Questo lavoro è stato sostenuto dal National Institute for Mental Health, e la National Science Foundation. Un ringraziamento speciale ai membri di laboratorio esclusivo per molte utili discussioni e critiche costruttive, e per Olivier Paugois per la zootecnia, esperto di pesce.

1. Preparazione per Microiniezione <ol><li> Preparare aghi microiniezione tirando tubi capillari con Sutter estrattore ago strumenti. </li><li> Carico ago microiniezione con colorante fluorescente (FITC-destrano). </li><li> Montare ago su micromanipolatore e apparecchi microiniezione. </li><li> Spezzare con cautela l&#39;ago microiniezione con pinze a circa 2 micron di larghezza, tuttavia, questo può variare a seconda della configurazione microiniettore. Per i nostri aghi microiniezione, questo corrisponde alla prima regione dell&#39;ago dalla punta che non si pieghi. </li><li> Misurare la dimensione delle goccioline di olio, la regolazione del tempo di iniezione e la pressione, in modo che ogni iniezione eroga 1 nl. Impostazioni di esempio per Harvard Apparatus microiniettore sono: equilibrio P = 1,4 psi, P out = 1,4 psi, iniettare P = 22,9 psi, P = 67,8 psi chiare con un tempo di iniezione di 0,4-0,7 sec. Il diametro del nostro ago utilizzando queste impostazioni è di circa 2 micron. Tuttavia, le impostazioni saranno microiniettore specifici e varianoecondo l&#39;ago di diametro. </li></ol> 2. Preparazione dei Embrioni <ol><li> Cappotto 2 piatti con 1% di agarosio in acqua per ogni condizione, fori curiosare in agarosio con una punta di pipetta 1-200 microlitri, e rimuovere i tappi di agarosio. Riempire i piatti con i media embrioni. </li><li> Con le pinzette, embrioni dechorionate che sono 18 anni o più hpf allo stereomicroscopio. Gli embrioni si svolgono secondo Kimmel et al. 11. </li><li> Trasferire gli embrioni dechorionated nel primo piatto rivestito di agarosio. </li><li> Per anestetizzare embrioni, aggiungere tricaine (0,1 mg / ml) al piatto fino embrioni interrompe il movimento (fatta secondo Westerfield 12). </li></ol> 3. Iniettare i ventricoli cerebrali <ol><li> Embrioni Orient così che stai cercando al loro fianco dorsale mettendo la coda dell&#39;embrione nel foro. Se la vostra micromanipolatore è sulla destra, quindi spostare l&#39;embrione in modo che il prosencefalo è a sinistra e romboencefalo verso destra. </li><li> Ago Situazione al wipunto dest del ventricolo rombencefalo. </li><li> Attenzione forare lamiera del tetto del ventricolo romboencefalo assicurandosi di non passare attraverso la profondità del cervello nel tuorlo (Figura 1A). </li><li> Iniettare 1-2 nl di colorante fluorescente nei ventricoli assicurandosi che il colorante riempie l&#39;intera lunghezza dei ventricoli cerebrali. </li><li> Trasferire gli embrioni al secondo piatto agarosio rivestita riempito con i media embrioni e ri-anestetizzare, come descritto al punto 2.4. </li><li> Immediatamente iniziare imaging, come descritto nella sezione 4 per ottenere una immagine tempo zero. </li></ol> 4. Imaging <ol><li> Embrioni Orient con la coda nel buco, come descritto al punto 3.1. </li><li> Utilizzare un microscopio da dissezione con la luce sia trasmessa e fluorescenza permette di registrare un&#39;immagine dorsale campo chiaro. Mantenere l&#39;ingrandimento costante tra immagini di embrioni differenti. In questo modo per un confronto diretto di analisi eseguite con Image J (5,2-6). </li><li> Senza spostare il, microscopio embrione o un piatto, prendere uncorrispondente immagine fluorescente. </li><li> Ripetere l&#39;operazione per ogni embrione in momenti desiderati. </li></ol> 5. Quantificazione del Movimento Dye <ol><li> Unire chiaro e immagini fluorescenti in Photoshop come precedentemente descritto da Gutzman e Sive 10. </li><li> Misurare la distanza si muove fronte del colorante in software Image J disponibili <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> . </li><li> Apri il file unito in J Immagine e utilizzare lo strumento Linea per disegnare una linea dal prosencefalo cerniera-point per tingere frontale con un angolo di 10-20 ° da neuroepitelio (Figura 1A). Questa regione è stata scelta perché è sul primo e più evidente di colorante fuoriuscita del neuroepitelio tipo selvatico. </li><li> Selezionare lo strumento di misurazione per calcolare la lunghezza della linea. </li><li> Ripetere per ogni time-point. </li><li> Calcolare distanza netta il fronte del colorante spostato nel tempo sottraendo distanza per t = 0 da altri punti temporali. </li><li> Tracciate sugrafico. </li></ol> 6. Risultati rappresentativi Un esempio dei risultati ottenuti in un saggio di permeabilità neuroepiteliale utilizzando embrioni di tipo selvatico è mostrata in Figura 1B-D. Per differenziare accuratamente permeabilità, è utile per testare con coloranti diversi weightsto molecolare identificare una dimensione che è solo leggermente permeabile nel tipo selvatico o embrioni di controllo (Figura 2). Questo permette l&#39;identificazione di mutanti genetici o condizioni ambientali che aumentare o diminuire la permeabilità (Figura 1D, linee verde e rosso rispettivamente). Per il neuroepitelio 24 HPF zebrafish, 70 kDa FITC perdite Destrano lentamente nel corso di 2 ore, mentre 2.000 kDa non e 10 kDa quasi subito perdite fuori. Quindi 70 kDa è il peso ideale molecolare per identificare le condizioni che aumentano e diminuiscono la permeabilità neuroepiteliale. Se l&#39;ago non trova la lume ventricolare, fluorescenza wmalato apparire al di fuori del cervello per t = 0 (per un esempio vedere Gutzman e Sive, 2009 10). Questi embrioni dovrebbero essere scartata poiché il colorante non è stato iniettato inizialmente contenuta all&#39;interno del cervello e nessuna conclusione chiara riguardo movimento del colorante e la permeabilità della neuropeithelium può essere fatto. Infine, se gli embrioni hanno ventricoli piccoli o non gonfiati ventricoli cerebrali, pre-iniezione di ventricoli con una soluzione salina può essere fatto prima della iniezione del colorante fluorescente. Questo gonfia i ventricoli che fanno successiva visualizzazione dei ventricoli più facile quando si inietta con il colorante fluorescente. Opportuni controlli devono essere eseguiti per determinare se l&#39;iniezione di soluzione salina sconvolge il normale sviluppo del tubo neurale. <img alt="Figura 1" src="/files/ftp_upload/4242/4242fig1.jpg" /> Figura 1. Naturalmente il tempo di diversi coloranti peso molecolare. (A) Diagramma sperimentale. Prima, Colorante fluorescente viene iniettato nei ventricoli. X = posizione di ago per l&#39;iniezione. Successivo immagini dorsali vengono acquisite nel corso del tempo. Infine, la distanza percorsa dal fronte del colorante del proencefalo viene misurata cerniera-point (rappresentata da una linea rossa). (BC) Fusione chiaro e fluorescenti immagini dorsali a 22 HPF (t = 0 min, B) e 24 HPF (t = 120 min, C). Linea bianca indica la distanza del fronte del colorante dal ventricolo proencefalo. (D) di dati ipotetici permeabilità del campione. Blu = tipo selvaggio o di controlli, rosso = campione con permeabilità ridotta rispetto al controllo, e il verde del campione = con aumento della permeabilità rispetto al controllo. <img alt="Figura 2" src="/files/ftp_upload/4242/4242fig2.jpg" /> Figura 2. La misura della permeabilità neuroepiteliale a diversi coloranti peso molecolare. (AE) brightfield dorsale dalla fusione e immagini fluorescenti di 22 embrioni di tipo HPF selvatico al tempo t = 0 min dopo l&#39;iniezione con FITC-dExtran dei pesi molecolari seguenti: 10 kDa (A), 40 kDa (B), 70 kDa (C), 500 kDa (D) e (E 2000 kDa). (A&#39;-E &#39;) stesso embrione come in (AE) a t = 120 minuti a 24 HPF. Anteriore a sinistra. F = proencefalo, M = mesencefalo, H = rombencefalo. Asterisk = orecchio.

<ol>
	<li>Harrington, M. J., 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=Comparative+analysis+of+neurulation:+first+impressions+do+not+count.">Comparative analysis of neurulation: first impressions do not count.</a> Mol. Reprod. Dev. 76, 954-965 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+vertebrate+neurulation+and+a+re-evaluation+of+teleost+neural+tube+formation.">Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation.</a> Mech. Dev. 121, 1189-1197 (2004).</li><li>Salehi, Z., Mashayekhi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cerebrospinal+fluid+on+neural+cell+survival+in+the+developing+chick+cerebral+cortex:+an+in+vivo+study.">The role of cerebrospinal fluid on neural cell survival in the developing chick cerebral cortex: an in vivo study.</a> Eur. J. Neurol. 13, 760-764 (2006).</li><li>Martin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryonic+brain+development+in+rats+requires+the+trophic+influence+of+cerebrospinal+fluid.">Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid.</a> Int. J. Dev. Neurosci. 27, 733-740 (2009).</li><li>Lehtinen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebrospinal+fluid+provides+a+proliferative+niche+for+neural+progenitor+cells.">The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.</a> Neuron. 69, 893-905 (2011).</li><li>Gato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cerebrospinal+fluid+regulates+neuroepithelial+survival,+proliferation,+and+neurogenesis+in+chick+embryos.">Embryonic cerebrospinal fluid regulates neuroepithelial survival, proliferation, and neurogenesis in chick embryos.</a> Anat. Rec. A. Discov. Mol. Cell Evol. Biol. 284, 475-484 (2005).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Totally+tubular:+the+mystery+behind+function+and+origin+of+the+brain+ventricular+system.">Totally tubular: the mystery behind function and origin of the brain ventricular system.</a> Bioessays. 31, 446-458 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+neuroepithelial+barrier+by+Claudin5a+is+essential+for+zebrafish+brain+ventricular+lumen+expansion.">Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 1425-1430 (2010).</li><li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+brain+ventricle+injection.">Zebrafish brain ventricle injection.</a> J. Vis. Exp. , (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M., Sprague, J., Doerry, E., Douglas, S., Grp, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Information+Network+(ZFIN):+a+resource+for+genetic,+genomic+and+developmental+research.">The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.</a> Nucleic Acids Research. 29, 87-90 (2001).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo dimostrato la capacità di quantificare permeabilità del cervello vivente zebrafish embrionale come determinato per un colorante iniettato di un dato peso molecolare. Nostra osservazione che neuroepitelio zebrafish embrionale è differenzialmente permeabile ai coloranti di differente peso molecolare suggerisce che il colorante si muove attraverso permeabilità paracellulare. Tuttavia, non si può escludere la possibilità di un contributo transcellulare alla permeabilità osservato. Questa tecnica può essere applicata a qualsiasi altra struttura tubolare purché sia ​​all&#39;interno che all&#39;esterno del tubo può essere visto e il lume può essere iniettato. Tuttavia, l&#39;identificazione del peso molecolare ideale per altri organi dovranno essere determinato come questo può variare tra tessuti e stadi di sviluppo. Questa analisi consentirà di ulteriori indagini sul ruolo della permeabilità epiteliale durante il gonfiaggio del lume e la regolazione della dimensione del lume. Inoltre, questa tecnica lo faràlp caratterizzare le alterazioni della permeabilità epiteliale associato a disturbi come l&#39;idrocefalo e la malattia del rene policistico.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Destrano, fluoresceina, anionico, Lisina fissabile </td><td> Invitrogen </td><td> D7136, D7137, D1822, D1820, D1845 </td></tr><tr><td> Tricaine polvere </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Tubi capillari </td><td> FHC Inc. </td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

Il sistema ventricolare cerebrale è conservata tra vertebrati ed è composto da una serie di cavità interconnesse chiamati ventricoli cerebrali, che si formano durante le prime fasi di sviluppo del cervello e si mantengono per tutta la vita dell&#39;animale. Il sistema ventricolare cerebrale si trova in vertebrati, e ventricoli svilupparsi dopo la formazione del tubo neurale, quando il lume centrale riempie di liquido cerebrospinale (CSF) 1,2. CSF è un liquido ricco di proteine, che è essenziale per il normale sviluppo del cervello e funzione 3-6. In zebrafish, l&#39;inflazione ventricolo del cervello inizia a circa 18 ore dopo la fecondazione (HPF), dopo che il tubo neurale è chiuso. Più processi sono associati con la formazione del cervello ventricolo, compresa la formazione di un neuroepitelio, formazione di giunzione a tenuta che regola la permeabilità e la produzione di CSF. Abbiamo mostrato che il Na, K-ATPasi è richiesto per gonfiaggio ventricolo cerebrale, impattando tutti questi processies 7,8, mentre la 5a claudina è necessario per la formazione di giunzione a tenuta 9. Inoltre, abbiamo dimostrato che &quot;rilassamento&quot; del neuroepitelio embrionale, attraverso l&#39;inibizione della miosina, è connesso all&#39;inflazione ventricolo cerebrale. Per studiare la regolazione della permeabilità durante il gonfiaggio ventricolo cerebrale zebrafish, abbiamo sviluppato un colorante test ventricolare ritenzione. Questo metodo utilizza l&#39;iniezione ventricolo del cervello in un embrione di zebrafish vivente, una tecnica già sviluppata nel nostro laboratorio 10, di etichettare fluorescente liquido cerebrospinale. Gli embrioni vengono poi ripresi nel corso del tempo come si muove il colorante fluorescente attraverso i ventricoli cerebrali e neuroepitelio. La distanza del fronte del colorante si allontana dal basale (non-luminale) lato del neuroepitelio nel tempo è quantificato ed è una misura della permeabilità neuroepiteliale (Figura 1). Osserviamo che i coloranti 70 kDa e piccoli si muoverà attraverso il neuroepitelio e può essere detected fuori del cervello zebrafish embrionale a 24 HPF (Figura 2). Questo saggio ritenzione colorante può essere utilizzato per analizzare permeabilità neuroepiteliale in una varietà di differenti contesti genetici, in momenti diversi durante lo sviluppo, e dopo perturbazioni ambientali. Può anche essere utile per esaminare accumulo patologico di CSF. Nel complesso, questa tecnica consente agli investigatori di analizzare il ruolo e la regolazione della permeabilità durante lo sviluppo e la malattia.

Watch this Scientific Journal Video about An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium at JoVE.com

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Descriviamo un animale vivo misurazione quantitativa per tutta la permeabilità del cervello zebrafish embrionale. La tecnica analizza la capacità di mantenere fluido cerebrospinale e molecole di differenti pesi molecolari all&#39;interno del lume del tubo neurale e quantifica loro movimento fuori dei ventricoli. Questo metodo è utile per determinare differenze di permeabilità epiteliale e maturazione durante lo sviluppo e la malattia.

Un saggio per permeabilità del neuroepitelio Zebrafish embrionali

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

an-assay-for-permeability-of-the-zebrafish-embryonic-neuroepithelium

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.4K Views.  Massachusetts Institute of Technology. Descriviamo un animale vivo misurazione quantitativa per tutta la permeabilità del cervello zebrafish embrionale. La tecnica analizza la capacità di mantenere fluido cerebrospinale e molecole di differenti pesi molecolari all'interno del lume del tubo neurale e quantifica loro movimento fuori dei ventricoli. Questo metodo è utile per determinare differenze di permeabilità epiteliale e maturazione durante lo sviluppo e la malattia.

Video: An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Ricerca

Neuroscienze

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Il saggio neuroblasti: un test per la generazione e arricchimento delle cellule progenitrici neuronali di differenziare i Progeny cellule staminali neurali in citometria a flusso

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imaging ad alta risoluzione dal vivo del comportamento delle cellule in via di sviluppo neuroepitelio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Drenaggio manuale dei ventricoli cerebrali embrionali zebrafish

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo Imaging in diretta di divisioni cellulari singoli in mouse neuroepitelio

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Bloccare Registrazioni patch da cellule embrionali Zebrafish Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un test per misurare gli effetti di etanolo sulla velocità di Locomotion<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sviluppo di analisi per la quantificazione alto contenuto di formazione aggregata di Sod1 mutante della proteina in cellule viventi

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...