ago nel area di destinazione per l&#39;iniezione. Per l&#39;espressione ampia gamma mGFP, iniettare DNA nel tuorlo o del citoplasma nella fase 1cell. Per limitare l&#39;espressione di mGFP per una regione più piccola, iniettare il DNA a 4 (o più) stadio delle cellule nel citoplasma di una cellula singola. </li><li> Premere il pedale dell&#39;apparato microiniezione e fornire circa 2 nl, assicurando che la soluzione (visibile a causa dell&#39;aggiunta di fenolo rosso) è correttamente iniettato. </li><li> Rimuovere con attenzione l&#39;ago dall&#39;embrione. </li><li> Ripetere l&#39;operazione per tutti gli embrioni nella scatola di Petri. </li><li> Permettono di sviluppare gli embrioni a 28,5 ° C di mettere in scena desiderata. </li><li> Embrioni Dechorionate delicatamente staccando il corion con una pinza sottile (se gli embrioni sono più giovani di 24hpf, dechorionating in un piatto di vetro Petri aumenta il tasso di sopravvivenza degli embrioni). </li><li> Rimuovere corion vuoto dalla piastra di Petri con una pipetta di vetro. </li></ol> 2. Imaging mGFP marcato sezioni vibratome <ol><li> Utilizzare una pipetta di vetro per il trasporto di embrioni in fissativo (paraformaldeide 4% in soluzione 1X tampone fosfato (PBS). Fissare gli embrioni a 4 ° C durante la notte. </li><li> Lavare gli embrioni in PBS 1X 3 volte per 5 minuti ciascuno. </li><li> Mettere gli embrioni in piastra di Petri e, utilizzando una pinza sottile, togliete il tuorlo senza danneggiare l&#39;embrione. Rimuovere il tuorlo più possibile. </li><li> Preparare il 4% bassa temperatura di fusione agarosio in H 2 O. </li><li> Luogo agarosio fuso in stampi di plastica sezionamento. </li><li> Utilizzare una pinza sottile per sollevare un embrione iniettato dalla piastra di Petri e di luogo e orientare nel agarosio. L&#39;embrione deve essere posizionato verso una delle estremità dello stampo. </li><li> Una volta che l&#39;agarosio è indurito, utilizzare uno strumento superficie piana, come una spatola, per rimuovere il blocco di agarosio dallo stampo. </li><li> Tagliare l&#39;estremità inferiore del blocco di agarosio con una lametta in modo che sia pari. </li><li> Aggiungere una goccia di colla super per la fase vibratome e posizionare il blocco di agarosio su di esso (il lato del blocco che è stato stabilizzato con il rasoio deve essere rivolta la colla). Per sezionamento più embrioni, montare diversi agarosio-embedded campioni sul palco. </li><li> Collegare la fase vibratome al vibratome (Vibratome, Inc.). </li><li> Impostare i parametri del vibratome (impostazioni comuni: velocità-3;-spessore 50 micron). Se sezionare embrioni multipli, allineare i blocchi agarosio sul palco e in modo che la lama attraversa tutta l&#39;embrione ma non attraverso l&#39;intera larghezza dei blocchi agarosio (in modo tale che sezioni corrispondenti per l&#39;embrione stesso rimarrà insieme). </li><li> Versare PBS 1X nella fase vibratome. </li><li> Sezione. </li><li> Una volta che gli embrioni sono SEZIONEned, rimuovere il blocco di agarosio (s) dal palco vibratome. </li><li> Accuratamente separati ogni sezione, tagliando la parte posteriore del blocco di agarosio (s). </li><li> Utilizzare una pinza sottile per selezionare le sezioni desiderata e posto in un pieno di PBS 1X e multi-piatto (uso pozzi diversi per sezioni corrispondenti alle diverse embrioni). </li><li> Ripetere per ogni embrione. </li><li> Procedere con l&#39;etichettatura degli anticorpi, anche se i passaggi 19 23, per migliorare mGFP immagini (questo passo potrebbe non essere necessario se le sezioni sono esposte all&#39;interno di uno o due giorni di preparazione). </li><li> Trattare sezioni per 30 minuti a temperatura ambiente con una soluzione di blocco in PBS 1X per bloccare legame non specifico. </li><li> Sezioni incubare con l&#39;anticorpo primario (coniglio a-GFP, Invitrogen Cat. No. A11122) @ diluito 1:200 nella soluzione bloccante su un agitatore per 5 ore a temperatura ambiente o durante la notte a 4 ° C. </li><li> Lavare quattro volte (10 min, 15 min, 30 min e 60 min ciascuna) in 1% BSA/1X PBS / 1% di soluzione di DMSO. </li><li> Trattare con adeguate sezioni anticorpo secondario in soluzione di blocco per 5 ore a temperatura ambiente o durante la notte a 4 ° C. </li><li> Lavare quattro volte (10 min, 15 min, 30 min e 60 min ciascuna) in 1% BSA/1X PBS / 1% di soluzione di DMSO. </li><li> Trattare le sezioni con DAPI (0,1% DAPI in 50ml di PBS 1X; Invitrogen Cat. No. D1306) per 30 minuti per colorare i nuclei. </li><li> Lavare tre volte in PBS 1X per 5 minuti ciascuno. </li><li> Montaggio sul vetrino. </li><li> Immagine mediante microscopia confocale. </li></ol> 3. Vivere l&#39;imaging <ol><li> Dechorionate vivi, embrioni iniettata 30 minuti prima che raggiungano lo stadio di sviluppo desiderato. Mettere gli embrioni in soluzione sedativo (0,01% in media Tricaine embrioni) per 15 min. </li><li> Mettere una goccia di agarosio all&#39;1% su un piatto di 35 millimetri cultura fondo di vetro (MatTek; Codice P35G-o-20-C). </li><li> Lasciare agarosio a solidificarsi. </li><li> Calore capillare sul fuoco per fare 3 fori in agarosio (altri oggetti come punte micropipetta può essere usato per fare i fori). Garantire che non c&#39;è residuo agarosio nel fondo foro. </li><li> Riempire la piastra Petri con Media embrione. </li><li> Utilizzare una pipetta di vetro al posto dell&#39;embrione nel foro. </li><li> Quindi, orientare l&#39;embrione utilizzando una pinza sottile in modo che l&#39;area da acquisire (testa dorsale) è in contatto con il vetro. </li><li> Cura il trasporto scatola di Petri alla fase microscopio (posizionare il coperchio sulla piastra di Petri per prevenire l&#39;evaporazione). </li><li> Mentre imaging, in modo che gli embrioni sono a 28.5 ° C. </li></ol> 4. Risultati Descriviamo qui un approccio diretto alle cellule singola immagine nel tubo neurale utilizzando l&#39;espressione zebrafish mosaico. Nonostante la trasparenza ottica dell&#39;embrione zebrafish, abbiamo trovato che la visualizzazione di cellule neurali è notevolmente migliorata in sezioni ottenuti utilizzando un vibratome. Queste sezioni sono di spessore (50 mm) che consente di estensione per l&#39;imaging cellulare da una data cellula in diversi piani focali utilizzando un microscopio confocale. I risultati di tale metodologia sono stati pubblicati altrove 2, ma immagini rappresentative delle cellule del tubo neurale di un embrione WT (Figura 1) e di un N-caderina (N-cad) mutante (Figura 2) sono forniti. Quest&#39;ultimo rivela che le cellule nelle regioni laterali della piastra neurale in N-cad mutanti non riescono ad orientarsi correttamente verso la linea mediana (Figura 2). In contrasto con questa etichettatura mosaico, immunostaining il neuroepitelio con un indicatore generale di superficie cellulare, come il beta-catenina non consente la morfologia delle singole cellule da visualizzare (Figura 3). Lasso di tempo di imaging di embrioni vivi completato l&#39;etichettatura mosaico di campioni fissati, permettendo una migliore comprensione delle dinamiche cellulari che avvengono durante neurulazione. Movie 1 ha rivelato che le cellule WT attivamente migrano verso la linea mediana, estendendo medialmente orientata sporgenze membrana. <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="Figura 1" /> Figura 1. MGFP espressione nel tubo neurale di un embrione di zebrafish WT. <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="Figura 2" /> Figura 2. MGFP espressione nel tubo neurale di un N-caderina embrione zebrafish. <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="Figura 3" /> Figura 3. B-catenina colorazione immuno del neuroepitelio.

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Practical approach series. , (2002).</li><li>Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+is+required+for+the+polarized+cell+behaviors+that+drive+neurulation+in+the+zebrafish.">N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.</a> Development. 133, 3895-3905 (2006).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Motility+and+Actin+Cytoskeleton+of+Individual+Neurons+and+Neural+Crest+Cells+in+Zebrafish+Embryos.">Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos.</a> J Vis Exp. , (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

In conclusione, le tecniche descritte qui di etichettatura consentono di analisi singola cella dei processi morfogenetici nell&#39;embrione zebrafish. L&#39;enfasi principale di questo protocollo è sui metodi di imaging per le cellule etichettate nel tubo neurale utilizzando mGFP sotto il controllo di un promotore ubiquitario. Per ulteriori applicazioni di questa analisi lettori transitoria espressione deve fare riferimento ad un recente articolo da Andersen et al. 3.

A correction was made to <a href="http://www.jove.com/index/Details.stp?ID=1976">Labeling and Imaging Cells in the Zebrafish Hindbrain</a>. There was an error in the authors affiliations. The authors have been corrected to:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster1 1Department of Biological Sciences, University of Maryland, Baltimore County 2Center for Neuroscience, Children's National Medical Center
instead of:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster2 1Center for Neuroscience, Children's National Medical Center 2Department of Biological Sciences, University of Maryland Baltimore County

A correction to the author affiliations has been made for the article Labeling and Imaging Cells in the Zebrafish Hindbrain.

Erratum: Labeling and Imaging Cells in the Zebrafish Hindbrain

Soluzioni <ul><li> Archivi di Hank # 1: 8,0 g NaCl, 0,4 g di KCl in 100 ml gg H 2 O </li><li> Archivi di Hank # 2: 0,358 g di Na 2 HPO 4 anidro, 0,60 g di KH 2 PO 4 in 100 ml DDH 2 O </li><li> Archivi di Hank # 4: 0,72 g di CaCl 2 in 50 ml DDH 2 O </li><li> Archivi di Hank # 5: 1,23 g MgSO 4 x7H 2 O in 50 ml gg H 2 O </li><li> 1 X tampone fosfato: 0,8% di NaCl, 0,02% KCl, 0.02 M PO4, pH 7,3 </li><li> Soluzione di bloccaggio: 2% di siero normale di capra, 1% di sieroalbumina bovina (BSA), dimethysulfoxide 1% (DMSO) </li></ul>

labeling and imaging cells in the zebrafish hindbrain

Chiave per comprendere i processi morfogenetici che forma l&#39;embrione precoce vertebrati è la capacità di celle di immagine ad alta risoluzione. In embrioni di zebrafish, l&#39;iniezione di risultati del DNA plasmide di espressione mosaico, consentendo la visualizzazione di singole cellule o piccoli gruppi di cellule<sup&gt; 1</sup&gt;. Ci descrivono come iniezione di codifica del DNA plasmidico membrana mirati proteina verde fluorescente (mGFP) sotto il controllo di un promotore ubiquitario può essere utilizzata per le celle di imaging in fase neurulazione. Al centro di questo protocollo è la metodologia di imaging per le cellule etichettate ad alta risoluzione e anche nelle sezioni in tempo reale. Questo protocollo prevede l&#39;iniezione di DNA mGFP in embrioni di zebrafish giovani. Gli embrioni vengono poi elaborati per il sezionamento vibratome, l&#39;etichettatura degli anticorpi e di imaging con un microscopio confocale. In alternativa, embrioni vivi esprimendo mGFP è possibile creare immagini utilizzando time-lapse microscopia confocale. Abbiamo già utilizzato questo metodo semplice per analizzare i comportamenti cellulari che guidano la formazione del tubo neurale nella regione romboencefalo di embrioni di zebrafish<sup&gt; 2</sup&gt;. I preparativi fisso permesso per la visualizzazione senza precedenti di forme e di organizzazione delle cellule del tubo neurale, mentre immagini dal vivo accompagnato questo approccio consente una migliore comprensione delle dinamiche cellulari che avvengono durante neurulazione.

Watch this Scientific Journal Video about Labeling and Imaging Cells in the Zebrafish Hindbrain at JoVE.com

Labeling and Imaging Cells in the Zebrafish Hindbrain

Chiave per comprendere i processi morfogenetici che forma l&#39;embrione precoce è la capacità di celle di immagine ad alta risoluzione. Descriviamo qui una tecnica per l&#39;etichettatura di singole cellule o piccoli gruppi di cellule in embrioni di zebrafish intero con membrana mirati proteina verde fluorescente.

Etichettatura e cellule Imaging nel romboencefalo Zebrafish

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

labeling-and-imaging-cells-in-the-zebrafish-hindbrain

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11.2K Views.  University of Maryland, Baltimore County. Chiave per comprendere i processi morfogenetici che forma l'embrione precoce è la capacità di celle di immagine ad alta risoluzione. Descriviamo qui una tecnica per l'etichettatura di singole cellule o piccoli gruppi di cellule in embrioni di zebrafish intero con membrana mirati proteina verde fluorescente.

Video: Labeling and Imaging Cells in the Zebrafish Hindbrain

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

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Neuroscienze

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Elettroporazione del romboencefalo tracciare traiettorie assonale e Obiettivi Synaptic in embrione di pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etichettatura retrograda di cellule gangliari della retina in Zebrafish adulti con coloranti fluorescenti

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

Il Hindbrain Mouse come modello di studio della neurogenesi embrionale

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Nel calcio in Vivo Imaging delle cellule ciliate linea laterale in Zebrafish larvale

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificazione dei marcatori di superficie cellulare delle cellule staminali primarie e delle cellule progenitrici mediante l'etichettatura metabolica di Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Etichettatura e imaging di placche amiloidi nel tessuto cerebrale utilizzando la curcumina polifenolo naturale

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Imaging in vivo del tessuto cerebrale completamente attivo in larve di zebrafish svegli e giovani mediante rimozione del cranio e della pelle

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...